WO2015025054A1

WO2015025054A1 - Dye-specific antibodies for prestained molecular weight markers and methods producing the same

Info

Publication number: WO2015025054A1
Application number: PCT/EP2014/067948
Authority: WO
Inventors: Egon OGRIS; Stefan SCHÜCHNER
Original assignee: Medizinische Universität Wien
Priority date: 2013-08-22
Filing date: 2014-08-22
Publication date: 2015-02-26

Abstract

The present invention relates to a method for producing an antibody that specifically recognizes a dye which is a portion of a dye/protein complex by selectively eliciting an immune response against the dye. The present invention also relates to antibodies produced by the method of the invention, in particular to antibodies that specifically recognize a dye which is a portion of a dye/protein (marker) complex. Further, an antibody which specifically recognizes the dye Remazol Brilliant Blue R as well as an antibody that is produced by the hybridoma 2D2-F11 deposited at the Leibnitz-lnstitut DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen) Braunschweig under No: DSM ACC3212 is provided herein. The present invention also relates to uses of an antibody of the invention, inter alia in the detection of one or more dye-prestained protein molecular weight marker proteins. Finally, a kit comprising the antibodies of the present invention is provided.

Description

Dye-specific antibodies for prestained molecular weight markers and methods producing the same

The present invention relates to a method for producing an antibody that specifically recognizes a dye which is a portion of a dye/protein complex by selectively eliciting an immune response against the dye, wherein the method comprises the steps of (i) immunizing a non-human animal with a first dye/protein complex, (ii) subsequently immunizing said non-human animal with a second dye/protein complex wherein the protein portion of the dye/protein complex is similar or different than the protein portion of the dye/protein complex used in step (i); (iii) optionally repeating step (ii) at least once with a further dye/protein complex wherein the protein portion of the dye/protein complex is similar or different than the protein portion of the dye/protein complex used in step (i) and any subsequent step; (iv) optionally immunizing said non-human animal with a mixture of all dye/protein complexes used in step (i) and/or (ii) and/or (iii); (v) obtaining/isolating said dye-specific antibody, wherein said dye- specific antibody is obtained/isolated by screening using a dye/protein complex that has not been used in the elicitation of the immune response and wherein the protein portion of the dye/protein complex is similar or different than the protein portion of the dye/protein complex used in step (i) and/or (ii) and/or (iii) and/or (iv). The present invention also relates to antibodies produced by the method of the invention, in particular to antibodies that specifically recognize a dye which is a portion of a dye/protein (marker) complex. Further, an antibody which specifically recognizes the dye Remazol Brilliant Blue R as well as an antibody that is produced by the hybridoma 2D2-F11 deposited at the Leibnitz-lnstitut DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen) Braunschweig under No: DSM ACC3212 is provided herein. The present invention also relates to uses of an antibody of the invention, inter alia in the detection of one or more dye-prestained protein molecular weight marker proteins. Finally, a kit comprising the antibodies of the present invention is provided.

A key technique in molecular biology is the electrophoretic separation of molecules, like e.g. proteins, nucleic acids, lipids or carbohydrates with the help of carrier matrices like agarose or polyacrylamide. The most frequently adopted method for the separation of proteins is the so called SDS polyacrylamide gel electrophoresis (SDS- PAGE), by which proteins are separated depending on/according to their molecular weight. To determine or at least estimate the molecular weight of a given protein, it is necessary to compare the migration distance of the protein of unknown molecular weight with the migration distance of proteins of known molecular weights. These proteins are so called protein molecular weight markers or standards and are electrophoretically separated together with the proteins to be analysed. A non- stained protein size marker ladder is, e.g., described in US 5,449,758. Moreover, in DE 102 44 502 B4 molecular weight markers and methods for producing such markers are described while it is mentioned that said protein markers can be transferred onto a membrane and be detected by antibodies against the protein marker. To be able to monitor the migration of the molecular weight markers during electrophoresis, these proteins are commonly covalently coupled to the blue dye Remazol Brilliant Blue R or the vinyl sulfone derivative of Remazol Brilliant Blue R, i.e., Uniblue A (Sigma). These dyes are recognized by the human eye as colour (or as black or as white) upon illumination with visible light which ranges from approximately 380 to 800 nm.. Less often, these proteins are commonly covalently coupled to other different-coloured Remazol derivatives like e.g. Remazol Turquoise, Brilliant Red F3B, Brilliant Orange 3R, or Golden Yellow RNL. As an example, a protein marker and a ladder that contains a series of different markers is described in WO 2006/138366 A2 wherein the described protein marker is a product of a protein covalently bound to dye(s). Antibodies or antisera, which are specifically directed against a particular protein, are used to analyze this protein in a protein mixture (e.g. a whole cell lysate), which has been electrophoretically separated. For this purpose, the SDS-PAGE separated proteins are electro-transferred to a carrier membrane (e.g. nitrocellulose or polyvinyliden fluoride [PVDF]), where they can be detected with a specific antibody. This technique is called Western blot or immunoblotting. Immunoblotting is not always required if an in-gel Western blot is carried out. A particular protein is made visible by incubation of the membrane with a primary antibody (in most cases a mouse, rat, goat or a rabbit antibody), which in turn is detected by a secondary antibody, which is directed against mouse, rat, goat or rabbit antibodies and which is coupled to the enzyme horseradish peroxidase (HRP) (or, alternatively, to a fluorescence dye). This enzyme catalyzes the oxidation of luminol leading to the emission of light (chemoluminescence), which then can be detected on X-ray films or with the help of CCD camera-based systems. However, the blue prestained molecular weight markers do not emit any light and are therefore not displayed on the X-ray films. To determine/estimate the molecular weight of the protein recognized by the antibody, it is necessary afterwards (after the emitted light has been detected on the X-ray film) to manually mark the marker protein bands on the X-ray film. This is done by placing the film on the membrane and requires the perfect positioning of the two components. This carries the difficulty that the contours of the membrane are mostly not apparent on the film and thus reference points are lacking. Another source of error is the experimenter and his/her accuracy in mapping the shape of the molecular weight markers on the film. Recently, the company Abeam has put on the market a so called luminol pen (Optiblot Luminol Membrane Pen), with which the marker protein bands can be manually marked on the membrane and subsequently be detected on an X-ray film. The disadvantage of this is again the fact that it requires to manually mark the molecular weight markers, which - as described above - is one of the most common sources of error. Thermo Fisher Scientific on the other hand offers molecular weight markers (Thermo Scientific PageRuler Prestained NIR Protein Ladder), which are marked with a blue dye as well as a fluorescence dye and which can therefore be directly detected by a Western blot analysis. To do so, however, one needs a scanner (e.g. LiCOR, Odyssey, or GE Healthcare Life Sciences, Typhoon), which is a very expensive acquisition (> 50K€) and only available in few laboratories. Hence, the detection of proteins by X-ray films still represents the most popular Western blot method used today.

Antibodies that specifically and directly detect the dye(s) of a protein molecular weight marker that is recognized, perceived and/or detected by the human eye as (a) colour(s) (or as black or as white) upon illumination with visible light which ranges from approximately 380 to 800 nm.have not been disclosed in the prior art. This is not entirely surprising, since Saoji et al. (Clin. Chem. 30/7: 1252-1254 (1984)) raised monospecific (= protein specific) antibodies by using protein components which were prestained with Remazol Brilliant Blue and discussed in this publication that the dye itself is non-antigenic. In humans, the generation of antibodies against reactive dye- human serum albumin conjugates has been described in workers of dye factories, wherein this immunological response is correlated with asthmatic reactions (Park et al., Journal of Korean Medical Science 6(1 ):63-68 (1991 )). Moreover, respiratory allergy has been correlated with specific IgE and IgG antibodies against reactive dye/albumin-conjugates in workers in the wool industry while the antibodies detect dye-albumin conjugates (Topping et al., Journal of Occupational Medicine 31(10): 857-862 (1989)). Moreover, antibodies against conjugates between reactive dyes and human serum albumin have been detected in workers of textile plants with dyehouses, who have been exposed to reactive dyes and who suffer from asthma, rhinitis and dermatitis. (Nielsson et al., British Journal of Industrial Medicine 50: 65- 70( 993)). However, antibodies that specifically detect the dye(s) of a protein/dye complex which are perceived by the human eye as colour (or as black or as white) upon illumination with visible light which ranges from approximately 380 to 800 nm have not been disclosed in the prior art.

Thus, in the light of the above disadvantages of the presently available systems there is a need to improve the determination and estimation of the molecular weight of (a) given protein(s) recognized by the antibody in immunological and in immuno- research methods, in particular on solid phase technologies, like on Western blots and/or on other immunoblots. Accordingly, the problem underlying the present invention is the provision of means and methods for a more precise and reliable determination and estimation of the molecular weight of (a) given protein(s) recognized by the antibody in, inter alia, solid phase immunological technologies, in particular immunological methods like Western blots or immunoblots. These means and methods are suitable for routine biomedical or biological research or other research involving solid phase immunological methods, like in Western blot analysis, in chemoluminescence and/or immunoblotting analysis in an averagely equipped laboratory.

The present invention addresses this need by providing the embodiments as defined in the claims. Thus, it is the gist of the present invention to provide antibodies which are specifically raised against a certain dye of (a) marker protein(s), said dye being recognized, perceived and/or detected by the human eye as colour (or as black or as white) upon illumination with visible light which ranges from approximately 380 to approximately 800 nm. The antibodies as provided herein and/or as generated by the methods of this invention allow to directly display (involving, e.g., a luminol-based light reaction) the marker bands, for example on X-ray films. In particular, the present invention allows the generation of antibodies, in particular (a) monoclonal antibody(ies), which specifically recognize(s) the dye of prestained protein molecular weight markers. Such antibodies may be used, inter alia, in the simultaneous detection of prestained protein molecular weight markers as well as of any (other) protein of choice, for example, by a second specific antibody in solid phase immunological technologies, like Western blotting and/or chemoluminescence analysis.

The present invention is not only limited to dyes of marker proteins and, accordingly, not only limited to antibodies and methods for producing antibodies that specifically recognize a dye which is a portion of a covalently linked dye/protein marker complex by specifically eliciting an immune response against the dye. Rather, the present invention also in more general terms relates to other dye/protein complexes which do not necessarily have to be marker proteins. As such, antibodies and methods for producing an antibody are also provided wherein the antibodies specifically recognize a dye which is a portion of a dye/protein complex by selectively eliciting an immune response against the dye. Accordingly, the present invention provides methods for producing antibodies that specifically recognize a dye which is a portion of a dye/protein complex by selectively eliciting an immune response against the dye.

The present invention in particular also provides a monoclonal antibody (clone 2D2- F11 ), which is raised against the dye of prestained molecular weight marker proteins. Surprisingly, as it is exemplified in the examples, the antibody does not show a reactivity with the (unstained) protein of the prestained molecular weight marker. Thus, the present invention provides means (i.e. antibodies) which specifically recognize a dye which is a portion of a covalently dye/protein complex by selectively eliciting an immune response against the dye/protein complex but not against the unstained protein of the dye/protein complex. This is entirely unexpected in the light of the prior art since Saoji et al. (Clin. Chem. 30/7: 1252-1254 (1984)) raised monospecific antibodies by using protein components which are prestained with Remazol Brilliant Blue and it was emphasized that the dye itself is non-antigenic.

As it is illustrated in the examples, the dye-specific (monoclonal) antibodies of the present invention, like the 2D2-F11 antibody, which specifically recognizes a dye which is a portion of a covalently dye/protein complex can be used to detect the said prestained molecular weight marker proteins in a Western blot analysis and can be made visible by a horseradish peroxidase catalyzed chemoluminescence reaction followed by detection on X-ray films (or with the help of CCD camera systems).

As illustrated in more detail in the examples, the dye-specific monoclonal antibodies of the present invention which specifically recognize a dye which is a portion of a covalently dye/protein complex is generated by selectively eliciting an immune response against the dye. Selectively eliciting an immune response against the dye is achieved by immunizing mice with blue dye prestained (covalently coupled to) marker proteins of the BioRad Precision Plus Protein™ All Blue Standard (161-0373). In order to obtain an antibody that specifically detects the dye the proteins of the BioRad Precision Plus Protein Standard marker mixture, which consists of 10 different proteins (10, 15, 20, 25, 37, 50, 75, 100, 150, 250 kD), are separated by SDS-PAGE, the bands corresponding to the 25, 50 and 75 kD marker proteins are cut out and electro-eluted in separate electro-elution chambers. To evoke in non- human animals an immune response specific for the blue dye of the marker proteins, the first immunization is performed with the 50 kD marker protein. This is followed by a booster immunization with the 75 kD and a third boost with the 25 kD marker. Surprisingly, as shown in the examples, it has been demonstrated that this immunization schedule impedes a secondary immune response against particular marker proteins and promotes a strong secondary immune response against the dye common to all marker proteins. A final boost with a mixture of all three (25, 50 and 75 kD) marker proteins may further enhance the specificity.

As demonstrated in the examples, the immune sera of the immunized mice contained antibodies which advantageously not only recognize the 25, 50 and 75 kD marker proteins, which had been used for the immunizations, but which also detected all other marker proteins of the BioRad Precision Plus Protein™ All Blue Standard. The mouse with the highest antibody titer was selected for the fusion of splenocytes with X63-Ag8.653 myeloma cells, from which the hybridoma single clone 2D2-F11 (deposited at the Leibnitz-lnstitut DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) Braunschweig, Inhoffenstr. 7B, D-38124 Braunschweig under No: DSM ACC3212 on July 24, 2013) was established.

As demonstrated in the examples, the dye-specific monoclonal antibodies of the present invention, like the 2D2-F11 antibody, which specifically recognize a dye which is a portion of a covalently dye/protein complex exhibit numerous advantageous properties which are briefly summarized. Firstly, the dye-specific monoclonal antibodies of the present invention, like the 2D2-F11 antibody, which specifically recognize a dye which is a portion of a covalently dye/protein complex advantageously detect specifically all blue dye-stained but not any red dye- or green dye-stained or unstained protein marker bands of various commercially available protein marker mixtures Moreover, importantly, the antibodies of the present invention do not show any unspecific cross-reactivity with unstained proteins in whole cell lysates of a wide range of organisms like bacteria, yeast, monkey, mouse, rat, hamster, chicken or man. The lack of any cross-reactivity with unstained proteins together with the detection of prestained protein molecular weight markers is the indispensable prerequisite for the routine application of this antibody in Western blot analysis. Furthermore, it is demonstrated that a Western blot can at the same time be incubated with a dye-specific monoclonal antibodies of the present invention, like the 2D2-F11 antibody, which specifically recognize a dye which is a portion of a covalently dye/protein complex in combination with other mono- as well as polyclonal antibodies against other proteins, e.g. against the hemagglutinin (HA) tag, myc-tag or endogenous proteins like protein phosphatase 2A (PP2A) methyl esterase PME-1 , Lamin A/C, PP2A catalytic subunit C (PPP2CA), PP2A regulatory subunit B56 (PPP2R5A), or the yeast proteins Cdc55 (Cell Division Cycle 55) or Net1 (Nucleolar silencing Establishing factor and Telophase regulator 1) without any adverse cross- reactions. Thus, the dye-specific monoclonal antibodies of the present invention, like the 2D2-F11 antibody, which specifically recognizes a dye which is a portion of a covalently dye/protein complex is capable of selectively detecting the dye without interfering with the detection of these proteins. It is demonstrated that the blue dye used by the companies BioRad, Fermentas, Thermo Fisher and New England Biolabs for their prestained protein molecular weight markers is Remazol Brilliant Blue R, because 2D2-F11 detected Remazol Brilliant Blue R stained bovine serum albumin (BSA). Surprisingly, as demonstrated in the examples and in contrast to the teaching of the prior art, the dye-specific monoclonal antibodies of the present invention, like the 2D2-F11 antibody, which specifically recognizes a dye which is a portion of a covalently dye/protein complex are capable of detecting the dye only when covalently bound to a protein but not the free dye. Finally, the dye-specific monoclonal antibodies of the present invention, like the 2D2-F11 antibody, which specifically recognizes a dye which is a portion of a covalently dye/protein complex beneficially and advantageously does not recognize Coomassie Brilliant Blue R-250, another blue dye used for staining proteins. Moreover, it has been demonstrated herein and in the appended examples that the blue dye in several prestained protein molecular weight markers (i.e., like in the markers of Expedeon and Life Tech's SeeBlue prestained molecular weight markers) is Remazol Brilliant Blue R, because 2D2-F11 detected the respective blue bands; data not shown.

In accordance with the methods of the present invention, not only the above specific antibody against Remazol Brilliant Blue R has been generated with the inventive specific immunization protocol (which is also illustrated in the examples of the present invention). As exemplified further below, in another example, by applying the inventive immunization protocol, a further antibody (monoclonal antibody clone 6F4- F6) against Remazol Brilliant Blue R has been generated by first immunizing with Remazol Blue coupled to BSA followed by Remazol Blue coupled to alcohol dehydrogenase (ADH), subsequently by immunizing with Remazol Blue coupled to lysozyme and finally by boosting with a mix comprising all three Remazol blue/protein complexes. Appended Figure 14 shows the testing of the antisera from mice immunized with Remazol Blue stained BSA, ADH and lysozyme. The generation and characterization of clone 6F4-F6 is further described in appended Examples 15-19. Appended Figures 15 and 16 show the performance of both clones 2D2-F11 and 6F4-F6 in the immunoblot analysis. In addition mice were also immunized with orange stained marker proteins shown in appended Figure 11 and described in appended Example 12. In appended Figure 12 and appended Example 13 the immunoblot testing (towards Remazol Brilliant Orange stained proteins) of the antisera from mice immunized with orange prestained marker proteins further demonstrates that this approach also works for another dye. This demonstrated the general applicability of the means and methods provided herein. The inventive methods lead to the generation of antibodies that specifically recognize a visible dye as defined herein, i.e., inter alia, a dye which is a portion of a dye/protein complex. The methods and means provided herein lead to the selective elucidation an immune response against the dye.

Thus, the above technical problem of providing means and methods for a more precise and reliable determination and estimation of the molecular weight of (a) given protein(s) recognized by the antibody in Western blots or immunoblots which are, moreover, suitable for routine Western blot analysis in an averagely equipped laboratory is solved by the embodiments provided herein and as characterized in the claims.

The present invention is not only limited to dyes of marker proteins and, accordingly, not only limited to antibodies and methods for producing antibodies that specifically recognize a dye which is a portion of a covalently linked dye/protein marker complex by specifically eliciting an immune response against the dye. Rather, in accordance with the examples of the present invention, this rationale also applies in more general terms and relates to other dye/protein complexes which do not necessarily have to be marker proteins. As such, antibodies and methods for producing an antibody are also provided wherein the antibodies specifically recognize a dye which is a portion a dye/protein complex by selectively eliciting an immune response against the dye. The term "eliciting an immune response against the dye" also includes the possibility that, while the immune response is predominantly directed against the dye, the immune response is also directed against (flanking) parts of the protein, e.g., amino acid side chain(s) of the protein to which the dye is coupled. Accordingly, the present invention provides methods for producing antibodies that specifically recognize a dye which is a portion of a dye/protein complex by selectively eliciting an immune response against the dye. As will be defined in more detail in further below, the term "dye" against which the inventive antibodies of the present invention are directed are dyes which are recognized, perceived and/or detected by the human eye as colour (or black or white) upon illumination with visible light which ranges from approximately 380 to 800 nm.. The dye to be specifically recognized, detected and/or bound by the antibodies as described herein is a dye that is visible for the human eye upon illumination with visible light which ranges from approximately 380 to 800 nm. The term "dye" not only comprises a colored dye visible to the human eye upon illumination with visible light which ranges from approximately 380 to 800 nm, but also comprises a black and a white dye. A black dye is a dye which completely absorbs light. The color of a dye recognized, perceived and/or detected by the human eye has a complementary relationship with respect to the dye's absorbed part of the visible light: whereby a colored dye is recognized, perceived and/or detected by the (human) eye as the reflected light by the dye, the human eye does not detect, recognize or perceive the wavelengths which are absorbed by the dye. In case all wavelengths are absorbed the dye appears black or white if the light is totally reflected. The term "visible light to /for the human eye" is very well known in the art and is defined in common text books. For example, Sheehan (2000) in Physical Biochemistry: Principles and Applications (Univ. College Cork, Ireland), John Wiley & Sons Ltd, states "Light visible to humans as the colour-range red to violet (lambda = 720 -400nm) represents only a tiny part of the spectrum called the visible range." Curtis, Barnes. Invitation to Biology: Fifth Edition. New York: Worth Publishers, 1994: 163, explains: "For the human eye, the visible radiations range from violet light, in which the shortest rays are about 380 nanometers, to red light, in which the longest rays are about 750 nanometers." Accordingly, a color reflects the wavelengths in the nm range that the human retinal cones respond to and Visible light is the very small portion of the electromagnetic spectrum that human eyes are sensitive to.

Thus, in one aspect of the invention, a method is provided which is a method for producing an antibody that specifically recognizes a dye which is a portion of a dye/protein complex by selectively eliciting an immune response against said dye, wherein said method for producing an antibody comprises the steps of:

(i) immunizing a non-human animal with a first dye/protein complex,

(ii) subsequently immunizing said non-human animal with a second dye/protein complex wherein the protein portion of the dye/protein complex is similar or different than the protein portion of the dye/protein complex used in step (i);

(iii) optionally repeating step (ii) at least once with a further dye/protein complex wherein the protein portion of the dye/protein complex is similar or different than the protein portion of the dye/protein complex used in step (i) and any subsequent step;

(iv) optionally immunizing said non-human animal with a mixture of all dye/protein complexes used in step (i) and/or (ii) and/or (iii);

(v) obtaining/isolating said dye-specific antibody, wherein said dye-specific antibody is obtained/isolated by screening using a dye/protein complex that has not been used in the elicitation of the immune response and wherein the protein portion of the dye/protein complex is similar or different than the protein portion of the dye/protein complex used in step (i) and/or (ii) and/or (iii) and/or (iv).

In context of the present invention, the term "antibody" or "antibody molecule" relates to full immunoglobulin molecules, preferably IgMs, IgDs, IgEs, IgAs or IgGs, more preferably lgG1 , lgG2, lgG2b, lgG3 or lgG4 as well as to parts of such immunoglobulin molecules. Furthermore, the term relates to modified and/or altered antibody molecules, like chimeric and bovinized or humanized antibodies. In a preferred embodiment, the antibody is bovinized. The term also relates to monoclonal or polyclonal antibodies as well as to recombinantly or synthetically generated/synthesized antibodies. In a preferred embodiment, the dye-specific antibody is a monoclonal antibody. The term also relates to intact antibodies as well as to antibody fragments thereof, like, separated light and heavy chains, Fab, Fab/c, Fv, Fab', F(ab')₂. The term antibody also comprises but is not limited to fully-human antibodies, chimeric antibodies, humanized antibodies, CDR-grafted antibodies and antibody constructs, like single chain Fvs (scFv) or antibody-fusion proteins. "Single- chain Fv" or "scFv" antibody fragments have, in the context of the invention, the V_H and V[_ domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. Techniques described for the production of single chain antibodies are described, e.g., in Pliickthun in The Pharmacology of Monoclonal Antibodies, Rosenburg and Moore eds. Springer- Verlag, N.Y. (1994), 269-315. A "Fab fragment" as used herein is comprised of one light chain and the CH1 and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. An "Fc" region contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains. A "Fab' fragment" contains one light chain and a portion of one heavy chain that contains the VH domain and the CH1 domain and also the region between the CH1 and C H2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form a F(ab')2 molecule. A "F(ab')2 fragment" contains two light chains and two heavy chains containing a portion of the constant region between the CH1 and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains. A F(ab')2 fragment thus is composed of two Fab' fragments that are held together by a disulfide bond between the two heavy chains. The "Fv region" comprises the variable regions from both the heavy and light chains, but lacks the constant regions.

Techniques for the production of antibodies and the elicitation of an immune response against a specific antigen are well known in the art and described, e.g. in Howard and Bethell (2000) Basic Methods in Antibody Production and Characterization, Crc. Pr. Inc. With the normal skill of the person skilled in the art and by routine methods, the person skilled in the art can easily deduce from the sequences provided herein relevant epitopes (also functional fragments) of the polypeptides of the present invention which are useful in the generation of antibodies like polyclonal and monoclonal antibodies. However, the person skilled in the art is readily in a position to also provide for engineered antibodies like CDR-grafted antibodies or also bovinized or humanized and fully human or bovine antibodies and the like.

The terms "recognizing", "binding" and "detecting" as used in the context of the present invention are interchangeably used in the context of the present invention and define a binding (interaction) of at least two "antigen-interaction-sites" with each other. The term "antigen-interaction-site" defines, in accordance with the present invention, a motif of a polypeptide of the antibody which shows the capacity of specific interaction with a specific antigen or a specific group of antigens of the dye. Said "recognition", "binding" and "detection" is also understood to define a "specific recognition". The term "specifically recognizing" means in accordance with this invention that the antibody is capable of specifically interacting with and/or binding to a dye which is a portion of a dye/protein complex but not with the dye alone. It is, however, also envisaged that the antibody may also be capable of specifically interacting with and/or binding to a dye with the dye alone. Antibodies can recognize, interact and/or bind to different epitopes on the same target molecule. This term relates to the specificity of the antibody molecule, i.e., to its ability to discriminate between the specific regions of the target molecule, i.e., the dye as defined herein. Thus, a specific motif in the amino acid sequence of the antigen-interaction-site and the antigen bind to each other as a result of their primary, secondary or tertiary structure as well as the result of secondary modifications of said structure.

Thus, the terms "recognizing", "binding" and "detecting" as used in the context of the antibodies of the present invention and the method of generating such antibodies of the present invention refers in particular to a binding reaction that is determinative of the presence of the particular dye in the presence of a heterogeneous population of dyes and, e.g., other biologies like proteins.

Thus, under designated assay conditions, the specified antibodies and the specific dye bind to one another and do not bind in a significant amount to other components present in a sample. A variety of immunoassay formats may be used to select antibodies specifically reactive with a particular antigen, i.e., the dye which is a portion of the above dye/protein complex. Such immunoassay formats and methods for identifying whether a specific immune reaction has been elicited are well-known to the person skilled in the art. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with an analyte. See Shepherd and Dean (2000), Monoclonal Antibodies: A Practical Approach, Oxford University Press and/ or Howard and Bethell (2000) Basic Methods in Antibody Production and Characterization, Crc. Pr. Inc. for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity. Typically a specific or selective reaction will be at least twice background signal to noise and more typically more than 10 to 100 times greater than background. The person skilled in the art is in a position to provide for and generate specific antibodies directed against the dye in terms of the method for the production of an antibody that specifically recognizes a dye which is a portion of a dye/protein complex of the invention as defined above and further below. For specific binding-assays it can be readily employed to avoid undesired cross-reactivity, for example polyclonal antibodies can easily be purified and selected by known methods (see Shepherd and Dean, loc. cit.). Thus, the person skilled in the art is readily in a position to determine whether a specific immune response against the dye has been elicited.

Preferably, the above step (iii) of the method of the invention, i.e., the optional step of repeating at least once the immunization with a further dye/protein complex wherein the protein portion of the dye/protein complex is similar or different than the protein portion of the dye/protein complex used in the first immunization step and any subsequent step is repeated as long until such a specific immune response against the dye has been elicited. Thus, the term "at least once" means in this context that the optional step is repeated preferably once, twice, three times or even four times. In case no specific immune response can be detected the step is repeated until a specific immune response against the dye is detected, i.e., more than twice, three or four times mentioned above, i.e., five, six, seven or oven eight times. As mentioned, the skilled person has numerous methods at hand that can be used to determine whether a specific immune response against the dye has been elicited.

The term "specifically recognizing a dye which is a portion of a dye/protein complex" means in accordance with this invention that the antibody molecule is capable of specifically recognizing or specifically interacting with and/or binding to the dye as defined herein. Said term relates to the specificity of the antibody molecule, i.e. to its ability to discriminate between the specific dye defined herein and other dyes. Accordingly, specificity can be determined experimentally by methods known in the art and methods as disclosed and described herein. Such methods comprise, but are not limited to Western blots, ELISA-, RIA-, ECL-, IRMA-tests and peptide scans. The term "specifically recognizing a dye which is a portion of a dye/protein complex" also includes the possibility that the antibody not only recognizes the dye part of a dye/protein complex but also (flanking) parts of the protein, e.g., amino acid side chain(s) of the protein to which the dye is coupled. Moreover, the possibility is included that the antibody does not recognize the dye alone but only in the context of (surrounding) parts of the protein part of the protein/dye complex.

The term "recognizing", "binding" and "detecting" as used in accordance with the present invention means that the antibody of the invention does not or does not essentially cross-react with dyes of similar structures. Accordingly, the antibody of the invention specifically binds to/interacts with the dye as defined further below.

Cross-reactivity of the antibodies of the invention may be tested, for example, by assessing binding of said antibodies under conventional conditions (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1988) and Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1999)) to the (poly)peptide of interest as well as to a number of more or less (structurally and/or functionally) closely related dyes. Only those antibodies that bind to the dye of interest but do not or do not essentially bind to any other related or unrelated dye are considered specific for the dye of interest and selected for further studies in accordance with the method provided herein. These methods may comprise, inter alia, binding studies, blocking and competition studies with structurally and/or functionally closely related molecules. These binding studies also comprise FACS analysis, surface plasmon resonance (SPR, e.g. with BIAcore®), analytical ultracentrifugation, isothermal titration calorimetry, fluorescence anisotropy, fluorescence spectroscopy or by radiolabeled ligand binding assays. The term "binding to", "binding" and "detecting" does not only relate to a linear epitope but may also relate to a conformational epitope, a structural epitope or a discontinuous epitope consisting of two regions of the dye or parts thereof. In the context of this invention, a conformational epitope is defined by two or more discrete parts separated in the dye. The term "specifically recognizing a dye which is a portion of a dye/protein complex" also includes the possibility that the antibody not only recognizes the dye part of a dye/protein complex but also (flanking) parts of the protein, e.g., amino acid side chain(s) of the protein to which the dye is coupled. Moreover, the possibility is included that the antibody does not recognize the dye alone but only in the context of (surrounding) parts of the protein part of the protein/dye complex.

Accordingly, specificity can be determined experimentally by methods known in the art and methods as described herein. Such methods comprise, but are not limited to Western Blots, ELISA-, RIA-, ECL-, IRMA-tests and peptide scans.

The term "selectively eliciting an immune response against the dye" relates to the provocation of an immune response in a non-human animal, in particular the provocation of an antibody response to/against dye. Said antibody response comprises primary as well as secondary antibody responses to the antigenic challenge with said dye/protein complex, wherein, as described above, the immunization scheme comprises (i) immunizing a non-human animal with a first dye/protein complex, (ii) subsequently immunizing said non-human animal with a second dye/protein complex wherein the protein portion of the dye/protein complex is similar or different than the protein portion of the dye/protein complex used in step (i); (iii) optionally repeating step (ii) at least once with a further dye/protein complex wherein the protein portion of the dye/protein complex is similar or different than the protein portion of the dye/protein complex used in step (i) and any subsequent step; and (iv) optionally immunizing said non-human animal with a mixture of all dye/protein complexes used in step (i) and/or (ii) and/or (iii). The term "eliciting an immune response", accordingly, relates to the provocation of an immune reaction involving the production of antibodies directed towards the dye. The term "eliciting an immune response against the dye" also includes the possibility that, while the immune response is predominantly directed against the dye, the immune response is also directed against (flanking) parts of the protein, e.g., amino acid side chain(s) of the protein to which the dye is coupled.

The term that the protein portion of the dye/protein complex is "similar or different" than the protein portion of the dye/protein complex means in the context of the present invention that the protein portion of the dye/protein complex used in the initial immunization round is not identical (i.e., at least only similar or even (completely) different) to the protein portion of the dye/protein complex used in the subsequent round(s) of immunization. In other words, while the dye is identical in all the immunization rounds, the protein portion of the dye/protein complex is not identical and displays at least some variations or is completely different. It has been surprisingly found in the present invention that this scheme of subsequent immunization rounds leads to an immune response selectively or predominantly eliciting a response against the dye. In accordance with the present invention, the term "similar" refers to a protein portion of the dye/protein complex for which antibodies cross-reactive to similar proteins exist, i.e., antibodies which are generated during the method for producing an antibody in accordance with the invention whereas the term "different" refers to a protein portion of the dye/protein complex for which no cross-reactive antibodies exist. Thus, for a "different protein" as used in the context of the present invention no antibodies are obtained during the immunization scheme of the present invention. In other words, "similar" protein portions of the dye/protein complex as used in the context of the present invention are capable of eliciting antibodies which cross-react with "similar" proteins. In contrast, "different" protein portions of the dye/protein complex as used in the context of the present invention are not capable of eliciting antibodies which cross-react with the "different" protein.

The disclaimer "non-human" also excludes prior art antibodies against reactive dyes used in the textile industry disclosed. As mentioned, in humans, the generation of antibodies against reactive dye-human serum albumin conjugates has been described in workers of dye factories which are correlated with asthmatic responses (Park et al., Journal of Korean Medical Science 6(1 ):63-68 (1991 )). Moreover, respiratory allergy has been correlated with specific IgE and IgG antibodies against reactive dyes/albumin-conjugates in workers in the wool industry while the antibodies detect dye-albumin conjugates (Topping et al., Journal of Occupational Medicine 31(10): 857-862 (1989)). Moreover, antibodies against conjugates between reactive dyes and human serum albumin have been detected in workers of textile plants with dyehouses, who have been exposed to reactive dyes and who suffer from asthma, rhinitis and dermatitis. (Nielsson et al., British Journal of Industrial Medicine 50: 65- 70(1993)). These authors have detected Remazol antibodies in patients but have not shown how these antibodies are generated in those patients. It has only been hypothesized that the hapten Remazol has been linked to serum proteins such as human serum albumin. Experiments, in which they determined the hapten/carrier specificity of the patients' IgE antibodies, support their hypothesis. However, no direct experimental evidence for the presence of such Remazol-HSA complexes in patients has been provided nor did they immunize animals with such conjugates. Importantly, patient IgE did not recognize the molecular weight marker in the Western blot analysis shown in Figure 4 of Park et ai., Journal of Korean Medical Science 6(1 ):63-68 (1991 ). Moreover, in humans, the generation of antibodies against reactive dye-human serum albumin conjugates has also been described in Luczynska CM, Topping MD. J Immunol Methods. 1986 Dec 24;95(2):177-86; and Hagmar L, et al., Scand J Work Environ Health. 1986 Jun;12(3):221-2. However, as mentioned, whenever reference is made to an "antibody" in the context of the present invention, this antibody is not generated in humans. Thus, antibodies that specifically recognize a dye which is a portion of a dye/protein complex of the present invention is not an antibody generated as part of an allergic reaction against textile dyes in human.

Methods for the production of antibodies and for immunization of non-human animals in accordance with the present invention are well known in the art, see, e.g. Harlow and Lane, "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988.

Once a desired immune response has specifically been elicited against the dye the dye-specific antibody can be "obtained or isolated" as described in step (v) of the above method. Thus, said dye-specific antibody is obtained/isolated, using in a screening approach a dye/protein complex that has not been used in the elicitation of the immune response (wherein in said preceding immune response, said protein portion of the dye/protein complex was similar or different than the protein portion of the dye/protein complex used in step (i) and/or (ii) and/or (iii) and/or (iv)). Thus, for the screening approach, a dye/protein complex is used wherein the protein portion is different to the protein portion used in the immunization step(s) while the dye which is used in the obtaining/isolating/screening approach is identical to the dye used in the immunization step(s). The person skilled in the art is aware of numerous methods for the specific isolation or generation of a specific antibody. As an example, immunoaffinity purification procedures may be used which is a method of separating biochemical mixtures, i.e., the serum of the immunized non-human animal, based on a highly specific interaction between the antigen, i.e., the dye and the antibody, i.e., the desired dye-specific antibody. Such a method may be used to specifically isolate or obtain the dye-specific antibodies from, e.g., blood serum or from the supernatant of a hybridoma cell expressing/secreting the desired dye-specific antibody. If serum is known to contain antibodies against a specific antigen against the dye (i.e., if the serum comes from an organism immunized against the dye antigen concerned and/or has positively been tested that a corresponding immune response against the dye has been elicited as outlined above) then it can be used for the affinity purification of that antibody, i.e., the dye-specific antibody. This is also known as Immunoaffinity Chromatography. The dye (alone or coupled to a protein in the form of a dye/protein complex) can, e.g., be covalently coupled to a solid support such as agarose and used as an affinity ligand in purifications of antibody from immune serum or from the supernatant of a hybridoma cell expressing/secreting the specific antibody. The serum (or supernatant of a hybridoma cell expressing/secreting the specific antibody) is initially allowed to bind to the dye (/protein) affinity matrix. This affinity binding step will remove the dye-specific antibodies from the serum. Antibodies, however, against the protein part of the dye/protein complex(es) used in the immunization and other non-related antibodies will remain in the unbound fraction of the serum (or supernatant of a hybridoma cell expressing/secreting the specific antibody). Elution of the antibodies of interest from the affinity matrix may be achieved using a low pH buffer such as glycine pH 2.8. The eluate is collected into a neutral Tris or phosphate buffer, to neutralize the low pH elution buffer and halt any degradation of the antibody's activity.

Moreover, it is preferred that the dye-specific antibody according to the present invention is obtained/isolated in a screening approach by using a double-screening wherein the serum or supernatant of a hybridoma cell expressing/secreting the specific antibody is screened in a first step with a coupled, stained protein portion (i.e., a carrier or a protein that corresponds to or is different to the protein part of the dye/protein complex that has been used in the elicitation of the immune response, i.e., in the immunization step(s) as outlined above carrying the respective dye). In a second, separate screening step the serum or supernatant of a hybridoma cell expressing/secreting the specific antibody is screened with an uncoupled, unstained protein portion (i.e., a carrier or a protein that corresponds to the protein part of the dye/protein complex that has been used in the elicitation of the immune response, i.e., in the immunization step(s) as outlined above, however, without the respective dye). Only those antibodies that positively react with the carrier or protein which is coupled to the dye (i.e., antibodies that recognize and bind to the carrier or protein coupled to the dye) and which do not react with the carrier or protein alone (i.e., antibodies that do not recognize and bind to the dye-uncoupled carrier or protein) are characterized as antibodies which "specifically recognize a dye which is a portion of a dye/protein complex" in accordance with the present invention. Accordingly, in a corresponding screening method, only the latter antibodies which fulfil both criteria (i.e., the ones which are characterized as antibodies which "specifically recognize a dye which is a portion of a dye/protein complex") are selected, i.e., obtained or isolated as dye-specific antibodies in accordance with the present invention, while the others are discarded.

Without being bound to theory, for the immunization, the antigen may be prepared as follows which is merely for the sake of illustration as explained for the Precision Plus Protein™ All Blue Standards marker protein mixture. However, any other antigen may be prepared accordingly or by other means and methods known to the person skilled in the art: One 500μΙ aliquot of Precision Plus Protein™ All Blue Standards marker protein mixture (Biorad, 161-0373) is separated by preparative 10% w/v sodium-dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) in 0.025 M Tris/0.2 M Glycine/0.01 % w/v SDS pH 8.5 running buffer using a 16.5 cm wide and 22 cm long slab gel unit (C.B.S. Scientific, ASU-250) with a 1.5 mm thick and 13 cm wide preparative Teflon comb (Hoefer Scientific). The prestained bands corresponding to the 75 kD, 50 kD and 25 kD, respectively, marker proteins are excised from the gel with a sterile stainless steel surgical blade (Swann-Morton, Size 22, Ref 0308) and cut into small pieces of approximately 1-2 mm³. The polyacrylamide gel pieces containing the respective prestained marker proteins are filled into three separate chambers of an S&S Elutrap electro-separation system (Schleicher&Schull) and the prestained proteins are electro-eluted from the polyacrylamide gel pieces for 6 hours at a constant voltage of 200 V at 22°C in 0.025M Tris/0.2 M Glycine/0.01% w/v SDS pH 8.5 running buffer. Each electro-eluted protein is collected in a volume of 600 μΙ of running buffer between a BT2 Elutrap- Membrane (Whatman, 10404092) and a BT1 Elutrap-Membrane (Whatman, 10404090) as suggested by the manufacturer. Electro-eluted proteins are dialyzed for 14 hours at 4°C against 600 ml of Tris-buffered saline (TBS; 0.137 M sodium chloride [NaCI], 0.0027 M potassium chloride [KCI], 0.025 M Tris, adjusted to pH 7.4 with hydrochloric acid [HCI]) for the 25 kD marker protein or 1000 ml of TBS for the 50 kD and 75 kD marker proteins in Slide-A-Lyzer Dialysis Cassettes (Extra Strength) with a cut-off of 10 kD and a volume capacity of 0.5-3 ml (Thermo Scientific, Product # 66380). The dialyzed proteins are collected from the dialysis cassettes by diluting in approximately 1 ml of 37°C warm TBS and concentrated to 400 μΙ by centrifugation through Amicon Ultra-4 10K centrifugal filters with a nominal molecular weight limit of 10 kD (Millipore, UFC801024).

The immunization in accordance with the present invention may, e.g., be performed as follows. For the sake of illustration the immunization is explained for the Precision Plus Protein™ All Blue Standards marker protein mixture. However, any other antigen may be used for the immunization and any other method may be used for the immunization since methods for the production of antibodies and for immunization of non-human animals are well-known to the person skilled in the art; see, e.g. Harlow and Lane, "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988. Accordingly, 5 μΙ of dialyzed 75, 50, and 25 kD prestained marker proteins may be separated by 12.5% SDS-PAGE in 0.025 M Tris/0.2 M Glycine/0.01 % w/v SDS pH 8.5 running buffer together with 4 μΙ of Precision Plus Protein™ All Blue Standards marker protein mixture as provided by the manufacturer to estimate the amount of prestained proteins present in the purified samples. 50μΙ of dialyzed 50 kD prestained marker protein are diluted with 50 μΙ phosphate buffered saline (PBS; 0.137 M NaCI, 0.0027 M KCI, 0.0015 M KH₂P0₄, 0.0081 M Na₂HP0₄, pH 7.4) and mixed with 100 μΙ of Freund^'s Complete adjuvant (Sigma, F5881 ). Alternatively, in accordance with the present invention, other adjuvants like e.g. Gerbu Adjuvant MM (Gerbu, 3001 ) can be used. The aqueous antigen solution and the adjuvant oil are emulsified by repeated cycles of sucking-up and pushing-out the oil-water mixture through a 23G (0.6mm diameter) needle until a stable emulsion was formed. Blood samples are collected from the tail veins of three female cByJ.RBF-Rb(8.12)5Bnr/J mice at the age of 10 weeks ("preimmune sera"), incubated for 1 h at 37°C and centrifuged for 5 min at 22°C at 14,000 rpm in a Beckman&Coulter Microfuge 18. The cleared blood sera are collected, sodium azide is added to a final concentration of 0.02% w/v, and the sera are stored at 4°C. Immediately after the collection of blood, the mice may be immunized with 200 μΙ of antigen-adjuvant emulsion per mouse injected subcutaneously at the abdomen. 14 days after the first immunization, the mice are boosted with 50 μΙ of dialyzed 75 kD prestained marker protein mixed with 50 μΙ PBS and emulsified with 100 μΙ of Freund^'s Incomplete adjuvant (Sigma, F5506) per mouse injected subcutaneously at the abdomen. Alternatively, other adjuvants like e.g. Gerbu Adjuvant MM (Gerbu, 3001) may be used. 35 days after the first immunization, the mice are boosted a second time with 50 μΙ of dialyzed 25 kD prestained marker protein mixed with 50 μΙ PBS and emulsified with 100 μΙ of Freund^'s Incomplete adjuvant (or alternatively Gerbu Adjuvant MM 3001 ) per mouse injected subcutaneously at the abdomen. 10 days after the second boost, blood samples of all mice are taken ("immune sera") from the tail veins, incubated for 1 h at 37°C and centrifuged for 5 min at 22°C at 14,000 rpm in a Beckman&Coulter Microfuge 18.

The immune sera may be tested as follows: The cleared blood sera may be collected, sodium azide can be added to a final concentration of 0.02% w/v, and the sera are tested for the presence of dye-specific IgG antibodies by immunoblotting against 10% SDS-PAGE separated Precision Plus Protein™ All Blue Standards marker proteins. 85 x 73 mm SDS polyacrylamide gels with 1 mm thick preparative combs (Bio-Rad, 165-2928) may be casted with Bio-Rad Mini PROTEAN II electrophoresis cell systems. 40 μΙ of Precision Plus Protein™ All Blue Standards marker proteins (BioRad, 161-0373) may be separated in 0.025 M Tris/0.2 M glycine/0.01% w/v SDS pH 8.5 running buffer at a constant voltage of 100 V at 22°C. Proteins are transferred to Protran BA 83 nitrocellulose membrane (Whatman, 10401396) in 0.025 M Tris/0.19 M glycine/20% methanol pH 8.5 transfer buffer for 2h at a constant current of 0.5 A at 4°C in Hoefer TE-Series Transphor Electrophoresis Units (Pharmacia Biotech, TE42). Membranes are washed with deionised water and stored dry between two 3MM paper sheets at 22°C. Prior to usage, membranes are rehydrated by incubation for 2 min at 22°C in PBS + 0.1% Tween-20 (PBS-T). Membranes are blocked by incubation for 1 h at 22°C in PBS-T + 3% w/v skim milk powder (Merck, 1.15363). Blots are incubated with preimmune and immune sera diluted 1 :500 in PBS-T + 0.5% skim milk powder in a Miniblotter system 28 channels dual blot MN28 unit (Immunetics, 168830) over night at 4°C. Membranes are washed 3x 5 min with PBS-T at 22°C. For detection of primary mouse antibodies, membranes are incubated with peroxidase-conjugated AffiniPure goat anti-mouse IgG Fey fragment specific (Jackson ImmunoResearch, 115-035-008) secondary antibody diluted 1 :10,000 in PBS-T + 0.5% skim milk powder for 1 h at 22°C. Membranes are washed three times 10 min with PBS-T at 22°C and bound antibodies are visualized by enhanced chemoluminescence with ECL Western Blotting Detection Reagents (GE Healthcare, RPN2106) and exposure of Fuji Medical X-ray films (FUJIFILM Corporation, Super HR-E, 47410 08471 ).

As already mentioned above, the dye-specific antibody can be obtained or isolated in step (v) by screening methods known to the person skilled in the art by taking advantage of the specific interaction of the antibody to be screened, isolated or to be obtained with the respective dye. It is preferred that the dye-specific antibody can be obtained or isolated in a screening approach by using a dye/protein complex that has not been used in the elicitation of the immune response and wherein the protein portion of the dye/protein complex is similar or different than the protein portion of the dye/protein complex used in step (i) and/or (ii) and/or (iii). At the same time, it is preferred that the dye is identical to the respective dye used in the elicitation of the immune response by taking advantage of the specific interaction between the resepective dye and the antibody to be isolated which is the dye-specific antibody. As demonstrated in the appended examples the screening method is a key feature of the present invention for obtaining such antibodies that specifically recognize the dye (but not any of the protein portions of the dye/protein complexes). In contrast, although Saoji et al. (Clin. Chem. 30/7: 1252-1254 (1984)) raised monospecific (i.e., protein specific) antibodies by using protein components which are prestained with Remazol Brilliant Blue, it is described that the dye itself is non-antigenic. It may be speculated that this conclusion is based on the fact that Saoji missed the dye specific antibodies. In contrast, in order to specifically isolate/obtain the antibodies that specifically detect the dye, it is a preferred aspect of the present invention the screening for such dye specific antibodies is done with a dye-protein conjugate that was not or at least not repeatedly used in the immunizations. As demonstrated in the appended examples, it has surprisingly found that this screening assay successfully and selectively isolates the antibodies that are specific for the dye while antibodies that might also have been generated against the protein portion(s) are not screened for (identified). Without being bound to theory, for example, as demonstrated in the examples, the Remazol-stained MW markers have been screened that consist of 10 different proteins, 7 of which were not used in the immunizations. With such a screening procedure one can identify dye-specific antibodies also in the serum of an animal that was immunized with one and the same dye-protein conjugate.

Alternatively to the above immunization protocol by selectively eliciting an immune response against the dye by immunizing with different dye/protein complexes it is also envisaged that dye specific antibodies can also be generated by immunizing one or more times (i.e., once, twice, three times or even four or five times) with the same (i.e., identical) dye/protein complex (i.e., a hapten-carrier complex). While the immune response may be less efficient against the dye compared to the above immunization procedure using different dye/protein complexes, the non-human animal nevertheless generates antibodies specific for the dye which can then, subsequently, be screened and isolated or obtained in line with the screening procedure as outlined above. Accordingly, there is also provided a method for producing an antibody that specifically recognizes a dye which is a portion of a dye/protein complex by selectively eliciting an immune response against the dye, wherein the method comprises the steps of:

(i) immunizing a non-human animal with a first dye/protein complex,

(ii) subsequently immunizing said non-human animal with a second dye/protein complex wherein the protein portion of the dye/protein complex is identical to the protein portion of the dye/protein complex used in step (i);

(iii) optionally repeating step (ii) at least once with the same (i.e., idential) dye/protein complex wherein the protein portion of the dye/protein complex is identical to the protein portion of the dye/protein complex used in step (i) and any subsequent step;

(iv) obtaining/isolating said dye-specific antibody, wherein said dye-specific antibody is obtained/isolated by screening using a dye/protein complex that has not been used in the elicitation of the immune response and wherein the protein portion of the dye/protein complex is similar or different than the protein portion of the dye/protein complex used in step (i) and/or (ii) and/or (iii). The latter "obtaining/isolating" step has already been further defined in the context of the immunization procedure using different dye/protein complexes which here applies mutatis mutandis.

With the exception that in the above method the dye/protein complex is identical in all steps, all other embodiments as outlined above in the context of the method utilizing different dye/protein complexes apply here, mutatis mutandis. Inter alia, the above step (iii) of the method of the invention, i.e., the optional step of repeating at least once the immunization with a further dye/protein complex is repeated as long until such a specific immune response against the dye has been elicited. Thus, the term "at least once" means in this context that the optional step is repeated preferably once, twice, three times or even four times. In case no specific immune response can be detected the step is repeated until a specific immune response against the dye is detected, i.e., more than twice, three or four times mentioned above, i.e., five, six, seven or oven eight times. As mentioned, the skilled person has numerous methods at hand that can be used to determine whether a specific immune response against the dye has been elicited.

The above described methods may generate polyclonal antibodies. The term "polyclonal antibody" as used herein, refers to an antibody which was produced among or in the presence of one or more other, non-identical antibodies. In general, polyclonal antibodies are produced from a B-lymphocyte in the presence of several other B-lymphocytes which produced non-identical antibodies. Usually, polyclonal antibodies are obtained directly from an immunized animal.

However, it is also in particular a preferred embodiment of the invention that the antibody that specifically detects a dye which is a portion of a dye/protein complex is a monoclonal antibody. Thus, the present invention relates in particular to the above methods, wherein the antibody is a monoclonal antibody which is obtained in step (v) by

(i) fusing a B-cell or a plasma-cell or a progenitor cell thereof, from a non-human animal which has been immunized according to steps (ii) to (iv) as defined in claim 1 with myeloma cells in order to obtain hybridoma cells, wherein the non- human animal which has been immunized is a mouse, rabbit or rat

(ii) culturing said hybridoma cells;

(iii) identifying a hybridoma cell that produces a dye-specific antibody; and

(iv) isolating the dye-specific antibody wherein said dye-specific antibody is obtained/isolated by screening using a dye/protein complex that has not been used in the elicitation of the immune response and wherein the protein portion of the dye/protein complex is similar or different than the protein portion of the dye/protein complex used in step (i) and/or (ii) and/or (iii) of claim 1.

The term "monoclonal antibody" as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Monoclonal antibodies are advantageous in that they may be synthesized by a hybridoma culture, essentially uncontaminated by other immunoglobulins. The modified "monoclonal" indicates the character of the antibody as being amongst a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. As mentioned above, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method described by Kohler, Nature 256 (1975), 495. Thus, particularly preferred in the context of the present invention are monoclonal antibodies. For the preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples for such techniques include the hybridoma technique, the trioma technique, the human B-cell hybridoma technique and the EBV-hybridoma technique to produce human monoclonal antibodies (Shepherd and Dean (2000), Monoclonal Antibodies: A Practical Approach, Oxford University Press, Goding and Goding (1996), Monoclonal Antibodies: Principles and Practice - Production and Application of Monoclonal Antibodies in Cell Biology, Biochemistry and Immunology, Academic Pr Inc, USA).

The antibody derivatives can also be produced by peptidomimetics. Further, techniques described for the production of single chain antibodies (see, inter alia, US Patent 4,946,778) can be adapted to produce single chain antibodies specifically recognizing the antigen of the invention. Also, transgenic animals may be used to express humanized or bovinized antibodies to the polypeptide of the invention.

It is preferred that the B-cell, plasma-cell or the progenitor cell thereof is a cell derived from spleen, lymph nodes or peyer^s patches.

In the generation of monoclonal antibodies, without being bound to theory, in accordance with the present invention, the fusion of splenocytes may be performed as follows: a non-human animal like a mouse which shows a robust immune response as, e.g., determined by immunblotting as outlined above may receive a final boost injected intravenously into the tail vein as it has been done in the present examples in the generation of blue dye-specific antibodies wherein the final boost consists of a mixture of 5 μΙ of dialyzed 25 kD marker protein from the same batch as used for the 2^nd boost, 5 μΙ of dialyzed 50 kD marker protein from the same batch as used for the first immunization and 5 μΙ of dialyzed 75 kD marker protein from the same batch as used for the 1^st boost diluted in 85 μΙ of PBS. 88 hours post injection the mouse is sacrificed by cervical dislocation and the spleen removed surgically. The spleen is placed in 10 ml of 37°C warm Dulbecco^'s Modified Eagle^'s medium (DMEM; Sigma, D5671 ), cut in small pieces with a sterile pair of scissors and grinded between two sterile frosted microscope slides (Menzel Glaser Superfrost Plus, Thermo Scientific, J1800AMNZ) until no macroscopic pieces of splenic tissue is visible. The cell suspension may then be filtered through a 100 m nylon cell strainer (BD Falcon, Ref. 352360) and the filter may be washed two times with 10 ml of 37°C warm DMEM. Cells are centrifuged for 5 min at 1200 rpm in a Heraeus Megafuge 1.0 at 22°C, resuspended in 3 ml of ice-cold red blood cell lysis buffer (Sigma, R7757) and incubated for 90 sec. The cell suspension is filled up to 30 ml with 37°C warm DMEM and centrifuged for 5 min at 1200 rpm in Heraeus Megafuge 1.0 at 22°C. The splenocytes are counted with a 0.0025 mm² glass counting chamber (0,100 mm depth; Biirker, Labor Optik). X63-Ag8.653 mouse myeloma cells are grown at 37°C in a 5% C0₂ atmosphere on Vents Nunclon TC 140/20 petri dishes (Nunc, 168381) for a minimum of 3 passages after thawing in DMEM + 10% fetal bovine serum (Sigma, F7524) + 2 mM Glutamax (Gibco, 35050-038) + 100 units/ml Penicillin/0.1 mg/ml Streptomycin (Sigma, P4333) + 1 mM sodium pyruvate (Sigma, S8636). X63- Ag8.653 cells are harvested by rinsing off the petri dish, centrifuged for 5 min at 1200 rpm in a Heraeus Megafuge 1.0 at 22°C, resuspended in 30 ml of 37°C warm DMEM, counted with a 0.0025 mm² glass counting chamber (0,100 mm depth; Biirker, Labor Optik) and centrifuged again for 5 min at 1200 rpm in a Heraeus Megafuge 1.0 at 22°C. Splenocytes and myeloma cells are mixed at a ratio of 2.5:1 , centrifuged for 5 min at 1200 rpm and fused by resuspending and incubating for 90 sec at 37°C the cells in 1 ml of polyethylenglycol (PEG) 1450 (50% w/v solution in PBS; Sigma, P7181). After 90 sec, the cell suspension is diluted stepwise with 1 ml of 37°C warm DMEM, followed by 5 ml of 37°C warm DMEM and followed again by 10 ml of 37°C warm DMEM and is then incubated at 37°C for 5 min. Cells are centrifuged for 5 min at 1200 rpm in a Heraeus Megafuge 1.0 at 22°C and are resuspended in DMEM + 10% HyClone Fetal Clone I (Thermo Scientific, SH30080.03) + 2 mM Glutamax + 100 units/ml Penicillin/0.1 mg/ml Streptomycin + 1 mM sodium pyruvate + 5% BM Condimed H1 Hybridoma Cloning Supplement (Roche, 11088947001 ) + 0.1 mM hypoxanthine/0.4 μΜ aminopterin/16 μΜ thymidine (provided as HAT 50x stock; Life Technologies, 21060-017) (referred to as "Hybridoma growth medium"). 10⁵ cells per well are seeded onto 96-well petri-dishes (TC Microwell 96F, Nunc, 167000). Cells are grown for 7 days at 37°C in a 5% CO2 atmosphere and the supernatants are tested for the presence of dye-specific IgG antibodies by immunoblotting.

Subsequently, as an example, the hybridoma supernatants may be screened as follows followed by the establishment of a hybridoma single clone:

For antibody screening of splenic fusion hybridoma supernatants, 85 x 73 mm SDS polyacrylamide gels with 1 mm thick preparative combs (Bio-Rad, 165-2928) are casted with Bio-Rad Mini PROTEAN II electrophoresis cell systems. 40 μΙ of Precision Plus Protein™ All Blue Standards marker proteins (BioRad, 161-0373) is separated in 0.025 M Tris/0.2 M glycine/0.01% w/v SDS pH 8.5 running buffer at a constant voltage of 100 V at 22°C. Proteins are transferred to Protran BA 83 nitrocellulose membrane (Whatman, 10401396) in 0.025 M Tris/0.19 M glycine/20% methanol transfer buffer for 2h at a constant current of 0.5 A at 4°C in Hoefer TE- Series Transphor Electrophoresis Units (Pharmacia Biotech, TE42). Membranes are washed with deionised water and stored dry between two 3MM paper sheets at 22°C. Prior to usage, membranes are rehydrated by incubation for 2 min at 22°C in PBS + 0.1% Tween-20 (PBS-T). Membranes are blocked by incubation for 1 h at 22°C in PBS-T + 3% w/v skim milk powder (Merck, 1.15363). Blots are incubated with undiluted supernatants in Miniblotter system 28 channels dual blot MN28 units (Immunetics, 168830) over night at 4°C. Membranes are washed 3x 5 min with PBS- T at 22°C. For detection of primary mouse antibodies, membranes are incubated with peroxidase-conjugated AffiniPure goat anti-mouse IgG Fey fragment specific (Jackson ImmunoResearch, 115-035-008) secondary antibody diluted 1 :10,000 in PBS-T + 0.5% skim milk powder for 1 h at 22°C. Membranes are washed three times 10 min with PBS-T at 22°C and bound antibodies are visualized by enhanced chemoluminescence with ECL Western Blotting Detection Reagents (GE Healthcare, RPN2106) and exposure of Fuji Medical X-ray films (FUJIFILM Corporation, Super HR-E, 47410 08471 ).

Cells growing in a tissue culture 96-well containing supernatant that is tested positive for the presence of antibodies specific for Precision Plus Protein™ All Blue Standards marker proteins are resuspended in "Hybridoma growth medium" and counted with a 0.0025 mm² glass counting chamber (0,100 mm depth; Biirker, Labor Optik). The appropriate volume of cell suspension is diluted in 30 ml of Hybridoma growth medium to yield a concentration of 1 cell in 200 μΙ of Hybridoma growth medium, and 300 μΙ of cell suspension per well is pipetted onto 96-well petri-dishes (TC Microwell 96F, Nunc, 167000). Cells are grown for 7 days at 37°C in a 5% CO₂ atmosphere and the supernatants are tested for the presence of dye-specific IgG antibodies by immunoblotting as described for the screening of the splenic fusion hybridoma supernatants. Wells containing supernatant that are tested positive for the presence of antibodies specific for Precision Plus Protein™ All Blue Standards marker proteins are examined under the microscope for the number of hybridoma clones growing. One well with a single clone growing is selected for expansion and further propagation using standard techniques.

However, the present invention is not limited to the above specific description of the generation of monoclonal antibodies. Rather, the skilled person recognizes that the above is only one example and for illustration purposes only.

The present invention also relates to the production of recombinant antibodies. A wide variety of recombinant antibody formats have been developed in the recent past, e.g. bivalent, trivalent or tetravalent bispecific antibodies. Examples include the fusion of an IgG antibody format and single chain domains (for different formats see e.g. Coloma, M.J., et al., Nature Biotech 15 (1997), 159-163; WO 2001/077342; Morrison, S.L., Nature Biotech 25 (2007), 1233-1234; Holliger, P., et. al, Nature Biotech. 23 (2005), 1126-1136; Fischer, N., and Leger, O., Pathobiology 74 (2007), 3-14; Shen, J., et. al., J. Immunol. Methods 318 (2007), 65-74; Wu, C, et al., Nature Biotech. 25 (2007), 1290-1297). The bispecific antibody or fragment herein also includes bivalent, trivalent or tetravalent bispecific antibodies described in WO 2009/080251 ; WO 2009/080252; WO 2009/080253; WO 2009/080254; WO 2010/112193; WO 2010/115589; WO 2010/136172; WO 20 0/145792; WO 2010/145793 and WO 2011/117330. Thus, the present invention also relates to recombinant human antibodies, heterologous antibodies and heterohybrid antibodies. The term "recombinant antibody" includes all sequence antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes; antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial human and non-human combinatorial antibody library, or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant antibodies have variable and constant regions (if present) derived from germline immunoglobulin sequences. Such antibodies can, however, be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to germline VH and VL sequences, may not naturally exist within the antibody germline repertoire in vivo.

A "heterologous antibody" is defined in relation to the transgenic non-human organism producing such an antibody. This term refers to an antibody having an amino acid sequence or an encoding nucleic acid sequence corresponding to that found in an organism not consisting of the transgenic non-human animal, and generally from a species other than that of the transgenic non-human animal.

The term "heterohybrid antibody" refers to an antibody having light and heavy chains of different organismal origins. For example, an antibody having a human heavy chain associated with a murine light chain is a heterohybrid antibody. Examples of heterohybrid antibodies include chimeric and humanized antibodies.

The term antibody also relates to humanized antibodies. "Humanized" forms of non- human (e.g. murine or rabbit) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Often, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibody may comprise residues, which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see: Jones, Nature 321 (1986), 522-525; Reichmann Nature 332 (1998), 323-327 and Presta Curr Op Struct Biol 2 (1992), 593-596. A popular method for humanization of antibodies involves CDR grafting, where a functional antigen-binding site from a non-human 'donor' antibody is grafted onto a human 'acceptor' antibody. CDR grafting methods are known in the art and described, for example, in US 5,225,539, US 5,693,761 and US 6,407,213. Another related method is the production of humanized antibodies from transgenic animals that are genetically engineered to contain one or more humanized immunoglobulin loci which are capable of undergoing gene rearrangement and gene conversion (see, for example, US 7,129,084). Inventive antibody molecules can easily be produced in sufficient quantities, inter alia, by recombinant methods known in the art, see, e.g. Bentley, Hybridoma 17 (1998), 559-567; Racher, Appl. Microbiol. Biotechnol. 40 (1994), 851- 856; Samuelsson, Eur. J. Immunol. 26 (1996), 3029-3034.

Further methods for the production of antibodies are well known in the art, see, e.g. Harlow and Lane, "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988.

The inventive antibodies/antibody molecules can readily be recombinantly constructed and expressed. Preferably, the antibody molecule of the invention comprises at least one, more preferably at least two, preferably at least three, more preferably at least four, more preferably at least five and most preferably at least six CDRs of the herein defined antibodies. The person skilled in the art can readily employ the information given herein to deduce corresponding CDRs of the antibodies. The term "CDR" as employed herein relates to "complementary determining region", which is well known in the art. The CDRs are parts of immunoglobulins that determine the specificity of said molecules and make contact with a specific ligand. The CDRs are the most variable part of the molecule and contribute to the diversity of these molecules. There are three CDR regions CDR1 , CDR2 and CDR3 in each V domain. CDR-H depicts a CDR region of a variable heavy chain and CDR-L relates to a CDR region of a variable light chain. VH means the variable heavy chain and VL means the variable light chain. The CDR regions of an Ig-derived region may be determined as described in Kabat "Sequences of Proteins of Immunological Interest", 5th edit. NIH Publication no. 91-3242 U.S. Department of Health and Human Services (1991); Chothia J. Mol. Biol. 196 (1987), 901-9 7 or Chothia Nature 342 (1989), 877-883.

In a further aspect of the present invention the above described methods for producing an antibody that specifically recognizes a dye which is a portion of dye/protein complex includes a step preceding the immunization wherein the dye is coupled to a protein. As an example, the dye like, e.g., Remazol, may be coupled to a protein, e.g., a protein which is commonly used in experiments as standard proteins which are easily available like, e.g., BSA, lysozyme or alcohol dehydrogenase. However, these proteins are only mentioned as examples and the present invention is not bound to these proteins. Rather, any possible protein may be used to couple or to link it to the dye of interest.

Methods for coupling dyes to a protein are well-known to the person skilled in the art. Without being bound to specific procedures, the following is only described for illustration purposes. For an overview of methods for (covalently) coupling a dye to a protein reference is made, e.g., to the review article of Brinkley M. Bioconjug Chem. 1992 Jan-Feb; 3(1 ): 2-13 and to the article of Lopez-Jaramillo, et al., in chapter 16 entitled "Vinyl Sulfone: A Multi-Purpose Function in Proteomics" the book Biochemistry, Genetics and Molecular Biology "Integrative Proteomics" edited by Hon-Chiu Eastwood Leung, Subject editors: Tsz-Kwong Man and Ricardo J. Flores , ISBN 978-953-51-0070-6, Published: February 24, 2012. The reactive dyes of the Remazol (via vinyl sulfone-reactive group), Levafix (via dichloroquinoxalines-reactive group) and Procion (via the dichlorotriazine-reactive group) group couple covalently to functional groups-Nhb (the ε-amine group of the lysine side chain or the a-amino group of the N-terminal amino acid) > -SH (the thiol group of cysteine) >-OH of proteins.

According to Puchtler H, Meloan SN, Waldrop FS. Histochemistry. 1988;88(3-6):243- 56 direct dyes such as Sirius red bind via non-ionic interactions, i.e. van der Waals and dispersion forces and hydrophobic bonding to collagen.

Without being bound to theory, remazol Brilliant Blue R-staining of proteins can, e.g., be achieved by the following protocol: Remazol Brilliant Blue R (Sigma, R8001) can be dissolved at a concentration of 10 mg/ml in 10% w/v SDS. Bovine serum albumin (BSA; Sigma, A9647) is dissolved at a concentration of 10 mg/ml in 0.15 M sodium chloride. Alcohol dehydrogenase (ADH; Sigma, A8656) is dissolved at a concentration of 10 mg/ml in 0.15 M sodium chloride. Lysozyme (Serva, 28262) is dissolved at a concentration of 10 mg/ml in 0.15 M sodium chloride. 200 μΙ of each protein solution is mixed with 50 μΙ Remazol Brilliant Blue R solution and 50 μΙ of 1 M disodium hydrogen phosphate (Na₂HP0₄) pH 9.6 solution and incubated for 20 min at 65°C. Remazol Brilliant Blue R stained BSA is then diluted with 1 ml of water and purified to a final volume of 400 μΙ by centrifugation through an Amicon Ultra-4 10K centrifugal filter with a nominal molecular weight limit of 10 kD (Millipore, UFC801024). Remazol Brilliant Blue R stained ADH and lysozyme are separated by preparative 15% SDS-PAGE in 0.025 M Tris/0.2 M Glycine/0.01% w/v SDS pH 8.5 running buffer using a 16.5 cm wide and 22 cm long slab gel unit (C.B.S. Scientific, ASU-250) with a 1.5 mm thick preparative Teflon comb with two 6 cm wide slots (CBS Scientific, VGC-1503M). The bands corresponding to ADH or lysozyme, respectively, are then excised from the gel with sterile stainless steel surgical blades (Swann-Morton, Size 22, Ref 0308) and cut into small pieces of approximately 1-2 mm³. The polyacrylamide gel pieces containing the respective prestained proteins are subsequently filled into two separate chambers of an S&S Elutrap electro- separation system (Schleicher&Schiill) and the proteins were electro-eluted from the polyacrylamide gel pieces for 6 hours at a constant voltage of 200 V at 22°C in 0.025M Tris/0.2 M Glycine/0.01% w/v SDS pH 8.5 running buffer. Each electro-eluted protein was collected in a volume of 600 pi of running buffer between a BT2 Elutrap- Membrane (Whatman, 10404092) and a BT1 Elutrap-Membrane (Whatman, 10404090) as suggested by the manufacturer.

The methods of the present invention also relate to methods for producing an antibody/antibodies that specifically recognize(s) a dye (being a portion of dye/protein complex in accordance with the foregoing) wherein said dye is recognized, perceived and/or detected by the human eye as colour (or black or white) upon illumination with visible light which ranges from approximately 380 to 800 nm.. More specifically, the dye to be specifically recognized by the antibodies as described herein is a dye that is visible for the human eye upon illumination with visible light which ranges from approximately 380 to 800 nm. The term "dye" not only comprises a colored dye visible to the human eye upon illumination with visible light which ranges from approximately 380 to 800 nm, but also comprises a black and white dye. A black dye is a dye which completely absorbs light. The color of a dye recognized, perceived and/or detected by the human eye has a complementary relationship with respect to the dye's absorbed part of the visible light: whereby a colored dye is recognized, perceived and/or detected by the (human) eye as the reflected light by the dye, the human eye does not detect, recognize or perceive the wavelengths which are absorbed by the dye. In case all wavelengths are absorbed the dye appears black or white if the light is totally reflected. Thus, in terms of the present invention, a "visually detectable dye"means that the dye can be detected by the human eye upon illumination with visible light which ranges from approximately 380 to 800 nm and includes colored and black and white dyes

The methods of the present invention also relate to methods for producing (an) antibody that specifically recognizes a dye which is a portion of dye/protein complex in accordance with the foregoing wherein the visually detectable dye is a Remazol dye, a Levafix dye, a Procion dye, Sirius dyes, Coomassie Brilliant Blue, Ponceau red, AuroDye, FerriDye, India ink, Amido black or Kongo red. While, e.g., Levafix dyes and Procion dyes are covalently couples dyes, Sirius dyes are direct dyes and do not covalently couple to dyes as outlined above. The above dyes are well known to the person skilled in the art and can, e.g., be purchased from DyStar Colours Deutschland GmbH. Examples of Remazol dyes are:

Remazol RR (Yellow RR, Orange RR, Red RR, Blue RR), Remazol RGB (Brilliant Yellow RGB, Golden Yellow RGB, Orange RGB, Deep Red RGB, Red RGB, Navy RGB), Remazol (Luminous Yellow FL, Brilliant Yellow 4GL, Brilliant Yellow 3GL, Brilliant Yellow 3GL, Yellow GL, Yellow GR, Golden Yellow RNL, Yellow 3RS, Orange BN, Brilliant Orange 3R spec, Brilliant Red BB, Red RB, Red GWF, Brilliant Red F3B, Brilliant Red 3BS, Red 3B, Brilliant Violet 5R, Brilliant Blue R spec, Brilliant Blue RN, Brilliant Blue BB, Turquoise Blue G, Brilliant Green 6B, Black RL, Navy Blue GG, Black B, Black A, Black NF, Deep Black N 150).

Levafix dyes are, e.g., described in (Waldrop FS, Puchtler H., Arch Pathol. 1975 Oct;99(10):529-32 and in Waldrop FS, Puchtler H, Akamatsu Y., Stain Technol. 1976 Jul;51(4):219-25.) Examples of Levafix dyes are Levafix CA (Brilliant Yellow CA, Yellow CA, Orange CA, Scarlet CA, Red CA, Blue CA, Navy CA), Levafix (Golden Yellow E-G, Yellow E-3RL, Orange E-3GA, Scarlet E-2GA, Brilliant Red E-BA, Brilliant Red E-4BA, Brilliant Red E-RN, Brilliant Red E-6BA, Brilliant Blue E-FFN, Royal Blue E-FR, Brilliant Blue E-BRA, Brilliant Blue E-B, Blue E-GRN, Navy Blue E- BNA, Brown E-RN, Brown E-2R, Olive E-G LA).

Procion dyes are, e.g., described in Fazekas de St Groth S, Webster RG, Datyner A., Biochim Biophys Acta. 1963 May 14;71 :377-91. Examples of Procion dyes: Procion H-EXL (Yellow H-EXL, Brilliant Orange H-EXL, Deep Red H-EXL, Brilliant Red H- EGXL, Crimson H-EXL, Blue H-EXL, Royal Blue H-EXL, Sapphire H-EXL, Navy H- EXL, Amber H-EXL, Red Brown H-EXL, Dark Blue H-EXL), Procion XL+ (Yellow XL+, Brilliant Red XL+, Rubine XL+, Cyan XL+, Dark Blue XL+, Navy XL+), Procion H-E (Yellow H-E6G, Yellow H-E4R, Orange H-ER, Red H-E3B, Red H-E7B, Blue H- ERD, Blue H-EGN, Turquoise H-A, Navy H-ER).

Sirius dyes are, e.g., described in Junqueira LC, Bignolas G, Brentani RR., Histochem J. 1979 Jul;11(4):447-55. Examples of Sirius dyes: Sirius L (Yellow K- GRL, Orange 3GDL, Red F4BL, Rubine K-2BL, Red Violet RL, Grey K-CGL), Sirius (Yellow S-2G, Yellow R, Yellow K-CF, Orange K-CF, Orange K-CFN, Scarlet S-G, Scarlet K-CF, Scarlet BN, Red F3B, Red K-BE, Blue K-CFN, Blue K-GRLN, Blue GN, Blue K-BE, Blue S-BRR, Royal Blue S, Turquoise S-FBL, Green S-4B, Brown BRK- N, Brown 3RL, Brown RL, Dark Brown G-V), Sirius Black (Black VSF h/c, Black S- VSF, Black OB-V, Black L-V, Black FRB-V, Black G).

An example of a black dye would be "Amido Black 10B" ( as, inter alia, provided by Sigma-Aldrich), whereas an example of a white dye would be "MX 010 Winter White" ( as, inter alia, provided by PRO Chemical& Dye)

In a further embodiment, the methods of the present invention also relate to methods for producing an antibody that specifically recognizes a dye which is a portion of dye/protein complex in accordance with the foregoing wherein the visually detectable dye is a Remazol dye, wherein said Remazol dye is selected from the group consisting of Remazol Turquoise, Brilliant Red F3B, Brilliant Orange 3R, Golden Yellow RNL, The following Remazol dyes taken from Nilsson et al; Table 3: Remazol Marine Blue GG, Remazol Brilliant Blue BB, Remazol Black B, Remazol Brilliant Green 6B, Remazol Brilliant Blue R spec, Remazol Gold Yellow RNL, Remazol Brilliant Orange FR, Remazol Brilliant Yellow 4GL, Remazol Black GF; The following Remazol dyes taken from page 6 of WO2006/138366: Remazol Brown GR, Remazol Brilliant Red 5BA, Remazol Brilliant Red BB, Remazol Red F3B, Remazol Red 3B, Remazol Brilliant Orange 3R, Remazol Brilliant Yellow GR, Remazol Yellow FG, Remazol Yellow GR, Remazol Brilliant Green 5GA, Remazol Green B, Remazol Brilliant Blue R, Coomassie Blue R-250, Reactive Blue Remazol Turquoise P, Remazol Brilliant Violet 5R, Remazol Red Violet R, Remazol Yellow RR, Remazol Orange RR, Remazol Red RR, Remazol Blue RR, Remazol Brilliant Yellow RGB, Remazol Golden Yellow RGB, Remazol Orange RGB, Remazol Deep Red RGB, Remazol Red RGB, Remazol Navy RGB), Remazol Luminous Yellow FL, Remazol Brilliant Yellow 4GL, Remazol Brilliant Yellow 3GL, Remazol Brilliant Yellow 3GL, Remazol Yellow GL, Remazol Yellow GR, Remazol Golden Yellow RNL, Remazol Yellow 3RS, Remazol Orange BN, Remazol Brilliant Orange 3R spec, Remazol Brilliant Red BB, Red RB, Remazol Red GWF, Remazol Brilliant Red F3B, Remazol Brilliant Red 3BS, Remazol Red 3B, Remazol Brilliant Violet 5R, Remazol Brilliant Blue R spec, Remazol Brilliant Blue RN, Remazol Brilliant Blue BB, Remazol Turquoise Blue G, Remazol Brilliant Green 6B, Remazol Black RL, Remazol Navy Blue GG, Remazol Black B, Remazol Black A, Remazol Black NF and Remazol Deep Black N 150.

As mentioned above, the methods of the present invention also relate to methods for producing an antibody that specifically recognizes a dye which is a portion of dye/protein complex in accordance with the foregoing wherein the dye is covalently linked to the protein of the dye/protein complex. However, the dye does not necessarily have to be covalently coupled to the proteins. As an example, as already outlined above, the Sirius dye is a direct dye and does not covalently couple to proteins while the other dyes, i.e., Remazol, Levafix and Procion are covalently coupled to the protein. However, the dye does not necessarily have to be coupled to the protein, in particular, in applications which are not related to the detection of marker proteins. For marker proteins, however, it commonly is the case that the dye/protein complex is covalently coupled in order to remain intact during the SDS PAGE.

As mentioned above, in humans, the generation of antibodies against reactive dye- human serum albumin conjugates has been described in workers of dye factories which are correlated with asthmatic responses. However, the methods of the present invention explicitly exclude such antibodies. Thus, methods are provided which relate to methods for producing an antibody that specifically recognizes a dye which is a portion of dye/protein complex in accordance with the foregoing with the proviso that the antibody that specifically recognizes a dye which is a portion of a dye/protein complex is not an antibody generated as part of an allergic reaction against (textile) dyes in human. In other words, the present invention does not provide for anti-dye antibodies that are polyclonal antibodies generated in humans in particular not in humans that are exposed to (textile) dyes.

The methods for producing an antibody that specifically recognizes a dye which is a portion of dye/protein complex in accordance with the foregoing are particularly preferred for the generation an antibody that specifically recognizes a dye which is a portion of dye/protein marker complex. Marker proteins or dye/protein marker complexes in accordance with the present invention are well known to the person skilled in the art and these proteins are so called protein molecular weight markers or standards which are used in the estimation of the molecular weight of a certain protein and are electrophoretically separated together with the proteins to be analysed. Examples of such molecular weight markers which are all commercially available are PageRuler^* Plus Prestained Protein Ladder 10-250kD (Thermo Scientific, 26619), ColorPlus Prestained Protein Ladder 10-230kD (New England Biolabs, P7711 ), Spectra Multicolor High Range Protein Ladder (Fermentas, SM1859), and Precision Plus Protein™ All Blue Standards (Biorad, 161-0373). By applying the method of the present invention antibodies can be generated against all the different dyes of these and other marker proteins.

However, the present invention is not only limited to dyes of marker proteins and, accordingly, not only limited to antibodies and methods for producing antibodies that specifically recognize a dye which is a portion of a covalently linked dye/protein marker complex by specifically eliciting an immune response against the dye. Rather, as outlined in great detail above, in accordance with the examples of the present invention, this rationale also applies in more general terms and relates to other dye/protein complexes which do not necessarily have to be marker proteins. As such, antibodies and methods for producing an antibody are also provided wherein the antibodies specifically recognize a dye which is a portion a dye/protein complex by selectively eliciting an immune response against the dye. Accordingly, the present invention provides methods for producing antibodies that specifically recognize a dye which is a portion of a dye/protein complex by selectively eliciting an immune response against the dye.

Moreover, the above rationale also applies in even more general terms and relates to other dye complexes wherein, e.g., the dye is complexed, coupled to or linked to a nucleic acid, like a DNA- or an RNA-molecule, a lipid or a sugar. As such, antibodies and methods for producing an antibody are also provided wherein the antibodies specifically recognize a dye which is a portion a dye/nucleic acid complex (e.g., a dye/DNA-complex or a dye/RNA-complex), a dye/lipid-complex or a dye/sugar- complex by selectively eliciting an immune response against the dye. Accordingly, the present invention provides methods for producing antibodies that specifically

( recognize a dye which is a portion of a dye/nucleic acid complex (e.g., a dye/DNA- complex or a dye/RNA-complex), a dye/lipid-complex or a dye/sugar-complex by selectively eliciting an immune response against the dye.

Thus, in a further aspect of the invention, a method for producing an antibody that specifically recognizes a dye which is a portion of a dye/nucleic acid complex (e.g., a dye/DNA-complex or a dye/RNA-complex), a dye/lipid-complex or a dye/sugar- complex by selectively eliciting an immune response against the dye is provided, wherein the method comprises the steps of:

(i) immunizing a non-human animal with a first dye/nucleic acid complex (e.g., a dye/DNA-complex or a dye/RNA-complex), a dye/lipid-complex or a dye/sugar- complex,

(ii) subsequently immunizing said non-human animal with a second dye/nucleic acid complex (e.g., a dye/DNA-complex or a dye/RNA-complex), a dye/lipid- complex or a dye/sugar-complex wherein the DNA, RNA, lipid or sugar portion of the dye/nucleic acid complex (e.g., a dye/DNA-complex or a dye/RNA- complex), a dye/lipid-complex or a dye/sugar-complex is similar or different than the DNA, RNA, lipid or sugar portion of the dye/protein complex used in step (i);

(iii) optionally repeating step (ii) at least once with a further dye/nucleic acid complex (e.g., a dye/DNA-complex or a dye/RNA-complex), a dye/lipid- complex or a dye/sugar-complex wherein the DNA, RNA, lipid or sugar portion of the dye/nucleic acid complex (e.g., a dye/DNA-complex or a dye/RNA- complex), a dye/lipid-complex or a dye/sugar-complex is similar or different than the DNA, RNA, lipid or sugar portion of the dye/nucleic acid complex (e.g., a dye/DNA-complex or a dye/RNA-complex), a dye/lipid-complex or a dye/sugar-complex used in step (i) and any subsequent step;

(iv) optionally immunizing said non-human animal with a mixture of all dye/nucleic acid complexes (e.g., a dye/DNA-complexes or a dye/RNA-complexes), dye/lipid-complexes or dye/sugar-complexes used in step (i) and/or (ii) and/or (iii); (v) obtaining/isolating said dye-specific antibody, wherein said dye-specific antibody is obtained/isolated by screening using a dye/nucleic acid complex (e.g., a dye/DNA-complex or a dye/RNA-complex), a dye/lipid-complex or a dye/sugar-complex that has not been used in the elicitation of the immune response and wherein the DNA, RNA, lipid or sugar portion of the dye/protein complex is similar or different than the DNA, RNA, lipid or sugar portion of the dye/protein complex used in step (i) and/or (ii) and/or (iii).

It is known in the prior art that DNA, RNA, lipids are poor immunogens. Accordingly, in order to nevertheless elicit an immune response against DNA, RNA or lipids in accordance with the present invention the DNA, RNA or lipid is coupled to a protein. Such coupling methods are known to the person skilled in the art and can easily performed. As an example, reference is made to Cerutti ML, Centeno JM, Goldbaum FA, de Prat-Gay G.₍ J Biol Chem. Apr 20;276( 16): 12769-73 (2001 ). The authors used a DNA/Protein complex as immunogen. Moreover, other strategies to obtain DNA binding antibodies are also envisaged by using, e.g., phage display technology. Further, in order to elicit an immune response towards lipids, the lipids may, e.g., be coupled to amino acids as carriers (Arnon R, Teitelbaum D., Chem Phys Lipids. Dec; 13(4):352-66 (1974)).

As regards other preferred embodiments, the same applies to the above method for producing an antibody that specifically recognizes a dye which is a portion of a dye/nucleic acid complex (e.g., a dye/DNA-complex or a dye/RNA-complex), a dye/lipid-complex or a dye/sugar-complex by selectively eliciting an immune response against the dye as has been set forth above in connection with the method for producing an antibody that specifically recognizes a dye which is a portion of a dye/protein (marker) complex by selectively eliciting an immune response against the dye. Alternatively to the above immunization protocol for producing an antibody that specifically recognizes a dye which is a portion of a dye/nucleic acid complex (e.g., a dye/DNA-complex or a dye/RNA-complex), a dye/lipid-complex or a dye/sugar-complex by selectively eliciting an immune response against the dye by immunizing with different dye/protein complexes it is also envisaged that dye specific antibodies can also be generated by immunizing one or more times (i.e., once, twice, three times or even four or five times) with the same dye/nucleic acid complex (e.g., dye/DNA-complex or a dye/RNA-complex), dye/lipid-complex or the same dye/sugar- complex as has been set forth above in connection with the method for producing an antibody that specifically recognizes a dye which is a portion of a dye/protein (marker) complex by selectively eliciting an immune response against the dye.

Moreover, the present invention provides an antibody produced by any of the above methods. Thus, the present invention relates to an antibody obtained by the above methods.

Moreover, in a preferred embodiment, the antibody produced by the method of the present invention is an antibody that specifically recognizes a dye which is a portion of a dye/protein complex, wherein said dye is perceived by the human eye as colour (or black or white) upon illumination with visible light which ranges from approximately 380 to 800 nm.

Accordingly, in a preferred embodiment, an antibody that specifically recognizes a dye which is a portion of a dye/protein complex or an antibody obtainable by any one the above methods is provided, with the proviso that the antibody that specifically recognizes a dye which is a portion of a dye/protein complex is not an antibody generated as part of an allergic reaction against textile dyes in human.

Preferably, the antibody of the invention is an antibody which specifically recognizes the dye Remazol Brilliant Blue R.

In other embodiments, the antibody of the invention is an antibody which specifically recognizes a dye which is a portion of dye/protein complex in accordance with the foregoing wherein the visually detectable dye is a Remazol dye, a Levafix dye, a Procion dye, Sirius dyes, Coomassie Brilliant Blue, Ponceau red, AuroDye, FerriDye, India ink, Amido black or Kongo red.

Thus, in preferred embodiments, the antibody of the invention is an antibody which specifically recognizes a dye which is a portion of dye/protein complex in accordance with the foregoing, wherein the visually detectable dye is Remazol RR (Yellow RR, Orange RR, Red RR, or Blue RR), Remazol RGB (Brilliant Yellow RGB, Golden Yellow RGB, Orange RGB, Deep Red RGB, Red RGB, or Navy RGB), or Remazol (Luminous Yellow FL, Brilliant Yellow 4GL, Brilliant Yellow 3GL, Brilliant Yellow 3GL, Yellow GL, Yellow GR, Golden Yellow RNL, Yellow 3RS, Orange BN, Brilliant Orange 3R spec, Brilliant Red BB, Red RB, Red GWF, Brilliant Red F3B, Brilliant Red 3BS, Red 3B, Brilliant Violet 5R, Brilliant Blue R spec, Brilliant Blue RN, Brilliant Blue BB, Turquoise Blue G, Brilliant Green 6B, Black RL, Navy Blue GG, Black B, Black A, Black NF, or Deep Black N 150).

Moreover, in preferred embodiments, the antibody of the invention is an antibody which specifically recognizes a dye which is a portion of dye/protein complex in accordance with the foregoing, wherein the visually detectable dye is Levafix CA (Brilliant Yellow CA, Yellow CA, Orange CA, Scarlet CA, Red CA, Blue CA or Navy CA), or Levafix (Golden Yellow E-G, Yellow E-3RL, Orange E-3GA, Scarlet E-2GA, Brilliant Red E-BA, Brilliant Red E-4BA, Brilliant Red E-RN, Brilliant Red E-6BA, Brilliant Blue E-FFN, Royal Blue E-FR, Brilliant Blue E-BRA, Brilliant Blue E-B, Blue E-GRN, Navy Blue E-BNA, Brown E-RN, Brown E-2R, or Olive E-GLA).

Further, in preferred embodiments, the antibody of the invention is an antibody which specifically recognizes a dye which is a portion of dye/protein complex in accordance with the foregoing, wherein the visually detectable dye is Procion H-EXL (Yellow H- EXL, Brilliant Orange H-EXL, Deep Red H-EXL, Brilliant Red H-EGXL, Crimson H- EXL, Blue H-EXL, Royal Blue H-EXL, Sapphire H-EXL, Navy H-EXL, Amber H-EXL, Red Brown H-EXL, or Dark Blue H-EXL), Procion XL+ (Yellow XL+, Brilliant Red XL+, Rubine XL+, Cyan XL+, Dark Blue XL+, or Navy XL+), or Procion H-E (Yellow H-E6G, Yellow H-E4R, Orange H-ER, Red H-E3B, Red H-E7B, Blue H-ERD, Blue H- EGN, Turquoise H-A, or Navy H-ER).

Moreover, in preferred embodiments, the antibody of the invention is an antibody which specifically recognizes a dye which is a portion of dye/protein complex in accordance with the foregoing, wherein the visually detectable dye is Sirius L (Yellow K-GRL, Orange 3GDL, Red F4BL, Rubine K-2BL, Red Violet RL, or Grey K-CGL), Sirius (Yellow S-2G, Yellow R, Yellow K-CF, Orange K-CF, Orange K-CFN, Scarlet S-G, Scarlet K-CF, Scarlet BN, Red F3B, Red K-BE, Blue K-CFN, Blue K-GRLN, Blue GN, Blue K-BE, Blue S-BRR, Royal Blue S, Turquoise S-FBL, Green S-4B, Brown BRK-N, Brown 3RL, Brown RL, or Dark Brown G-V), or Sirius Black (Black VSF h/c, Black S-VSF, Black OB-V, Black L-V, Black FRB-V, or Black G).

In yet another preferred embodiment, the antibody of the invention is an antibody which specifically recognizes a dye which is a portion of dye/protein complex in accordance with the foregoing, wherein the visually detectable dye is a Remazol dye, wherein said Remazol dye is selected from the group consisting of Remazol Turquoise, Brilliant Red F3B, Brilliant Orange 3R, Golden Yellow RNL, Remazol Marine Blue GG, Remazol Brilliant Blue BB, Remazol Black B, Remazol Brilliant Green 6B, Remazol Brilliant Blue R spec, Remazol Gold Yellow RNL, Remazol Brilliant Orange FR, Remazol Brilliant Yellow 4GL, Remazol Black GF, Remazol Brown GR, Remazol Brilliant Red 5BA, Remazol Brilliant Red BB, Remazol Red F3B, Remazol Red 3B, Remazol Brilliant Orange 3R, Remazol Brilliant Yellow GR, Remazol Yellow FG, Remazol Yellow GR, Remazol Brilliant Green 5GA, Remazol Green B, Remazol Brilliant Blue R, Coomassie Blue R-250, Reactive Blue Remazol Turquoise P, Remazol Brilliant Violet 5R, Remazol Red Violet R, Remazol Yellow RR, Remazol Orange RR, Remazol Red RR, Remazol Blue RR, Remazol Brilliant Yellow RGB, Remazol Golden Yellow RGB, Remazol Orange RGB, Remazol Deep Red RGB, Remazol Red RGB, Remazol Navy RGB, Remazol Luminous Yellow FL, Remazol Brilliant Yellow 4GL, Remazol Brilliant Yellow 3GL, Remazol Brilliant Yellow 3GL, Remazol Yellow GL, Remazol Yellow GR, Remazol Golden Yellow RNL, Remazol Yellow 3RS, Remazol Orange BN, Remazol Brilliant Orange 3R spec, Remazol Brilliant Red BB, Red RB, Remazol Red GWF, Remazol Brilliant Red F3B, Remazol Brilliant Red 3BS, Remazol Red 3B, Remazol Brilliant Violet 5R, Remazol Brilliant Blue R spec, Remazol Brilliant Blue RN, Remazol Brilliant Blue BB, Remazol Turquoise Blue G, Remazol Brilliant Green 6B, Remazol Black RL, Remazol Navy Blue GG, Remazol Black B, Remazol Black A, Remazol Black NF and Remazol Deep Black N 150. Moreover, the present invention provides a composition comprising two or more (preferably, two, three, four, five, six, seven or eight) antibodies obtainable by any one of the above methods, wherein each antibody in said composition recognizes a dye of an alternate dye/protein complex. Preferably, one of these antibodies of the composition is the antibody which specifically recognizes the dye Remazol Brilliant Blue R.

In yet another further embodiment, and in accordance with the above, the present invention relates to an antibody which is produced by the hybridoma cells of hybridoma 2D2-F11 deposited at the Leibnitz-lnstitut DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen) Braunschweig under No: DSM ACC3212. Said hybridoma cells (i.e. the clone 2D2-F11) are issued ,from the fusion of spleen lymphocytes with myeloma cells. This antibody is capable of specifically recognizing the dye Remazol Brilliant Blue R. The spleen cells were isolated from mice immunized with blue dye prestained (covalently coupled to) marker proteins of the BioRad Precision Plus Protein™ All Blue Standard (161-0373). For this purpose, the proteins of the BioRad Precision Plus Protein Standard marker mixture, which consists of 10 different proteins (10, 15, 20, 25, 37, 50, 75, 100, 150, 250 kD), were separated by SDS-PAGE, the bands corresponding to the 25, 50 and 75 kD marker proteins were cut out and electro-eluted in separate electro-elution chambers. To evoke in mice an immune response specific for the blue dye of the marker proteins, the first immunization was performed with the 50 kD marker protein. This was followed by a booster immunization with the 75 kD and a third boost with the 25 kD marker. This immunization schedule should impede a secondary immune response against particular marker proteins and should promote a strong secondary immune response against the dye common to all marker proteins. The final boost was performed with a mixture of all three (25, 50 and 75 kD) marker proteins. The mouse with the highest antibody titer was selected for the fusion of splenocytes with X63- Ag8.653 myeloma cells, from which the hybridoma single clone 2D2-F11 was established.

Thus, the present invention relates to the antibody or a derivative thereof that is produced by/obtainable from the hybridoma cells of hybridoma 2D2-F11 deposited at the Leibnitz-lnstitut DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen) Braunschweig under No: DSM ACC3212 which is capable of detecting the dye Remazol Brilliant Blue R. In the context of the disclosed and the descriptive terms, it is to be understood that the term "produced by" and "obtainable from" does not relate to the specific monoclonal antibodies but also to derivatives and variants of said deposited antibodies. Such derivatives and variants have at least parts of the CDR sequences of the deposited monoclonal antibodies. Derivatives and variants comprise but are not limited to CDR grafted, humanized antibodies, Fab, Fab', Fab'- SH, FV, scFV, F(ab')2, and a diabody.

The invention does not only relate to an antibody that is obtained or obtainable from the hybridoma cells of hybridoma 2D2-F11 deposited at the Leibnitz-lnstitut DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen) Braunschweig under No: DSM ACC3212 which is capable of detecting the dye Remazol Brilliant Blue R but also to variants thereof which are capable of detecting the dye Remazol Brilliant Blue R.

Such variants may have conservative substitutions. The term "conservative substitution" refers to substitutions of amino acids in a protein with other amino acids having similar characteristics (e.g. charge, side-chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.), such that the changes can frequently be made without altering the biological activity of the protein. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co. 4th Ed. (1987), 224 ). In addition, substitutions of structurally or functionally similar amino acids are less likely to disrupt biological activity. Within the context of the present invention the binding compounds/antibodies of the present invention comprise polypeptide chains with sequences that include up to 0 (no changes), 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20 or more conservative amino acid substitutions when compared with the specific amino acid sequences disclosed herein, for example, SEQ ID NOs: 4, 33, 43, 53, 63 (referring to the variable region of the antibody heavy chain of the antibody) and 6, 31 , 41 , 51 , 61 (referring to the variable of the light chain of the antibody). As used herein, the phrase "up to X" conservative amino acid substitutions includes 0 substitutions and any number of substitutions up to 10 and including 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 substitutions.

Accordingly, the present invention relates to an antibody that is obtained or obtainable from the hybridoma cells of hybridoma 2D2-F11 deposited at the Leibnitz- Institut DS Z (Deutsche Sammlung von Mikroorganismen und Zellkulturen) Braunschweig under No: DSM ACC32 2 which is capable of detecting the dye Remazol Brilliant Blue R and wherein said antibody comprises a light chain variable region comprising the sequence of SEQ ID NO: 6 having up to 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 conservative amino acid substitutions and a heavy chain variable region comprising the sequence of SEQ ID NO: 4 having up to 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 conservative amino acid substitutions.

Such exemplary substitutions are preferably made in accordance with those set forth in Table 1 as follows:

TABLE 1

Exemplary Conservative Amino Acid Substitutions

The present invention also relates to a nucleic acid, for example DNA, encoding an antibody that is obtained by any one of the methods of the present invention. Such a nucleic acid encoding an antibody comprises at least one antibody light chain variable region (VL) and at least one antibody heavy chain variable region (VH), or binding fragments of these domains, wherein the VL comprises specific complementarity determining regions (CDR).

The nucleic acid molecule may also encode one or both of the heavy and/or light chain variable regions. The nucleic acid molecule of the present invention may also encode the antibody that is obtained or obtainable from the hybridoma cells of hybridoma 2D2-F11 deposited at the Leibnitz-lnstitut DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen) Braunschweig under No: DSM ACC3212 which is capable of detecting the dye Remazol Brilliant Blue R.

The above nucleic acid molecule of the present invention may be a natural nucleic acid molecule as well as a recombinant nucleic acid molecule. The nucleic acid molecule of the invention may, therefore, be of natural origin, synthetic or semisynthetic. It may comprise DNA, RNA as well as PNA and it may be a hybrid thereof.

It is evident to the person skilled in the art that regulatory sequences may be added to the nucleic acid molecule of the invention. For example, promoters, transcriptional enhancers and/or sequences which allow for induced expression of the polynucleotide of the invention may be employed. A suitable inducible system is for example tetracycline-regulated gene expression as described, e.g., by Gossen and Bujard, Proc. Natl. Acad. Sci. USA 89 (1992), 5547-5551 ) and Gossen, Trends Biotech. 12 (1994), 58-62, or a dexamethasone-inducible gene expression system as described, e.g. by Crook, EMBO J. 8 (1989), 513-519.

Furthermore, said nucleic acid molecule may contain, for example, thioester bonds and/or nucleotide analogues. Said modifications may be useful for the stabilization of the nucleic acid molecule against endo- and/or exonucleases in the cell. Said nucleic acid molecules may be transcribed by an appropriate vector containing a chimeric gene which allows for the transcription of said nucleic acid molecule in the cell. In this respect, it is also to be understood that the nucleic acid molecule encoding the binding compound/antibody of the present invention can be used for "gene targeting". In the context of the present invention said nucleic acid molecules are labeled. Methods for the detection of nucleic acids are well known in the art, e.g., Southern and Northern blotting, PCR or primer extension.

The nucleic acid molecule(s) of the invention may be a recombinantly produced chimeric nucleic acid molecule comprising any of the aforementioned nucleic acid molecules either alone or in combination. Preferably, the nucleic acid molecule of the invention is part of a vector.

The present invention therefore also relates to a vector comprising the nucleic acid molecule of the present invention. Accordingly, the present invention relates to vectors, preferably expression vectors comprising the nucleic acids of the invention.

The vector of the present invention may be, e.g., a plasmid, cosmid, virus, bacteriophage or another vector used e.g. conventionally in genetic engineering, and may comprise further genes such as marker genes which allow for the selection of said vector in a suitable host cell and under suitable conditions.

Furthermore, the vector of the present invention may, in addition to the nucleic acid sequences of the invention, comprise expression control elements, allowing proper expression of the coding regions in suitable hosts. Such control elements are known to the skilled person and may include a promoter, a splice cassette, translation initiation codon, translation and insertion site for introducing an insert into the vector. Preferably, the nucleic acid molecule of the invention is operatively linked to said expression control sequences allowing expression in eukaryotic or prokaryotic cells. Accordingly, the present invention relates to a vector comprising the nucleic acids of the invention, wherein the nucleic acid is operably linked to control sequences that are recognized by a host cell when the eukaryotic and/or prokaryotic (host) cell is transfected with the vector.

Control elements ensuring expression in eukaryotic and prokaryotic (host) cells are well known to those skilled in the art. As mentioned herein above, they usually comprise regulatory sequences ensuring initiation of transcription and optionally poly- A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional as well as translational enhancers, and/or naturally-associated or heterologous promoter regions. Possible regulatory elements permitting expression in for example mammalian host cells comprise the CMV-HSV thymidine kinase promoter, SV40, RSV-promoter (Rous Sarcoma Virus), human elongation factor 1a-promoter, the glucocorticoid-inducible MMTV-promoter Mouse Mammary Tumor Virus), metallothionein- or tetracyclin- inducible promoters, or enhancers, like CMV enhancer or SV40-enhancer. For expression in neural cells, it is envisaged that neurofilament-, PGDF-, NSE-, PrP-, or thy-1 -promoters can be employed. Said promoters are known in the art and, inter alia, described in Charron, J. Biol. Chem. 270 (1995), 25739-25745. For the expression in prokaryotic cells, a multitude of promoters including, for example, the tac-lac-promoter or the trp promoter, has been described. Besides elements which are responsible for the initiation of transcription such regulatory elements may also comprise transcription termination signals, such as SV40-poly-A site or the tk-poly-A site, downstream of the polynucleotide. In this context, suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pRc/CMV, pcDNAI , pcDNA3 (ln-vitrogene), pSPORTI (GIBCO BRL), pX (Pagano, Science 255 (1992), 1144-1147), yeast two-hybrid vectors, such as pEG202 and dpJG4-5 (Gyuris, Cell 75 (1995), 791-803), or prokaryotic expression vectors, such as lambda gt11 or pGEX (Amersham-Pharmacia). Beside the nucleic acid molecules of the present invention, the vector may further comprise nucleic acid sequences encoding for secretion signals. Such sequences are well known to the person skilled in the art. Furthermore, depending on the expression system used leader sequences capable of directing the peptides of the invention to a cellular compartment may be added to the coding sequence of the nucleic acid molecules of the invention and are well known in the art. The leader sequence(s) is (are) assembled in appropriate phase with translation, initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein, or a protein thereof, into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein including a C- or N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and, as desired, the collection and purification of the antibody molecules or fragments thereof of the invention may follow.

Furthermore, the vector of the present invention may also be an expression vector. The nucleic acid molecules and vectors of the invention may be designed for direct introduction or for introduction via liposomes, viral vectors (e.g. adenoviral, retroviral), electroporation, ballistic (e.g. gene gun) or other delivery systems into the cell. Additionally, a baculoviral system can be used as eukaryotic expression system for the nucleic acid molecules of the invention.

The present invention also relates to a host cell transfected or transformed with the vector of the invention or a non-human host carrying the vector of the present invention, i.e. to a host cell or host which is genetically modified with a nucleic acid molecule according to the invention or with a vector comprising such a nucleic acid molecule. The term "genetically modified" means that the host cell or host comprises in addition to its natural genome a nucleic acid molecule or vector according to the invention which was introduced into the cell or host or into one of its predecessors/parents. The nucleic acid molecule or vector may be present in the genetically modified host cell or host either as an independent molecule outside the genome, preferably as a molecule which is capable of replication, or it may be stably integrated into the genome of the host cell or host.

The host cell of the present invention may be any prokaryotic or eukaryotic cell. Suitable prokaryotic cells are those generally used for cloning like E. coli or Bacillus subtilis. Furthermore, eukaryotic cells comprise, for example, fungal or animal cells. Examples for suitable fungal cells are yeast cells, preferably those of the genus Saccharomyces and most preferably those of the species Saccharomyces cerevisiae. Suitable animal cells are, for instance, insect cells, vertebrate cells, preferably mammalian cells, such as e.g. HEK293, NSO, CHO,COS-7, MDCK, U2- OSHela, NIH3T3, MOLT-4, Jurkat, PC-12, PC-3, I MR, NT2N, Sk-n-sh, CaSki, C33A. These host cells, e.g. CHO-cells, may provide posts-translational (secondary) modifications to the antibody molecules of the invention, including leader peptide removal, folding and assembly of H and C chains, glycosylation of the molecule at correct sides and secretion of the functional molecule. Further suitable cell lines known in the art are obtainable from cell line depositories, like, e.g., the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) or the American Type Culture Collection (ATCC). In accordance with the present invention, it is furthermore envisaged that primary cells/cell cultures may function as host cells. Said cells are in particular derived from insects (like insects of the species Drosophila or Blatta) or mammals (like human, swine, mouse or rat). Said host cells may also comprise cells from and/or derived from cell lines like neuroblastoma cell lines. The above mentioned primary cells are well known in the art and comprise, inter alia, primary astrocytes, (mixed) spinal cultures or hippocampal cultures.

In the context of the present invention, the host cell of the present invention may be the hybridoma cells of hybridoma 2D2-F11 deposited at the Leibnitz-lnstitut DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen) Braunschweig under No: DSM ACC3212 which is capable of detecting the dye Remazol Brilliant Blue R.

The present invention also relates to methods of producing the antibody of the present invention culturing a host cell harbouring an expression vector encoding the binding compounds in culture medium, and recovering the antibody from the host cell or culture medium. The present invention may also relate to a method for producing an antibody of the present invention comprising the cultivation of the host cell of the present invention and recovering the antibody from the culture. Accordingly, the present invention relates to a method for producing an antibody of the present invention, wherein said method comprises the cultivation of the host cell, for example the hybridoma cells of hybridoma 2D2-F11 deposited at the Leibnitz-lnstitut DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen) Braunschweig under No: DSM ACC3212 and recovering the antibody.

Host cells, e.g., CHO cells, may provide post-translational (secondary) modification on the expressed binding compounds of the present invention. These modifications comprise, inter alia, glycosylation and phosphorylation.

Moreover, in the present invention, it is preferred that the antibodies of the present invention are detectably labeled. A variety of techniques are available for labeling biomolecules (binding compounds) like antibodies, are well known to the skilled person in the art and are considered to be within the scope of the present invention. Such techniques are, e.g., described in Tijssen, "Practice and theory of enzyme immuno assays", Burden, RH and von Knippenburg (Eds), 15 (1985), "Basic methods in molecular biology"; Davis LG, Dibmer MD; Battey Elsevier (1990), Mayer et al., (Eds) "Immunochemical methods in cell and molecular biology" Academic Press, London (1987), or in the series "Methods in Enzymology", Academic Press, Inc. There are many different labels and methods of labeling known to those of ordinary skill in the art. Examples of the types of labels which can be used in the present invention include enzymes, radioisotopes, colloidal metals, fluorescent compounds, chemiluminescent compounds, and bioluminescent compounds. Commonly used labels comprise, inter alia, fluorochromes (like fluorescein, rhodamine, Texas Red, etc.), enzymes (like horse radish peroxidase, β- galactosidase, alkaline phosphatase), radioactive isotopes (like ³²P or ¹²⁵l), biotin, digoxygenin, colloidal metals, chemi- or bioluminescent compounds (like dioxetanes, luminol or acridiniums). Labeling procedures, like covalent coupling of enzymes or biotinyl groups, iodinations, phosphorylations, biotinylations, etc. are well known in the art. Detection methods comprise, but are not limited to, autoradiography, fluorescence microscopy, direct and indirect enzymatic reactions, etc. Commonly used detection assays comprise radioisotopic or non-radioisotopic methods. These comprise, inter alia, Westernblotting, overlay-assays, RIA (Radioimmuno Assay) and IRMA (Immune Radioimmunometric Assay), EIA (Enzyme Immuno Assay), ELISA (Enzyme Linked Immuno Sorbent Assay), FIA (Fluorescent Immuno Assay), and CLIA (Chemioluminescent Immune Assay).

As regards other preferred embodiments of the antibodies of the invention, the same applies as has been set forth above in connection with the methods of the present invention. Thus, inter alia, in a particular embodiment, the antibody of the present invention is an antibody that specifically recognizes a dye which is a portion of a dye/protein complex or an antibody obtainable by any one the above methods wherein the dye is covalently linked to the protein of the dye/protein complex. Moreover, in a particular embodiment, the antibody of the present invention is an antibody that specifically recognizes a dye which is a portion of a dye/protein complex or an antibody obtainable by any one the above methods wherein the dye/protein complex is a dye/protein marker complex.

The antibodies of the present invention can be used in many applications that require the detection of a particular dye.

As an example, without being bound to theory, the present invention, in accordance with the examples, relates to the use of an antibody that specifically recognizes a dye which is a portion of a covalently linked dye/protein marker complex or more than one antibodies that specifically recognize a dye which is a portion of a covalently linked dye/protein marker complex wherein each of said more than one antibodies recognizes a dye of an alternate covalently linked dye/protein marker complex in the detection of one or more dye-prestained protein molecular weight marker proteins.

Such a method can, e.g., beneficially be used in the estimation of the size of a sample protein. Accordingly, the present invention is preferably directed to the use of an antibody that specifically recognizes a dye which is a portion of a covalently linked dye/protein marker complex or more than one antibodies that specifically recognize a dye which is a portion of a covalently linked dye/protein marker complex wherein each of said more than one antibodies recognizes a dye of an alternate covalently linked dye/protein marker complex in the estimation of the size of a sample protein comprising the steps of:

(i) electrophoresing simultaneously in separate lanes on a gel a sample protein and a protein ladder comprising at least one dye-prestained protein molecular weight marker protein;

(ii) transferring the separated proteins on a membrane;

(iii) simultaneously detecting the sample protein and said at least one dye- prestained protein molecular weight marker protein comprising the incubation of the membrane with a protein sample specific antibody and an antibody that specifically recognizes a dye which is a portion of a covalently linked dye/protein marker complex or

more than one antibodies that specifically recognize/detect/bind a dye which is a portion of a covalently linked dye/protein marker complex wherein each of said more than one antibodies recognizes/detects/binds a dye of an alternate covalently linked dye/protein marker complex; and

(iv) comparing the size of the detected sample protein with the at least one detected dyes which is a portion of a covalently linked dye/protein marker complex.

The above step (ii) of transferring the separated proteins on a membrane is only optional and the detection of the sample protein in accordance with the present invention may also be performed directly in the gel in case an "in-gel Western blot" is performed.

Methods for performing the above steps are routine to the person skilled in the art and can be assessed easily applying standard techniques in molecular/biochemical research and applications. "More than one" antibodies that specifically recognize/detect/bind a dye which is a portion of a covalently linked dye/protein marker complex wherein each of said "more than one" antibodies recognizes/detects/binds a dye of an alternate covalently linked dye/protein marker complex not only includes one different antibodies that specifically recognize/detect/bind a dye which is a portion of a covalently linked dye/protein marker complex but may include two, three, four, five, six, seven, eight or even nine different antibodies that specifically recognize/detect/bind a dye which is a portion of a covalently linked dye/protein marker complex wherein each of said "more than one" antibody/antibodies recognizes/detects/binds a dye of an alternate covalently linked dye/protein marker complex. Preferably, the number of antibodies to be used equals the number of the different covalently linked dye/protein marker complexes of the respective dye-prestained protein molecular weight marker. Correspondingly, the protein ladder used in the above method comprises "at least one" dye-prestained protein molecular weight marker protein, i.e., one, two, three, four, five, six, seven, eight or even nine (different) dye-prestained protein molecular weight marker proteins.

Moreover, it is envisaged that the antibodies of the present invention may be used to visualize with high sensitivity co-immunoprecipitated proteins. In such a method, one first immunoprecipitates the protein of interest, then stains the entire immunoprecipitate with a certain dye that stains proteins like, e.g., the dye Remazol blue. Subsequently, the stained immunoprecipitate may be analyzed and separated by SDS-PAGE, followed by western blotting with an antibody of the invention. The pre-staining of the immunoprecipitates may, e.g., be performed as it is described by Mata-Gomez et al, PLoS ONE7(2): e31338 2012 which describes an accelerated identification of proteins by mass spectrometry by employing covalent pre-gel staining with the dye Uniblue A. Uniblue A (Sigma) is the vinyl sulfone derivate = an already activated version of Remazol. The antibody of the present invention, i.e., the one obtainable by the hybridoma cells of hybridoma 2D2-F11 deposited at the Leibnitz-lnstitut DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen) Braunschweig under No: DSM ACC3212. Said hybridoma cells (i.e. the clone 2D2- F11) recognize Uniblue A.

As regards other preferred embodiments of the uses of the invention, the same applies as has been set forth above in connection with the methods of the invention and in connection with the antibodies of the present invention. Accordingly, in a preferred embodiment, the present invention relates to the above uses, wherein the antibody that specifically recognizes a dye which is a portion of a covalently linked dye/protein marker complex is an antibody of the present invention as defined above.

In yet another embodiment, the present invention provides for a kit comprising at least one antibody molecule of the invention. Moreover, the kit may comprise the corresponding dye-prestained protein molecular weight marker protein(s) alone or in the form of (a) prestained protein ladder marker(s). Thus, in a preferred embodiment, the kit comprises "at least one" antibodies that recognizes/detects/binds a dye of an alternate covalently linked dye/protein marker complex, not only includes one different antibody that specifically recognizes/detects/binds a dye which is a portion of a covalently linked dye/protein marker complex but may include two, three, four, five, six, seven, eight or even nine different antibodies that specifically recognize/detect/bind a dye which is a portion of a covalently linked dye/protein marker complex wherein each of said "more than one" antibody/antibodies recognizes/detects/binds a dye of an alternate covalently linked dye/protein marker complex. Preferably, the number of antibodies to be used equals the number of the different covalently linked dye/protein marker complexes of the respective dye- prestained protein molecular weight marker wherein the latter may also be a preferred component of the kit. Advantageously, the kit of the present invention further comprises, optionally (a) buffer(s), storage solutions and/or remaining reagents or materials required for the conduct of the above uses and methods. Furthermore, parts of the kit of the invention can be packaged individually in vials or bottles or in combination in containers or multicontainer units. The kit of the present invention may be advantageously used, inter alia, for carrying out the methods of the invention and could be employed in a variety of applications referred herein, e.g., in the uses as outlined above. The manufacture of the kits follows preferably standard procedures which are known to the person skilled in the art. In a preferred embodiment, the kit comprises an antibody which specifically recognizes the dye Remazol Brilliant Blue R as described above. Moreover, the kit may comprise a composition comprising two or more antibodies obtainable by any one of the above methods, wherein each antibody in said composition recognizes a dye of an alternate dye/protein complex. Preferably, one of these antibodies of the composition part of the kit is the antibody which specifically recognizes the dye Remazol Brilliant Blue R.

Moreover, even more preferred, the kit of the present invention comprises an antibody which is produced by the hybridoma cells of hybridoma 2D2-F11 deposited at the Leibnitz-lnstitut DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen) Braunschweig under No: DSM ACC3212 as described in detail above.

Thus, the present invention relates to a kit comprising the antibody or a derivative thereof that is produced by/obtainable from the hybridoma cells of hybridoma 2D2- F11 deposited at the Leibnitz-lnstitut DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen) Braunschweig under No: DSM ACC3212 which is capable of detecting the dye Remazol Brilliant Blue R. In the context of the disclosed and the descriptive terms, it is to be understood that the term "produced by" and "obtainable from" does not relate to the specific monoclonal antibodies but also to derivatives and variants of said deposited antibodies. Such derivatives and variants have at least parts of the CDR sequences of the deposited monoclonal antibodies. Derivatives and variants comprise but are not limited to CDR grafted, humanized antibodies, Fab, Fab', Fab'-SH, FV, scFV, F(ab')2, and a diabody. The invention not only relates to a kit comprising the antibody that is obtained or obtainable from the hybridoma cells of hybridoma 2D2-F11 deposited at the Leibnitz-lnstitut DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen) Braunschweig under No: DSM ACC3212 which is capable of detecting the dye Remazol Brilliant Blue R but also to variants thereof which are capable of detecting the dye Remazol Brilliant Blue R.

Thus, the present invention relates to a kit comprising the antibody of the invention as defined above or the composition as defined above. Optional components of the kit may be secondary antibodies suitable to be used in the above methods. Moreover, the antibodies of the present invention like, e.g., the 2D2-F11 antibody, may be directly coupled to horseradish peroxidase (or alkaline-phosphatase or to fluorescently detectable dyes) which may be a component of the kit. Moreover, the kit may also comprise as a component an anti-mouse-HRP coupled secondary antibody, or an (alkaline-phosphatase-coupled or fluorescently detectable dyes coupled secondary antibody).

Finally, in accordance with the foregoing, the present invention provides the use of a kit in the detection of one or more protein molecular weight marker proteins prestained with dyes or in the estimation of the size of a sample protein, said kit comprising the antibody of the invention or the composition of the invention.

Figure 1 : Purification of Precision Plus Protein™ All Blue Standards marker proteins. 5μΙ of gel eluted and dialyzed prestained marker proteins (Precision Plus Protein™ All Blue Standards, Biorad, 161-0373) with the size of 75, 50 and 25 kD, respectively, was separated by 12.5% SDS-PAGE together with 4μΙ of Precision Plus Protein™ All Blue Standards to estimate the amount of prestained proteins in the dialyzed samples used for immunizations of mice.

Figure 2: Immune sera of mice detect Precision Plus Protein™ All Blue Standards marker proteins. 40μΙ of Precision Plus Protein™ All Blue Standards was separated by 10% SDS-PAGE with a preparative comb, transferred to nitrocellulose membrane (GE Healthcare) and blocked in 3% Non-fat dry milk (NFDM) in PBS-Tween for 1h at RT. The membrane was then incubated in a Miniblotter 28 channel unit (Immunetics) with the indicated mouse sera diluted 1 :500 in 0.5% NFDM in PBS-T o/n at 4°C. After washing 3x 5min in PBS-T, incubation with anti-mouse-HRP coupled secondary antibody for 1 h at RT and 3x1 Omin washes in PBS-T, ECL was performed with GE Healthcare ECL reagents (RPN2106).

Figure 3: Clone 2D2-F11 detects blue prestained protein marker bands from different companies. 1 μΙ (left panels) or 4μΙ (right panels) of the indicated protein standards were separated by 10% SDS-PAGE, transferred to nitrocellulose membrane (GE Healthcare) and blocked in 3% Non-fat dry milk (NFDM) in PBS- Tween for 1 h at RT. The membrane was then incubated with anti-Marker, clone 2D2- F11 crude cell culture supernatant at a 1 :100 dilution in 0.5% NFDM in PBS-T o/n at 4°C. After washing 3x 5min in PBS-T, incubation with anti-mouse-HRP coupled secondary antibody for 1 h at RT and 3x1 Omin washes in PBS-T, ECL was performed with GE Healthcare ECL reagents (RPN2 06).

Figure 4: Clone 2D2-F11 does not cross-react with unstained proteins in whole cell lysates of different species. The indicated whole cell lysates were separated by 10% SDS-PAGE, transferred to nitrocellulose membrane (GE Healthcare) and incubated as described in Figure 3 with anti-Marker, clone 2D2-F11 crude cell culture supernatant at a :500 dilution. The left lane was loaded with 4μΙ of marker, the right lane was loaded with 2μΙ. The lower panel shows a "long exposure" of the film and the panel in the middle a correspondingly "short exposure".

Figure 5: Clone 2D2-F11 does not interfere with the detection of mammalian proteins by other monoclonal antibodies. The indicated mammalian cell lysates plus Biorad marker were loaded two times per gel in the same order, separated by 10% SDS-PAGE and transferred to nitrocellulose membrane (GE Healthcare). The membranes were stained with Ponceau S (exemplarily shown for one membrane), cut in two equal pieces and incubated either with one primary antibody (Mouse monoclonal anti-HA tag, anti-myc tag, or anti-PP2A methyl esterase [PME-1] antibodies) or with a mixture of the indicated antibody with clone 2D2-F11 (1 :500) as described above. The upper panel shows a "short exposure" of the film and, as indicated, the panel in the middle a correspondingly "long exposure".

Figure 6: Clone 2D2-F11 does not interfere with the detection of mammalian proteins by polyclonal antibodies raised in various species. The indicated mammalian cell lysates plus Biorad marker were loaded two times per gel in the same order, separated by 10% SDS-PAGE and transferred to nitrocellulose membrane (GE Healthcare). The membranes were stained with Ponceau S (exemplarily shown for one membrane), cut in two equal pieces and incubated either with one primary antibody (goat polyclonal anti-Lamin A/C [N-18, Santa Cruz], rabbit polyclonal anti-protein phosphatase 2A catalytic subunit [PP2A C; raised by Eurogentec for the lab of inventors], rabbit polyclonal anti-PP2A B56 regulatory subunit [raised in the lab of inventors] or with a mixture of the indicated antibody with clone 2D2-F1 (1 :500) as described above. As indicated, the upper panel shows a "short exposure" of the film and the panel in the middle a correspondingly "long exposure".

Figure 7: Clone 2D2-F11 does not interfere with the detection of yeast proteins by other monoclonal antibodies. The indicated yeast cell lysates plus Biorad marker were loaded two times per gel in the same order, separated by 10% SDS- PAGE and transferred to nitrocellulose membrane (GE Healthcare). The membranes were stained with Ponceau S (exemplarily shown for one membrane), cut in two equal pieces and incubated either with one primary antibody (Mouse monoclonal anti-Cdc55, or anti-Netl antibodies, raised in the lab of inventors) or with a mixture of the indicated antibody with clone 2D2-F11 (1 :500) as described above. As indicated, the upper panel shows a "short exposure" of the film and the panel in the middle a correspondingly "long exposure".

Figure 8: Clone 2D2-F11 detects Remazol Brilliant Blue R stained proteins. BSA was prestained with Remazol Brilliant Blue R dye (Sigma) as described (Griffith IP, Anal Biochem 46, 1972). Briefly, 200μΙ of BSA solution (10mg/ml in 0.15M NaCI) was incubated with 50μΙ of Remazol Brilliant Blue R (10mg/ml in 10% SDS) and 50μΙ Na2HP0₄ (1 M, pH 9.6) for 20min at^' 65°C. The indicated amounts of prestained as well as unstained BSA were separated by 10% SDS-PAGE, transferred to nitrocellulose and processed as described above.

Figure 9: Clone 2D2-F11 does not detect "free" Remazol Brilliant Blue R. 0.2μΙ of prestained Biorad protein marker or decreasing amounts of„free" Remazol Brilliant Blue R dye were spotted onto nitrocellulose (GE Healthcare) or PVDF (Amersham) membrane. Membranes were blocked in 3% NFDM in PBS-Tween for 30 min at RT, incubated for 45 min at RT with anti-Marker, clone 2D2-F1 crude cell culture supernatant at 1:500 in 0.5% NFDM in PBS-T, washed 2x 2 min in PBS-T, incubated for 30 min at RT with anti-mouse-HRP coupled secondary antibody and washed again 2x 2 min in PBS-T. ECL was performed with GE Healthcare ECL reagents (RPN2106).

Figure 10: Clone 2D2-F11 does not detect Coomassie Brilliant Blue stained proteins. The indicated amounts of prestained Biorad protein marker (lane 1), purified (by elution from a polyacrylamid gel) recombinant proteins (lanes 2 and 6), U20S whole cell lysate (lane 3), lysozyme (lane 4), bovine serum albumin (BSA; lane 5), and a mixture of gel eluted Biorad Precision Plus marker bands (lane 6) were spotted onto a PVDF membrane (Amersham). The U20S lysate, lysozyme and BSA spots were stained by spotting 1 μΙ of Coomassie Brilliant Blue (0.25g/l in 5% methanol/5% acetic acid) onto the dried protein spots. After drying, the membrane was blocked in 3% NFDM in PBS-Tween for 1 h at RT, incubated with anti-Marker, clone 2D2-F11 crude cell culture supernatant at 1 :500 in 0.5% NFDM in PBS-T o/n at 4°C, and processed for ECL detection as described for Figure 3. In the upper panel, the membrane is shown after spotting and a Western blot with the 2D2-F11 antibody is shown in the lower panel.

Figure 11: Purification of orange marker proteins for immunization of mice. 5μΙ of gel eluted and dialyzed orange prestained marker proteins (lane 1 : 80 kD protein from ColorPlusTM Prestained Protein Marker, Broad Range, New England Biolabs, P7709; lane 2 and 3: 70 kD and 25 kD, respectively, proteins from PageRuler Plus Prestained Protein Ladder, Thermo Scientific, 26619) was separated by 12.5% SDS- PAGE together with 4μΙ of Precision Plus ProteinTM All Blue Standards to estimate the amount of prestained proteins in the dialyzed samples used for immunizations of mice.

Figure 12: Immune sera of mice immunized with orange prestained marker proteins detect Remazol Brilliant Orange 3R (Reactive Orange 16, Sigma 306509) prestained proteins. 33pg of Remazol Brilliant Orange 3R-stained BSA, - ADH and -lysozyme were separated by 10% SDS-PAGE with a preparative comb, transferred to nitrocellulose membrane (GE Healthcare) and blocked in 3% Non-fat dry milk (NFDM) in PBS-Tween for 1 h at RT. The membrane was then incubated in a Miniblotter 28 channel unit (Immunetics) with the indicated mouse sera diluted 1 :500 in 0.5% NFDM in PBS-T o/n at 4°C. After washing 3x 5min in PBS-T, incubation with anti-mouse-HRP coupled secondary antibody for 1 h at RT and 3x10min washes in PBS-T, ECL was performed with GE Healthcare ECL reagents (RPN2106).

Figure 13: Purification of Remazol Brilliant Blue R stained proteins for immunization of mice. 3μΙ of purified Remazol Brilliant Blue R stained BSA (lane 1 ), ADH (lane 2), and lysozyme (lane 3) were separated by 15% SDS-PAGE together with 4μΙ of Precision Plus ProteinTM All Blue Standards to estimate the amount of prestained proteins in the dialyzed samples used for immunizations of mice. Figure 14: Immune sera of mice immunized with Remazol Brilliant Blue R stained proteins detect ColorPlus™ prestained marker proteins. 50 μΙ of

ColorPlus™ Prestained Protein Marker (NEB, P7709) was separated by 10% SDS- PAGE with a preparative comb, transferred to nitrocellulose membrane (GE Healthcare) and blocked in 3% Non-fat dry milk (NFDM) in PBS-Tween for 1 h at RT. The membrane was then incubated in a Miniblotter 28 channel unit (Immunetics) with the indicated mouse sera diluted 1 :500 in 0.5% NFDM in PBS-T o/n at 4°C. After washing 3x 5min in PBS-T, incubation with anti-mouse-HRP coupled secondary antibody for 1 h at RT and 3x10min washes in PBS-T, ECL was performed with GE Healthcare ECL reagents (RPN2106).

Figure 15: Clone 6F4-F6 detects blue prestained protein marker bands similarly to clone 2D2-F11. 1 μΙ, Ο.δμΙ, and 0.25 μΙ of the indicated protein standards were separated by 12.5% SDS-PAGE, transferred to nitrocellulose membrane (GE Healthcare) and blocked in 3% Non-fat dry milk (NFDM) in PBS-Tween for 1 h at RT. The membranes were then incubated with clone 6F4-F6 and clone 2D2-F11 crude cell culture supernatant at a 1 :2000 dilution in 0.5% NFDM in PBS-T together with a mouse-HRP coupled secondary antibody for 1 h at RT. After 3x10min washes in PBS-T, ECL was performed with GE Healthcare ECL reagents (RPN2106).

Figure 16: Dilution series of clone 6F4-F6 and clone 2D2-F11 detecting Precision Plus Protein™ All Blue Standards marker proteins. 20 μΙ of Precision Plus Protein™ All Blue Standards (Biorad, 161-0373) was separated by 15% SDS- PAGE, transferred to nitrocellulose membrane (GE Healthcare) and blocked in 3% Non-fat dry milk (NFDM) in PBS-Tween for 1 h at RT. The membrane was then incubated in a Miniblotter 28 channel unit (Immunetics) with the indicated dilutions of clone 6F4-F6 and clone 2D2-F11 in 0.5% NFDM in PBS-T o/n at 4°C. After washing 3x 5min in PBS-T, incubation with anti-mouse-HRP coupled secondary antibody for 1 h at RT and 3x10min washes in PBS-T, ECL was performed with GE Healthcare ECL reagents (RPN2106). Other aspects and advantages of the invention will be described in the following examples, which are given for purposes of illustration and not by way of limitation.

Examples

Example 1 : Antigen preparation

One 500μΙ aliquot of Precision Plus Protein™ All Blue Standards marker protein mixture (Biorad, 161-0373) was separated by preparative 10% w/v sodium-dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) in 0.025 M Tris/0.2 M Glycine/0.01% w/v SDS pH 8.5 running buffer using a 16.5 cm wide and 22 cm long slab gel unit (C.B.S. Scientific, ASU-250) with a 1.5 mm thick and 13 cm wide preparative Teflon comb (Hoefer Scientific). The prestained bands corresponding to the 75 kD, 50 kD and 25 kD, respectively, marker proteins were excised from the gel with a sterile stainless steel surgical blade (Swann- orton, Size 22, Ref 0308) and cut into small pieces of approximately 1-2 mm³. The polyacrylamide gel pieces containing the respective prestained marker proteins were filled into three separate chambers of an S&S Elutrap electro-separation system (Schleicher&Schull) and the prestained proteins were electro-eluted from the polyacrylamide gel pieces for 6 hours at a constant voltage of 200 V at 22°C in 0.025M Tris/0.2 M Glycine/0.01% w/v SDS pH 8.5 running buffer. Each electro-eluted protein was collected in a volume of 600 μΙ of running buffer between a BT2 Elutrap-Membrane (Whatman, 10404092) and a BT1 Elutrap-Membrane (Whatman, 10404090) as suggested by the manufacturer. Electro-eluted proteins were dialyzed for 14 hours at 4°C against 600 ml of Tris-buffered saline (TBS; 0.137 M sodium chloride [NaCI], 0.0027 M potassium chloride [KCI], 0.025 M Tris, adjusted to pH 7.4 with hydrochloric acid [HCI]) for the 25 kD marker protein or 1000 ml of TBS for the 50 kD and 75 kD marker proteins in Slide-A-Lyzer Dialysis Cassettes (Extra Strength) with a cut-off of 10 kD and a volume capacity of 0.5-3 ml (Thermo Scientific, Product # 66380). The dialyzed proteins were collected from the dialysis cassettes by diluting in approximately 1 ml of 37°C warm TBS and concentrated to 400 μΙ by centrifugation through Amicon Ultra-4 10K centrifugal filters with a nominal molecular weight limit of 10 kD (Millipore, UFC801024). Example 2: Immunization

5 μΙ of dialyzed 75, 50, and 25 kD prestained marker proteins were separated by 12.5% SDS-PAGE in 0.025 M Tris/0.2 M Glycine/0.01 % w/v SDS pH 8.5 running buffer together with 4 pi of Precision Plus Protein™ All Blue Standards marker ^* protein mixture as provided by the manufacturer to estimate the amount of prestained proteins present in the purified samples (Figure 1). 50μΙ of dialyzed 50 kD prestained marker protein was diluted with 50 μΙ phosphate buffered saline (PBS; 0.137 M NaCI, 0.0027 M KCI, 0.0015 M KH₂P0₄, 0.0081 M Na₂HP0₄, pH 7.4) and mixed with 100 μΙ of Freund^'s Complete adjuvant (Sigma, F5881 ). Alternatively, other adjuvants like e.g. Gerbu Adjuvant MM (Gerbu, 3001 ) can be used. The aqueous antigen solution and the adjuvant oil were emulsified by repeated cycles of sucking-up and pushing- out the oil-water mixture through a 23G (0.6mm diameter) needle until a stable emulsion was formed. Blood samples were collected from the tail veins of three female cByJ.RBF-Rb(8.12)5Bnr/J mice at the age of 10 weeks ("preimmune sera"), incubated for 1 h at 37°C and centrifuged for 5 min at 22°C at 14,000 rpm in a Beckman&Coulter Microfuge 18. The cleared blood sera were collected, sodium azide was added to a final concentration of 0.02% w/v, and the sera were stored at 4°C. Immediately after the collection of blood, the mice were immunized with 200 μΙ of antigen-adjuvant emulsion per mouse injected subcutaneously at the abdomen. 14 days after the first immunization, the mice were boosted with 50 μΙ of dialyzed 75 kD prestained marker protein mixed with 50 μΙ PBS and emulsified with 100 μΙ of Freund^'s Incomplete adjuvant (Sigma, F5506) per mouse injected subcutaneously at the abdomen. Alternatively, other adjuvants like e.g. Gerbu Adjuvant MM (Gerbu, 3001 ) can be used. 35 days after the first immunization, the mice were boosted a second time with 50 μΙ of dialyzed 25 kD prestained marker protein mixed with 50 μΙ PBS and emulsified with 100 μΙ of Freund^'s Incomplete adjuvant (or alternatively Gerbu Adjuvant MM 3001 ) per mouse injected subcutaneously at the abdomen. 10 days after the second boost, blood samples of all mice were taken ("immune sera") from the tail veins, incubated for 1 h at 37°C and centrifuged for 5 min at 22°C at 14,000 rpm in a Beckman&Coulter Microfuge 18. Example 3: Testing of immune sera

The cleared blood sera were collected, sodium azide was added to a final concentration of 0.02% w/v, and the sera were tested for the presence of dye-specific IgG antibodies by immunoblotting against 10% SDS-PAGE separated Precision Plus Protein™ All Blue Standards marker proteins (Figure 2). 85 x 73 mm SDS polyacrylamide gels with 1 mm thick preparative combs (Bio-Rad, 165-2928) were casted with Bio-Rad Mini PROTEAN II electrophoresis cell systems. 40 μΙ of Precision Plus Protein™ All Blue Standards marker proteins (BioRad, 61-0373) was separated in 0.025 M Tris/0.2 M glycine/0.01% w/v SDS pH 8.5 running buffer at a constant voltage of 100 V at 22°C. Proteins were transferred to Protran BA 83 nitrocellulose membrane (Whatman, 10401396) in 0.025 M Tris/0.19 M glycine/20% methanol pH 8.5 transfer buffer for 2h at a constant current of 0.5 A at 4°C in Hoefer TE-Series Transphor Electrophoresis Units (Pharmacia Biotech, TE42). Membranes were washed with deionised water and stored dry between two 3MM paper sheets at 22°C. Prior to usage, membranes were rehydrated by incubation for 2 min at 22°C in PBS + 0.1 % Tween-20 (PBS-T). Membranes were blocked by incubation for 1 h at 22°C in PBS-T + 3% w/v skim milk powder (Merck, 1.15363). Blots were incubated with preimmune and immune sera diluted 1 :500 in PBS-T + 0.5% skim milk powder in a Miniblotter system 28 channels dual blot MN28 unit (Immunetics, 168830) over night at 4°C. Membranes were washed 3x 5 min with PBS-T at 22°C. For detection of primary mouse antibodies, membranes were incubated with peroxidase-conjugated AffiniPure goat anti-mouse IgG Fey fragment specific (Jackson ImmunoResearch, 115-035-008) secondary antibody diluted 1 :10,000 in PBS-T + 0.5% skim milk powder for 1 h at 22°C. Membranes were washed three times 10 min with PBS-T at 22°C and bound antibodies were visualized by enhanced chemoluminescence with ECL Western Blotting Detection Reagents (GE Healthcare, RPN2106) and exposure of Fuji Medical X-ray films (FUJIFILM Corporation, Super HR-E, 47410 08471 ).

Example 4: Fusion of splenocytes

Mouse 3 and mouse 1 showed a robust immune response as determined by immunblotting (Figure 2). Mouse 3 received a final boost injected intravenously into the tail vein consisting of a mixture of 5 μΙ of dialyzed 25 kD marker protein from the same batch as used for the 2^nd boost, 5 μΙ of dialyzed 50 kD marker protein from the same batch as used for the first immunization and 5 μΙ of dialyzed 75 kD marker protein from the same batch as used for the 1^st boost diluted in 85 μΙ of PBS. 88 hours post injection the mouse was sacrificed by cervical dislocation and the spleen removed surgically. The spleen was placed in 10 ml of 37°C warm Dulbecco^'s Modified Eagle^'s medium (DMEM; Sigma, D5671 ), cut in small pieces with a sterile pair of scissors and grinded between two sterile frosted microscope slides (Menzel Glaser Superfrost Plus, Thermo Scientific, J1800AMNZ) until no macroscopic pieces of splenic tissue were visible. The cell suspension was filtered through a 100 m nylon cell strainer (BD Falcon, Ref. 352360) and the filter was washed two times with 10 ml of 37°C warm DMEM. Cells were centrifuged for 5 min at 1200 rpm in a Heraeus Megafuge 1.0 at 22°C, resuspended in 3 ml of ice-cold red blood cell lysis buffer (Sigma, R7757) and incubated for 90 sec. The cell suspension was filled up to 30 ml with 37°C warm DMEM and centrifuged for 5 min at 1200 rpm in Heraeus Megafuge 1.0 at 22°C. The splenocytes were counted with a 0.0025 mm² glass counting chamber (0,100 mm depth; Biirker, Labor Optik). X63-Ag8.653 mouse myeloma cells were grown at 37°C in a 5% C0₂ atmosphere on Vents Nunclon TC 140/20 petri dishes (Nunc, 168381 ) for a minimum of 3 passages after thawing in DMEM + 10% fetal bovine serum (Sigma, F7524) + 2 mM Glutamax (Gibco, 35050- 038) + 100 units/ml Penicillin/0.1 mg/ml Streptomycin (Sigma, P4333) + 1 mM sodium pyruvate (Sigma, S8636). X63-Ag8.653 cells were harvested by rinsing off the petri dish, centrifuged for 5 min at 1200 rpm in a Heraeus Megafuge 1.0 at 22°C, resuspended in 30 ml of 37°C warm DMEM, counted with a 0.0025 mm² glass counting chamber (0,100 mm depth; Biirker, Labor Optik) and centrifuged again for 5 min at 1200 rpm in a Heraeus Megafuge 1.0 at 22°C. Splenocytes and myeloma cells were mixed at a ratio of 2.5:1 , centrifuged for 5 min at 1200 rpm and fused by resuspending and incubating for 90 sec at 37°C the cells in 1 ml of polyethylenglycol (PEG) 1450 (50% w/v solution in PBS; Sigma, P7181 ). After 90 sec, the cell suspension was diluted stepwise with 1 ml of 37°C warm DMEM, followed by 5 ml of 37°C warm DMEM and followed again by 10 ml of 37°C warm DMEM and was then incubated at 37°C for 5 min. Cells were centrifuged for 5 min at 1200 rpm in a Heraeus Megafuge 1.0 at 22°C and were resuspended in DMEM +_.10% HyClone Fetal Clone I (Thermo Scientific, SH30080.03) + 2 mM Glutamax + 100 units/ml Penicillin/0.1 mg/ml Streptomycin + 1 mM sodium pyruvate + 5% BM Condimed H1 Hybridoma Cloning Supplement (Roche, 11088947001 ) + 0.1 mM hypoxanthine/0.4 μΜ aminopterin/16 μΜ thymidine (provided as HAT 50x stock; Life Technologies, 21060-017). 10⁵ cells per well were seeded onto 96-well petri-dishes (TC Microwell 96F, Nunc, 167000). Cells were grown for 7 days at 37°C in a 5% CO₂ atmosphere and the supematants were tested for the presence of dye-specific IgG antibodies by immunoblotting.

Example 5: Screening of hybridoma supematants by immunoblotting

For antibody screening of splenic fusion hybridoma supematants, 85 x 73 mm SDS polyacrylamide gels with 1 mm thick preparative combs (Bio-Rad, 165-2928) were casted with Bio-Rad Mini PROTEAN II electrophoresis cell systems. 40 μΙ of Precision Plus Protein™ All Blue Standards marker proteins (BioRad, 161-0373) was separated in 0.025 M Tris/0.2 M glycine/0.01% w/v SDS pH 8.5 running buffer at a constant voltage of 100 V at 22°C. Proteins were transferred to Protran BA 83 nitrocellulose membrane (Whatman, 10401396) in 0.025 M Tris/0.19 M glycine/20% methanol transfer buffer for 2h at a constant current of 0.5 A at 4°C in Hoefer TE- Series Transphor Electrophoresis Units (Pharmacia Biotech, TE42). Membranes were washed with deionised water and stored dry between two 3MM paper sheets at 22°C. Prior to usage, membranes were rehydrated by incubation for 2 min at 22°C in PBS + 0.1 % Tween-20 (PBS-T). Membranes were blocked by incubation for 1 h at 22°C in PBS-T + 3% w/v skim milk powder (Merck, 1.15363). Blots were incubated with undiluted supematants in Miniblotter system 28 channels dual blot MN28 units (Immunetics, 168830) over night at 4°C. Membranes were washed 3x 5 min with PBS-T at 22°C. For detection of primary mouse antibodies, membranes were incubated with peroxidase-conjugated AffiniPure goat anti-mouse IgG Fey fragment specific (Jackson ImmunoResearch, 115-035-008) secondary antibody diluted 1 :10,000 in PBS-T + 0.5% skim milk powder for 1 h at 22°C. Membranes were washed three times 10 min with PBS-T at 22°C and bound antibodies were visualized by enhanced chemoluminescence with ECL Western Blotting Detection Reagents (GE Healthcare, RPN2106) and exposure of Fuji Medical X-ray films (FUJIFILM Corporation, Super HR-E, 47410 08471 ). Example 6: Establishment of hybridoma single clone (2D2-F11)

Cells growing in a tissue culture 96-well containing supernatant that was tested positive for the presence of antibodies specific for Precision Plus Protein™ All Blue Standards marker proteins were resuspended in DMEM + 10% HyClone Fetal Clone I (Thermo Scientific, SH30080.03) + 2 mM Glutamax + 100 units/ml Penicillin/0.1 mg/ml Streptomycin + 1 mM sodium pyruvate + 5% BM Condimed H1 Hybridoma Cloning Supplement (Roche, 11088947001 ) + 0.1 mM hypoxanthine/0.4 μΜ aminopterin/16 μΜ thymidine (referred to as "Hybridoma growth medium") and counted with a 0.0025 mm² glass counting chamber (0,100 mm depth; Biirker, Labor Optik). The appropriate volume of cell suspension was diluted in 30 ml of Hybridoma growth medium to yield a concentration of 1 cell in 200 μΙ of Hybridoma growth medium, and 300 μΙ of cell suspension per well were seeded onto 96-well petri- dishes (TC Microwell 96F, Nunc, 167000). Cells were grown for 7 days at 37°C in a 5% CO₂ atmosphere and the supernatants were tested for the presence of dye- specific IgG antibodies by immunoblotting as described for the screening of the splenic fusion hybridoma supernatants. Wells containing supernatant that was tested positive for the presence of antibodies specific for Precision Plus Protein™ All Blue Standards marker proteins were examined under the microscope for the number of hybridoma clones growing. One well with a single clone growing was selected for expansion and further propagation.

Example 7: Preparation of cell lysates for immunoblot analysis (Figures 4, 5, 6, 7)

1. Preparation of mammalian cell lysates

U2OS human osteosarcoma cells, NIH3T3 mouse fibroblasts, Rati rat fibroblasts, BHK21 Syrian hamster kidney cells, CV1 African green monkey kidney cells and immortalized mouse embryo fibroblasts lacking the protein phosphatase 2A (PP2A) methyl esterase PME1 (MEF PME1^"A) (Ortega-Gutierrez S, Leung D, Ficarro S, Peters EC, Cravatt BF. PLoS One. 2008 Jul 2;3(7):e2486), were grown in DMEM + 10% FCS + 2 mM L-glutamine (Sigma, G2150) + 100 units/ml Penicillin/0.1 mg/ml Streptomycin at 37°C in a 7.5% CO₂ atmosphere. U2OS cells expressing 4x hemagglutinin (HA)-tagged human PPP2R5B (PP2A regulatory subunit Β56β) (Riedel CG, Katis VL, Katou Y, Mori S, Itoh T, Helmhart W, Galova M, Petronczki M, Gregan J, Cetin B, Mudrak I, Ogris E, Mechtler K, Pelletier L, Buchholz F, Shirahige K, Nasmyth K. Nature. 2006 May 4;441(7089):53-61) were grown in DMEM + 10% FCS + 2 mM L-glutamine + 100 units/ml Penicillin/0.1 mg/ml Streptomycin + 200 Mg/ml hygromycin B (PAA, P21-014) at 37°C in a 7.5% CO₂ atmosphere. NIH3T3 cells expressing myc-tagged human PPP2R1A (PP2A regulatory subunit Aa) (unpublished data, Ogris lab) were grown in DMEM + 10% FCS + 2 mM L-glutamine + 100 units/ml Penicillin/0.1 mg/ml Streptomycin + 5 pg/ml puromycin (Sigma, P7255) at 37°C in a 7.5% CO₂ atmosphere. NIH3T3 cells expressing myc-tagged human PPP2CA (PP2A catalytic subunit Ca) (unpublished data, Ogris lab) and HA- tagged rat PPP2R2A (PP2A regulatory subunit B55a) (Yeong FM, Hombauer H, Wendt KS, Hirota T, Mudrak I, Mechtler K, Loregger T, Marchler-Bauer A, Tanaka K, Peters JM, Ogris E. Curr Biol. 2003 Dec 2;13(23):2058-64) were grown in DMEM + 10% FCS + 2 mM L-glutamine + 100 units/ml Penicillin/0.1 mg/ml Streptomycin + 200 pg/ml hygromycin B + 5 pg/rnl puromycin at 37°C in a 7.5% CO₂ atmosphere. For cell lysis, cells were washed once with ice-cold PBS and lysed on the petri dish in 1 ml of 95°C hot protein loading buffer (0.112 M dithiothreitol, 2.22% w/v SDS, 11.1% glycerol, spatula tip of bromophenol blue dye; adjust pH with 1 M Tris pH 6.8 until solution is deep blue). Cell lysates were stored at -20°C.

2. Preparation of yeast cell lysates

Yeast cells were grown at 30°C to exponential growth phase in drop-out complete medium (2.3 g/l Bacto yeast nitrogen base [Difco, 233520], 20 mg/l adenine, 20 mg/l L-arginine, 15 mg/l L-tyrosine, 15 mg/l L-isoleucine, 25 mg/l L-phenylalanine, 50 mg/l L-glutamic acid, 50 mg/l L-aspartic acid, 100 mg/l L-threonine, 200 mg/l L-serine, 75 mg/l L-valine, 75 mg/l L-methionine, 90 mg/l L-lysine, 20 mg/l uracil, 30 mg/l L- histidine, 0.05 M ammoniumsulfate) lacking L-leucine and L-tryptophane and containing 2% w/v glucose. Yeast cells were collected by centrifugation at 3,500 rpm at 4°C in a Beckmann GS-6R centrifuge, washed with ice-cold water and lysed for 10 min on ice in 1.95 M sodium hydroxide/7.5% w/v β-mercaptoethanol. Proteins were precipitated by addition of an equal volume of 50% trichloroacetic acid (TCA) and incubation for 10 min on ice and were collected by centrifugation at 14,000 rpm for 5 min at 4°C in a Beckman&Coulter Microfuge 18. Proteins were dissolved in 90 μΙ protein loading buffer + 15 μΙ unbuffered 1 M Tris, incubated for 5 min at 95°C and stored at -20°C.

3. Preparation of bacterial lysate

Rosetta™(DE3)pLysS (Novagen) E.coli were grown at 37°C to exponential growth phase in lysogeny broth (LB; 1% w/v tryptone [AppliChem, A1553], 0.5% w/v yeast extract [AppliChem, A1552], 0.085 M NaCI), resuspended in 0.025 M Tris pH 8.4 + 0.025-0.05 trypsin inhibitor units Aprotinin [Sigma, A6279] + 1 mM phenylmethylsulfonyl fluoride [PMSF; Roche, #11836145001]) and lysed by sonication on ice for 3x 30 sec with a Bandelin UW70 sonicator and a Sonopius HD70 control unit at a continuous power of MS 72/D (-70% of maximum). Insoluble components were removed by centrifugation at 14,000 rpm for 20 min at 4°C, and the cleared lysate supernatant was stored at -80°C.

4. Preparation of chicken follicle lysate

Chicken follicles were surgically isolated, washed one time in ice cold PBS, resuspended in ice-cold RIPA buffer (1% Nonidet P-40, 0.1% w/v deoxycholic acid, 0.1 % w/v SDS, 0.15 M NaCI, 0.01 M sodium phosphate pH 7.2) + 1 mM sodium orthovanadate + 50 mM sodium fluoride + Complete Protease Inhibitor cocktail (Roche, 11836145001) and homogenized through a 18G needle followed by a 23G needle. The cells were lysed by sonication on ice for 15 sec with 1 sec pulses and 1 sec pauses with a Bandelin UW70 sonicator and a Sonopius HD70 control unit at a power of MS 72/D. Insoluble components were removed by centrifugation at 14,000 rpm for 15 min at 4°C, and the cleared lysate supernatant was stored at -80°C.

Example 8: Immunoblotting

For Figure 3, 1 μΙ (left panels) or 4 μΙ (right panels) of the following marker protein mixtures were loaded onto 85 x 73 mm 10% SDS polyacrylamide gels with 0.75 mm thick 10-slot combs (Bio-Rad, 165-2920), casted with a Bio-Rad Mini PROTEAN II electrophoresis cell system:

PageRuler* Plus Prestained Protein Ladder 10-250kD (Thermo Scientific, 26619), ColorPlus Prestained Protein Ladder 10-230kD (New England Biolabs, P7711 ), PageRuler Unstained Protein Ladder (Fermentas, SM0661 ), Spectra Multicolor High Range Protein Ladder (Fermentas, SM1859), and Precision Plus Protein™ All Blue Standards (Biorad, 161-0373).

For Figure 4, prior to loading on gels, the bacterial lysate and the chicken follicle lysate were mixed with a third of the lysate volume of 3x concentrated protein loading buffer (0.336 M dithiothreitol, 6.66% w/v SDS, 33.3% glycerol, spatula tip of bromophenol blue dye; adjust pH with 1 M Tris pH 6.8 until solution is deep blue) and heated at 95°C for 5 min.

For Figures 4, 5, 6, and 7, the mammalian cell lysates and yeast lysates were heated at 95°C for 5min. Similar amounts of all lysates corresponding to 20-30 pg of whole cell protein were loaded onto 85 x 73 mm 10% SDS polyacrylamide gels with 0.75 mm thick 10-slot combs (Bio-Rad, 165-2920), casted with Bio-Rad Mini PROTEAN II electrophoresis cell systems and proteins were separated in 0.025 M Tris/0.2 M glycine/0.01% w/v SDS pH 8.5 running buffer at a constant voltage of 100 V at 22°C.

For Figures 3, 4, 5, 6, and 7, proteins were transferred to Protran BA 83 nitrocellulose membrane (Whatman, 10401396) in 0.025 M Tris/0.19 M glycine/20% methanol transfer buffer for 2h at a constant current of 0.5 A at 4°C in Hoefer TE- Series Transphor Electrophoresis Units (Pharmacia Biotech, TE42). Membranes were washed with deionised water and transferred proteins were visualized by incubation for 1 min at 22°C with Ponceau S dye solution (2.6 mM Ponceau S, 0.184 M TCA, 0. 37 M sulfosalicylic acid). Membranes were washed 2x with water to remove excess Ponceau S dye and were scanned with a CanoScan 4200F scanner (Canon). Membranes were completely decolorized from the Ponceau S stain by incubation in PBS-T for 10 min at 22°C with gentle rocking. Membranes were blocked for 1 h at 22°C in PBS-T + 3% w/v skim milk powder. Blots were incubated with primary antibodies over night at 4°C.

For Figure 3, the membranes were incubated with clone 2D2-F11 cell culture supernatant diluted 1 :100 in PBS-T + 0.5% w/v skim milk powder over night at 4°C. For Figure 4, the membrane was incubated with clone 2D2-F11 cell culture supernatant diluted 1 :500 in PBS-T + 0.5% w/v skim milk powder over night at 4°C.

For Figure 5, the membranes were incubated over night at 4°C with the indicated antibodies as follows: mouse monoclonal anti-hemagglutinin (HA) tag clone 16B12 (Covance, HA.11 , MMS-101 R) diluted 1 :10,000 in PBS-T + 0.5% skim milk powder; mouse monoclonal anti-myc tag clone 4A6 (generated and produced in the inventors' lab) diluted 1 :2,000 in PBS- T + 0.5% skim milk powder; mouse monoclonal anti- PP2A methyl esterase (PME-1 ) clone 8A6-F3 (generated and produced in the inventors^' lab) diluted 1 :100 in PBS- T + 0.5% skim milk powder.

For Figure 6, the membranes were incubated over night at 4°C with -the indicated antibodies as follows: goat polyclonal anti-Lamin A/C (N-18, Santa Cruz, sc-6215) diluted 1 :1 ,000 in PBS-T + 0.5% skim milk powder; rabbit polyclonal anti-PP2A catalytic subunit (SAT20, generated at Eurogentec for lab of inventors) diluted 1 :5,000 in PBS- T + 0.5% skim milk powder; rabbit polyclonal anti-PP2A B56 subunit (generated in the inventors^' lab) diluted 1 :2,000 in PBS- T + 0.5% skim milk powder.

For Figure 7, the membranes were incubated over night at 4°C with the indicated antibodies as follows: mouse monoclonal anti-Cdc55 clone 9D3-H6 (generated and produced in the inventors^' lab) diluted 1 :300 in PBS-T + 0.5% skim milk powder; mouse monoclonal anti-Netl clone 7H2-C5 (generated and produced in the inventors^' lab) diluted 1 :500 in PBS- T + 0.5% skim milk powder.

In Figures 5, 6, and 7 where indicated, membranes were incubated with a mixture of the indicated primary antibodies and clone 2D2-F11 diluted 1 :1 ,000 in PBS-T + 0.5% skim milk powder over night at 4°C. Membranes were washed 3x 5 min with PBS-T at 22°C.

For detection of primary mouse antibodies, membranes were incubated with peroxidase-conjugated AffiniPure goat anti-mouse IgG Fey fragment specific (Jackson ImmunoResearch, 115-035-008) secondary antibody diluted 1 :10,000 in PBS-T + 0.5% skim milk powder for 1 h at 22°C. For detection of primary rabbit antibodies, membranes were incubated with peroxidase-conjugated AffiniPure goat anti-rabbit IgG Fc fragment specific (Jackson ImmunoResearch, 111-035-008) secondary antibody diluted 1 :10,000 in PBS-T + 0.5% skim milk powder for 1 h at 22°C.

For detection of primary goat antibodies, membranes were incubated with peroxidase-conjugated AffiniPure rabbit anti-goat IgG Fc fragment specific (Jackson ImmunoResearch, 305-035-008) secondary antibody diluted 1 :10.000 in PBS-T + 0.5% skim milk powder for 1 h at 22°C.

For the detection of two primary antibodies from two different species (Figure 6) membranes were incubated with a mixture of either peroxidase-conjugated AffiniPure goat anti-rabbit IgG Fc fragment specific (Jackson ImmunoResearch, 111-035-008) and peroxidase-conjugated AffiniPure goat anti-mouse IgG Fey fragment specific (Jackson ImmunoResearch, 1 5-035-008) secondary antibody both diluted 1:10,000 in PBS-T + 0.5% skim milk powder for 1 h at 22°C, or with a mixture of peroxidase- conjugated AffiniPure rabbit anti-goat IgG Fc fragment specific (Jackson ImmunoResearch, 305-035-008) and peroxidase-conjugated AffiniPure goat anti- mouse IgG Fey fragment specific (Jackson ImmunoResearch, 115-035-008) secondary antibody diluted 1 :10,000 in PBS-T + 0.5% skim milk powder for 1 h at 22°C. Membranes were washed three times 10 min with PBS-T at 22°C and bound antibodies were visualized by enhanced chemoluminescence with ECL Western Blotting Detection Reagents (GE Healthcare, RPN2106) and exposure of Fuji Medical X-ray films (FUJIFILM Corporation, Super HR-E, 47410 08471 ).

Example 9: Dye staining of proteins

Remazol Brilliant Blue R (Sigma, R8001 ) was dissolved at a concentration of 10 mg/ml in 10% w/v SDS. Bovine serum albumin (BSA; Sigma, A9647) was dissolved at a concentration of 10 mg/ml in 0.15 M sodium chloride. 200 pi of BSA solution was mixed with 50 μΙ Remazol Brilliant Blue R solution and 50 μΙ of 1 M disodium hydrogen phosphate (Na₂HP0₄) pH 9.6 solution and incubated for 20 min at 65°C. For Figure 8, 2 μΙ of Precision Plus Protein™ All Blue Standards and 0.66 pg, 1.33 g, and 2 pg of Remazol Brilliant Blue R stained BSA as well as 0.66 pg, 1.33 pg, and 2 pg of unstained BSA were separated by 10% SDS-PAGE on a 85 x 73 mm gel with a 0.75 mm thick 10-slot comb (Bio-Rad, 165-2920), casted with a Bio-Rad Mini PROTEAN II electrophoresis cell system. Proteins were transferred to Protran BA 83 nitrocellulose membrane (Whatman, 10401396) in 0.025 M Tris/0.19 M glycine/20% methanol transfer buffer for 2h at a constant current of 0.5 A at 4°C in a Hoefer TE- Series Transphor Electrophoresis Unit (Pharmacia Biotech, TE42). The membrane was washed with deionised water and scanned with a CanoScan 4200F scanner. Transferred proteins were visualized by incubation of the membrane for 1 min at 22°C with Ponceau S dye solution (2.6 mM Ponceau S, 0.184 M TCA, 0.137 M sulfosalicylic acid). The membrane was washed 2x with water to remove excess Ponceau S dye and was scanned again with a CanoScan 4200F scanner. The membrane was completely decolorized from the Ponceau S stain by incubation in PBS-T for 10 min at 22°C with gentle rocking and was blocked for 1 h at 22°C in PBS-T + 3% w/v skim milk powder. The blot was incubated with clone 2D2-F11 cell culture supernatant diluted 1 :1 ,000 in PBS-T + 0.5% w/v skim milk powder over night at 4°C, washed 3x 5 min with PBS-T and was incubated with peroxidase-conjugated AffiniPure goat anti-mouse IgG Fey fragment specific (Jackson ImmunoResearch, 115-035-008) secondary antibody diluted 1 :10,000 in PBS-T + 0.5% skim milk powder for 1 h at 22°C. The membrane was washed three times 0 min with PBS-T at 22°C and bound antibodies were visualized by enhanced chemoluminescence with ECL Western Blotting Detection Reagents (GE Healthcare, RPN2 06) and exposure of Fuji Medical X-ray films (FUJIFILM Corporation, Super HR-E, 47410 08471 ).

Example 10: Dot blotting (Figures 9 and 10)

Protran BA 83 nitrocellulose membrane was hydrated in PBS for 1 min at 22°C. Hybond™-P Polyvinylidenfluoride (PVDF) membrane (Amersham, RPK5025) was hydrated in methanol for 1 min at 22°C, washed 2x with deionised water and incubated in PBS for 10 min at 22°C. 1 μΙ of Precision Plus Protein™ All Blue Standards marker protein mixture (Biorad, 161-0373) was diluted with 4 μΙ of water and 1 μΙ of the dilution was spotted onto the wet nitrocellulose and PVDF membranes (spot 1 ). 1 μΙ of Remazol Brilliant Blue R solution (10 mg/ml in 10% w/v SDS) was spotted onto each wet membrane (spot 2). 1 μΙ of Remazol Brilliant Blue R solution (10 mg/ml in 10% w/v SDS) was serially diluted with 9 μΙ of water to give 1 :10, 1 : 00, 1 :1 ,000 and 1 :10,000 dilutions with Remazol Brilliant Blue R concentrations of 1 mg/ml, 0.1 mg/ml, 0.01 mg/ml, and 0.001 mg/ml, respectively. 1 μΙ of each diluted Remazol Brilliant Blue R solution was spotted onto each wet membrane (spots 3 - 6). The membranes were air-dried on 3MM paper sheets at 22°C for 2 h. The nitrocellulose membrane was rehydrated in PBS-T for 1 min. The PVDF membrane was rehydrated in methanol for 1 min, washed 2x with deionised water and incubated in PBS-T for 10 min. Membranes were blocked for 1 h at 22°C in PBS-T + 3% w/v skim milk powder and incubated with clone 2D2-F1 cell culture supernatant at a 1 :500 dilution in PBS-T + 0.5% w/v skim milk powder over night at 4°C. Membranes were washed 3x 5 min with PBS-T and were incubated with peroxidase-conjugated AffiniPure goat anti-mouse IgG Fey fragment specific (Jackson ImmunoResearch, 115-035-008) secondary antibody diluted 1 :10,000 in PBS-T + 0.5% skim milk powder for 1 h at 22°C. Membranes were washed three times 10 min with PBS-T at 22°C and bound antibodies were visualized by enhanced chemoluminescence with ECL Western Blotting Detection Reagents (GE Healthcare, RPN2106) and exposure of Fuji Medical X-ray films (FUJIFILM Corporation, Super HR-E, 47410 08471 ).

Hybond™-P Polyvinylidenfluoride (PVDF) membrane (Amersham, RPK5025) was hydrated in methanol for 1 min at 22°C, washed 2x with deionised water and incubated in PBS for 10 min at 22°C. 1 μΙ of Precision Plus Protein™ All Blue Standards marker protein mixture (Biorad, 161-0373) was diluted with 4 μΙ of water and 1 μΙ of the dilution was spotted onto the wet PVDF membrane (spot 1 ). 1 μΙ corresponding to 1 pg of protein of electro-eluted 6x histidine-tagged Net1 (amino acids 1-600) was spotted onto the wet PVDF membrane (spot 2). 1 μΙ corresponding to 1 pg of protein of whole cell lysate of U20S cells expressing 4xHA-tagged human PPP2R5D (PP2A regulatory subunit Β56δ) was spotted onto the wet PVDF membrane (spot 3). 1 μΙ corresponding to 1 pg of protein of lysozyme (Serva, 28262) was spotted onto the wet PVDF membrane (spot 4). 1 μΙ corresponding to 0.2 pg of protein of BSA was spotted onto the wet PVDF membrane (spot 5). 1 μΙ corresponding to 1 pg of protein of electro-eluted 12x HA-tag polypeptide was spotted onto the wet PVDF membrane (spot 6). 1 μΙ of a mixture of 1 μΙ of dialyzed 25 kD marker protein, 1 μΙ of dialyzed 50 kD marker protein and 1 μΙ of dialyzed 75 kD marker protein was spotted onto the wet PVDF membrane (spot 7). After complete disappearance of the droplets, 2 μΙ of 0.3 mM Coomassie Brilliant Blue R- 250 in 5% methanol/5% acetic acid/90% water was spotted onto spots 3, 4, and 5 and the membrane was air-dried at 22°C. The PVDF membrane was rehydrated in methanol for 1 min, washed 2x with deionised water and incubated in PBS-T for 10 min. The membrane was blocked for 1 h at 22°C in PBS-T + 3% w/v skim milk powder and incubated with clone 2D2-F11 cell culture supernatant at a 1 :500 dilution in PBS-T + 0.5% w/v skim milk powder over night at 4°C. The membrane was washed 3x 5 min with PBS-T and incubated with peroxidase-conjugated AffiniPure goat anti-mouse IgG Fey fragment specific (Jackson ImmunoResearch, 115-035-008) secondary antibody diluted 1 :10,000 in PBS-T + 0.5% skim milk powder for 1 h at 22°C. The membrane was washed three times 10 min with PBS-T at 22°C and bound antibodies were visualized by enhanced chemoluminescence with ECL Western Blotting Detection Reagents (GE Healthcare, RPN2106) and exposure of Fuji Medical X-ray films (FUJIFILM Corporation, Super HR-E, 47410 08471).

Example 11: Isotyping of 2D2-F11 antibody

The heavy chain and light chain isotypes of clone 2D2-F1 were determined with the ImmunoPure Monoclonal Antibody Isotyping Kit II (Pierce, 37502) by following the protocol for antigen-independent isotype determination as instructed by the manufacturer^'s manual. Absorption at 405 nm was determined with a Victor³V 1420 Multilabel Counter (Perkin Elmer). The isotype of 2D2-F11 is lgG1 κ.

Example 12: Preparation of orange dye stained marker proteins and immunization

Two 250μΙ aliquots of PageRuler* Plus Prestained Protein Ladder 10-250kD (Thermo Scientific, 26619) and one 1.05 ml aliquot of ColorPlus Prestained Protein Marker (New England Biolabs, P7709S) were separated by preparative 11% SDS-PAGE in 0.025 M Tris/0.2 M Glycine/0.01% w/v SDS pH 8.5 running buffer using 16.5 cm wide and 22 cm long slab gel units (C.B.S. Scientific, ASU-250) with 1.5 mm thick and 13 cm wide preparative Teflon combs (Hoefer Scientific). The orange-dye prestained bands corresponding to the 70 kD and 25 kD marker proteins of the PageRuler* Plus Prestained Protein Ladder and orange-dye prestained band corresponding to the 80 kD marker protein of the ColorPlus Prestained Protein Marker were excised from the gels with sterile stainless steel surgical blades (Swann-Morton, Size 22, Ref 0308) and cut into small pieces of approximately 1-2 mm³. The polyacrylamide gel pieces containing the respective prestained marker proteins were filled into three separate chambers of an S&S Elutrap electro-separation system (Schleicher&Schiill) and the prestained proteins were electro-eluted from the polyacrylamide gel pieces for 6 hours at a constant voltage of 200 V at 22°C in 0.025M Tris/0.2 M Glycine/0.01 % w/v SDS pH 8.5 running buffer. Each electro-eluted protein was collected in a volume of 600 μΙ of running buffer between a BT2 Elutrap-Membrane (Whatman, 10404092) and a BT1 Elutrap-Membrane (Whatman, 10404090) as suggested by the manufacturer. Electro-eluted proteins were dialyzed for 4 hours at 4°C against 900 ml of TBS in Slide-A-Lyzer Dialysis Cassettes (Extra Strength) with a cut-off of 10 kD and a volume capacity of 0.5-3 ml (Thermo Scientific, Product # 66380). The dialyzed proteins were collected from the dialysis cassettes by diluting in approximately 1 ml of 37°C warm TBS and concentrated to 400 μΙ by centrifugation through Amicon Ultra-4 10K centrifugal filters with a nominal molecular weight limit of 10 kD (Millipore, UFC801024).

5 μΙ of dialyzed 80, 70, and 25 kD orange prestained marker proteins were separated by 12.5% SDS-PAGE in 0.025 M Tris/0.2 M Glycine/0.01 % w/v SDS pH 8.5 running buffer together with 4 μΙ of Precision Plus Protein™ All Blue Standards marker protein mixture as provided by the manufacturer to estimate the amount of prestained proteins present in the purified samples (Figure 11). Prior to immunization blood samples were collected from the tail veins of mice at the age of 10 weeks ("preimmune sera"), incubated for 1 h at 37°C and centrifuged for 5 min at 22°C at 14,000 rpm in a Beckman&Coulter Microfuge 18. The cleared blood sera were collected, sodium azide was added to a final concentration of 0.02% w/v, and the sera were stored at 4°C. 50μΙ of dialyzed 70 kD orange prestained marker protein was diluted with 50 μΙ PBS, mixed with 100 μΙ of Freund^'s Complete adjuvant (Sigma, F5881) or alternatively Gerbu Adjuvant MM 3001 and was used for immunizations of mice as described above. For subsequent boosts, 50μΙ of dialyzed 25 kD orange prestained marker protein or 50μΙ of dialyzed 80 kD orange prestained marker protein, respectively, were diluted with 50 μΙ PBS, mixed with 100 pi of Freund^'s Incomplete adjuvant (Sigma, F5881) or alternatively Gerbu Adjuvant MM 3001 and used for immunizations of mice as described above. 10 days after the third immunization (= second boost), blood samples of mice were taken ("immune sera") from the tail veins, incubated for 1 h at 37°C and centrifuged for 5 min at 22°C at 14,000 rpm in a Beckman&Coulter Microfuge 18.

Example 13: Preparation of Remazol Brilliant Orange 3R-stained proteins and testing of immune sera of animals immunized with orange dye stained marker proteins

Remazol Brilliant Orange 3R (Reactive Orange 16, Sigma 306509) was dissolved at a concentration of 10 mg/ml in 10% w/v SDS. Bovine serum albumin (BSA; Sigma, A9647) was dissolved at a concentration of 10 mg/ml in 0.15 M sodium chloride. Alcohol dehydrogenase (ADH; Sigma, A8656) was dissolved at a concentration of 10 mg/ml in 0.15 M sodium chloride. Lysozyme (Serva, 28262) was dissolved at a concentration of 10 mg/ml in 0.15 M sodium chloride. 100 μΙ of each protein solution was mixed with 50 μΙ Remazol Brilliant Orange 3R solution and 30 μΙ of 1 M disodium hydrogen phosphate (Na₂HPO₄) pH 9.6 solution and incubated for 28 min at 65°C.

Immune sera were collected, sodium azide was added to a final concentration of 0.02% w/v, and the sera were tested for the presence of dye-specific IgG antibodies by immunoblotting against 10% SDS-PAGE separated Remazol Brilliant Orange 3R- stained BSA, ADH and lysozyme (Figure 12). 85 x 73 mm SDS polyacrylamide gels with 1 mm thick preparative combs (Bio-Rad, 165-2928) were casted with Bio-Rad Mini PROTEAN II electrophoresis cell systems. 33μg of Remazol Brilliant Orange 3R- stained BSA, -ADH and -lysozyme were separated in 0.025 M Tris/0.2 M glycine/0.01% w/v SDS pH 8.5 running buffer at a constant voltage of 100 V at 22°C. Proteins were transferred to Protran BA 83 nitrocellulose membrane (Whatman, 10401396) in 0.025 M Tris/0.19 M glycine/20% methanol pH 8.5 transfer buffer for 2h at a constant current of 0.5 A at 4°C in Hoefer TE-Series Transphor Electrophoresis Units (Pharmacia Biotech, TE42). Membranes were washed with deionised water and stored dry between two 3MM paper sheets at 22°C. Prior to usage, membranes were rehydrated by incubation for 2 min at 22°C in PBS + 0.1 % Tween-20 (PBS-T). Membranes were blocked by incubation for 1 h at 22°C in PBS-T + 3% w/v skim milk powder (Merck, 1.15363). Blots were incubated with preimmune and immune sera diluted 1 :500 in PBS-T + 0.5% skim milk powder in a Miniblotter system 28 channels dual blot MN28 unit (Immunetics, 168830) over night at 4°C. Membranes were washed 3x 5 min with PBS-T at 22°C. For detection of primary mouse antibodies, membranes were incubated with peroxidase-conjugated AffiniPure goat anti-mouse IgG Fey fragment specific (Jackson ImmunoResearch, 115-035-008) secondary antibody diluted 1 :10,000 in PBS-T + 0.5% skim milk powder for 1 h at 22°C. Membranes were washed three times 10 min with PBS-T at 22°C and bound antibodies were visualized by enhanced chemoluminescence with ECL Western Blotting Detection Reagents (GE Healthcare, RPN2106) and exposure of Fuji Medical X-ray films (FUJIFILM Corporation, Super HR-E, 47410 08471).

Example 14: Preparation of Remazol Brilliant Blue R-stained proteins for immunizing animals and immunization

Bovine serum albumin (BSA; Sigma, A9647) was dissolved at a concentration of 10 mg/ml in 0.15 M sodium chloride. Alcohol dehydrogenase (ADH; Sigma, A8656) was dissolved at a concentration of 10 mg/ml in 0.15 M sodium chloride. Lysozyme (Serva, 28262) was dissolved at a concentration of 10 mg/ml in 0. 5 M sodium chloride. 200 μΙ of each protein solution was mixed with 50 μΙ Remazol Brilliant Blue R solution and 50 μΙ of 1 M disodium hydrogen phosphate (Na₂HP0₄) pH 9.6 solution and incubated for 20 min at 65°C. Remazol Brilliant Blue R stained BSA was diluted with 1 ml of water and purified to a final volume of 400 μΙ by centrifugation through an Amicon Ultra-4 10K centrifugal filter with a nominal molecular weight limit of 10 kD (Millipore, UFC801024). Remazol Brilliant Blue R stained ADH and lysozyme were separated by preparative 15% SDS-PAGE in 0.025 M Tris/0.2 M Glycine/0.01% w/v SDS pH 8.5 running buffer using a 16.5 cm wide and 22 cm long slab gel unit (C.B.S. Scientific, ASU-250) with a 1.5 mm thick preparative Teflon comb with two 6 cm wide slots (CBS Scientific, VGC-1503M). The bands corresponding to ADH or lysozyme, respectively, were excised from the gel with sterile stainless steel surgical blades (Swann-Morton, Size 22, Ref 0308) and cut into small pieces of approximately 1-2 mm³. The polyacrylamide gel pieces containing the respective prestained proteins were filled into two separate chambers of an S&S Elutrap electro-separation system (Schleicher&Schiill) and the proteins were electro-eluted from the polyacrylamide gel pieces for 6 hours at a constant voltage of 200 V at 22°C in 0.025M Tris/0.2 M Glycine/0.01% w/v SDS pH 8.5 running buffer. Each electro-eluted protein was collected in a volume of 600 μΙ of running buffer between a BT2 Elutrap-Membrane (Whatman, 10404092) and a BT1 Elutrap-Membrane (Whatman, 10404090) as suggested by the manufacturer. 3 μΙ of Remazol Brilliant Blue R stained BSA, ADH and lysozyme were separated by 15% SDS-PAGE in 0.025 M Tris/0.2 M Glycine/0.01% w/v SDS pH 8.5 running buffer together with 4 μΙ of Precision Plus Protein™ All Blue Standards marker protein mixture as provided by the manufacturer to estimate the amount of prestained proteins present in the purified samples (Figure 13).

30μΙ of Remazol Brilliant Blue R stained BSA was diluted with with 70 μΙ phosphate buffered saline (PBS; 0.137 M NaCI, 0.0027 M KCI, 0.0015 M KH₂P0₄, 0.0081 M Na₂HP0₄, pH 7.4) and mixed with 100 μΙ of Freund^'s Complete adjuvant (Sigma, F5881 ). Alternatively, other adjuvants like e.g. Gerbu Adjuvant MM (Gerbu, 3001 ) can be used. The aqueous antigen solution and the adjuvant oil were emulsified by repeated cycles of sucking-up and pushing-out the oil-water mixture through a 23G (0.6mm diameter) needle until a stable emulsion was formed. Blood samples were collected from the tail veins of four female mice at the age of 10 weeks ("preimmune sera"), incubated for 1 h at 37°C and centrifuged for 5 min at 22°C at 14,000 rpm in a Beckman&Coulter Microfuge 18. The cleared blood sera were collected, sodium azide was added to a final concentration of 0.02% w/v, and the sera were stored at 4°C. Immediately after the collection of blood, the mice were immunized with 200 μΙ of antigen-adjuvant emulsion per mouse injected subcutaneously at the abdomen. 14 days after the first immunization, the mice were boosted with 15 μΙ of prestained ADH mixed with 85 μΙ PBS and emulsified with 100 μΙ of Freund^'s Incomplete adjuvant (Sigma, F5506) per mouse injected subcutaneously at the abdomen. Alternatively, other adjuvants like e.g. Gerbu Adjuvant MM (Gerbu, 3001 ) can be used. 35 days after the first immunization, the mice were boosted a second time with 15 μΙ of prestained lysozyme mixed with 85 μΙ PBS and emulsified with 100 μΙ of Freund^'s Incomplete adjuvant (or alternatively Gerbu Adjuvant MM 3001) per mouse injected subcutaneously at the abdomen. 10 days after the second boost, blood samples of all mice were taken ("immune sera") from the tail veins, incubated for 1 h at 37°C and centrifuged for 5 min at 22°C at 14,000 rpm in a Beckman&Coulter Microfuge 8.

Example 15: Testing of immune sera of animals immunized with Remazol Brilliant Blue R-stained proteins

The cleared blood sera were collected, sodium azide was added to a final concentration of 0.02% w/v, and the sera were tested for the presence of dye-specific IgG antibodies by immunoblotting against 10% SDS-PAGE separated Precision Plus Protein™ All Blue Standards marker proteins (Figure 14). 85 x 73 mm SDS polyacrylamide gels with 1 mm thick preparative combs (Bio-Rad, 165-2928) were casted with Bio-Rad Mini PROTEAN II electrophoresis cell systems. 50 μΙ of ColorPlus™ Prestained Protein Marker (NEB, P7709) was separated in 0.025 M Tris/0.2 M glycine/0.01% w/v SDS pH 8.5 running buffer at a constant voltage of 100 V at 22°C. Proteins were transferred to Protran BA 83 nitrocellulose membrane (Whatman, 10401396) in 0.025 M Tris/0.19 M glycine/20% methanol pH 8.5 transfer buffer for 2h at a constant current of 0.5 A at 4°C in Hoefer TE-Series Transphor Electrophoresis Units (Pharmacia Biotech, TE42). Membranes were washed with deionised water and stored dry between two 3MM paper sheets at 22°C. Prior to usage, membranes were rehydrated by incubation for 2 min at 22°C in PBS + 0.1% Tween-20 (PBS-T). Membranes were blocked by incubation for 1 h at 22°C in PBS-T + 3% w/v skim milk powder (Merck, 1.15363). Blots were incubated with preimmune and immune sera diluted 1 :500 in PBS-T + 0.5% skim milk powder in a Miniblotter system 28 channels dual blot MN28 unit (Immunetics, 168830) over night at 4°C. Membranes were washed 3x 5 min with PBS-T at 22°C. For detection of primary mouse antibodies, membranes were incubated with peroxidase-conjugated AffiniPure goat anti-mouse IgG Fey fragment specific (Jackson ImmunoResearch, 115-035-008) secondary antibody diluted 1 :10,000 in PBS-T + 0.5% skim milk powder for 1 h at 22°C. Membranes were washed three times 10 min with PBS-T at 22°C and bound antibodies were visualized by enhanced chemoluminescence with ECL Western Blotting Detection Reagents (GE Healthcare, RPN2106) and exposure of Fuji Medical X-ray films (FUJIFILM Corporation, Super HR-E, 47410 08471). Example 16: Fusion of splenocytes of animals immunized with Remazol Brilliant Blue R-stained proteins

Mouse 2 and mouse 1 showed a robust immune response as determined by immunblotting (Figure 14). Mouse 2 received a final boost injected intravenously into the tail vein consisting of a mixture of 1 μΙ of prestained BSA from the same batch as used for the first immunization, 2 μΙ of prestained ADH from the same batch as used for the 1^st boost and 2 μΙ of prestained Lysozyme from the same batch as used for the 2^nd boost diluted in 95 μΙ of PBS. 88 hours post injection the mouse was sacrificed by cervical dislocation and the spleen removed surgically. The spleen was placed in 10 ml of 37°C warm Dulbecco^'s Modified Eagle^'s medium (DMEM; Sigma, D5671 ), cut in small pieces with a sterile pair of scissors and grinded between two sterile frosted microscope slides (Menzel Glaser Superfrost Plus, Thermo Scientific, J1800AMNZ) until no macroscopic pieces of splenic tissue were visible. The cell suspension was filtered through a 100 μιη nylon cell strainer (BD Falcon, Ref. 352360) and the filter was washed two times with 10 ml of 37°C warm DMEM. Cells were centrifuged for 5 min at 1200 rpm in a Heraeus Megafuge 1.0 at 22°C, resuspended in 3 ml of ice-cold red blood cell lysis buffer (Sigma, R7757) and incubated for 90 sec. The cell suspension was filled up to 30 ml with 37°C warm DMEM and centrifuged for 5 min at 1200 rpm in Heraeus Megafuge 1.0 at 22°C. The splenocytes were counted with a 0.0025 mm² glass counting chamber (0,100 mm depth; Burker, Labor Optik). X63-Ag8.653 mouse myeloma cells were grown at 37°C in a 5% C0₂ atmosphere on Vents Nunclon TC 140/20 petri dishes (Nunc, 168381 ) for a minimum of 3 passages after thawing in DMEM + 10% fetal bovine serum (Sigma, F7524) + 2 mM Glutamax (Gibco, 35050-038) + 100 units/ml Penicillin/0.1 mg/ml Streptomycin (Sigma, P4333) + 1 mM sodium pyruvate (Sigma, S8636). X63- Ag8.653 cells were harvested by rinsing off the petri dish, centrifuged for 5 min at 1200 rpm in a Heraeus Megafuge 1.0 at 22°C, resuspended in 30 ml of 37°C warm DMEM, counted with a 0.0025 mm² glass counting chamber (0,100 mm depth; Biirker, Labor Optik) and centrifuged again for 5 min at 1200 rpm in a Heraeus Megafuge .0 at 22°C. Splenocytes and myeloma cells were mixed at a ratio of 2.5:1 , centrifuged for 5 min at 1200 rpm and fused by resuspending and incubating for 90 sec at 37°C the cells in 1 ml of polyethylenglycol (PEG) 1450 (50% w/v solution in PBS; Sigma, P7 81). After 90 sec, the cell suspension was diluted stepwise with 1 ml of 37°C warm DMEM, followed by 5 ml of 37°C warm DMEM and followed again by 10 ml of 37°C warm DMEM and was then incubated at 37°C for 5 min. Cells were centrifuged for 5 min at 1200 rpm in a Heraeus Megafuge 1.0 at 22°C and were resuspended in DMEM + 10% HyClone Fetal Clone I (Thermo Scientific, SH30080.03) + 2 mM Glutamax + 100 units/ml Penicillin/0.1 mg/ml Streptomycin + 1 mM sodium pyruvate + 5% BM Condimed H1 Hybridoma Cloning Supplement (Roche, 11088947001) + 0.1 mM hypoxanthine/0.4 μΜ aminopterin/16 μΜ thymidine (provided as HAT 50x stock; Life Technologies, 21060-017). 10⁵ cells per well were seeded onto 96-well petri-dishes (TC Microwell 96F, Nunc, 167000). Cells were grown for 7 days at 37°C in a 5% CO₂ atmosphere and the supernatants were tested for the presence of dye-specific IgG antibodies by immunoblotting.

Example 17: Immunoblot-screening of supernatants of hybridoma cells generated from animals immunized with Remazol Brilliant Blue R-stained proteins

For antibody screening of splenic fusion hybridoma supernatants, 85 x 73 mm SDS polyacrylamide gels with 1 mm thick preparative combs (Bio-Rad, 165-2928) were casted with Bio-Rad Mini PROTEAN II electrophoresis cell systems. 40 μΙ of Precision Plus Protein™ All Blue Standards marker proteins (BioRad, 161-0373) was separated in 0.025 M Tris/0.2 M glycine/0.01 % w/v SDS pH 8.5 running buffer at a constant voltage of 100 V at 22°C. Proteins were transferred to Protran BA 83 nitrocellulose membrane (Whatman, 10401396) in 0.025 M Tris/0.19 M glycine/20% methanol transfer buffer for 2h at a constant current of 0.5 A at 4°C in Hoefer TE- Series Transphor Electrophoresis Units (Pharmacia Biotech, TE42). Membranes were washed with deionised water and stored dry between two 3MM paper sheets at 22°C. Prior to usage, membranes were rehydrated by incubation for 2 min at 22°C in PBS + 0.1% Tween-20 (PBS-T). Membranes were blocked by incubation for 1 h at 22°C in PBS-T + 3% w/v skim milk powder (Merck, 1.15363). Blots were incubated with undiluted supernatants in Miniblotter system 28 channels dual blot MN28 units (Immunetics, 168830) over night at 4°C. Membranes were washed 3x 5 min with PBS-T at 22°C. For detection of primary mouse antibodies, membranes were incubated with peroxidase-conjugated AffiniPure goat anti-mouse IgG Fey fragment specific (Jackson ImmunoResearch, 115-035-008) secondary antibody diluted 1 :10,000 in PBS-T + 0.5% skim milk powder for 1 h at 22°C. Membranes were washed three times 10 min with PBS-T at 22°C and bound antibodies were visualized by enhanced chemoluminescence with ECL Western Blotting Detection Reagents (GE Healthcare, RPN2106) and exposure of Fuji Medical X-ray films (FUJIFILM Corporation, Super HR-E, 47410 08471).

Example 18: Establishment of a hybridoma single clone (6F4-F6) derived from an animal immunized with Remazol Brilliant Blue R-stained proteins

Cells growing in a tissue culture 96-well containing supernatant that was tested positive for the presence of antibodies specific for Precision Plus Protein™ All Blue Standards marker proteins were resuspended in DMEM + 10% HyClone Fetal Clone I (Thermo Scientific, SH30080.03) + 2 mM Glutamax + 100 units/ml Penicillin/0.1 mg/ml Streptomycin + 1 mM sodium pyruvate + 5% BM Condimed H1 Hybridoma Cloning Supplement (Roche, 11088947001) + 0.1 mM hypoxanthine/0.4 μΜ aminopterin/16 μΜ thymidine (referred to as "Hybridoma growth medium") and counted with a 0.0025 mm² glass counting chamber (0,100 mm depth; Burker, Labor Optik). The appropriate volume of cell suspension was diluted in 30 ml of Hybridoma growth medium to yield a concentration of 1 cell in 200 μΙ of Hybridoma growth medium, and 300 μΙ of cell suspension per well were seeded onto 96-well petri- dishes (TC Microwell 96F, Nunc, 167000). Cells were grown for 7 days at 37°C in a 5% CO₂ atmosphere and the supernatants were tested for the presence of dye- specific IgG antibodies by immunoblotting as described for the screening of the splenic fusion hybridoma supernatants. Wells containing supernatant that was tested positive for the presence of antibodies specific for Precision Plus Protein™ All Blue Standards marker proteins were examined under the microscope for the number of hybridoma clones growing. One well with a single clone growing was selected for expansion and further propagation. Example 19: Immunoblotting properties of hybridoma clones 6F4-F6 and 2D2- F11

For Figure 15, 1 μΙ of Precision Plus Protein™ All Blue Standards (Biorad, 161- 0373), 0.5μΙ of PageRuler* Plus Prestained Protein Ladder 10-250kD (Thermo Scientific, 26619), and 0.25 μΙ of RunBlue™ Dual Color SDS Marker (Expedeon NXA05160) were loaded onto each of two 85 x 73 mm 12.5 % SDS polyacrylamide gels with 0.75 mm thick 10-slot combs (Bio-Rad, 165-2920), casted with a Bio-Rad Mini PROTEAN II electrophoresis cell system. For Figure 16, 20 μΙ of Precision Plus Protein™ All Blue Standards (Biorad, 161-0373) was loaded onto a 85 x 73 mm 15 % SDS polyacrylamide gel with a 1 mm thick preparative comb (Bio-Rad, 165-2928), casted with a Bio-Rad Mini PROTEAN II electrophoresis cell systems. Proteins were separated in 0.025 M Tris/0.2 M glycine/0.01% w/v SDS pH 8.5 running buffer at a constant voltage of 100 V at 22°C and transferred to Protran BA 83 nitrocellulose membrane (Whatman, 10401396) in 0.025 M Tris/0.19 M glycine/20% methanol transfer buffer for 2h at a constant current of 0.5 A at 4°C in Hoefer TE-Series Transphor Electrophoresis Units (Pharmacia Biotech, TE42). Membranes were washed with deionised water and blocked for 1 h at 22°C in PBS-T + 3% w/v skim milk powder.

For Figure 15 one membrane was incubated with clone 6F4-F6 cell culture supernatant and the other membrane with clone 2D2-F11 cell culture supernatant. Both supernatants were diluted 1 :2000 in PBS-T + 0.5% w/v skim milk powder. All blots were incubated with the primary antibodies together with a peroxidase- conjugated AffiniPure goat anti-mouse IgG Fey fragment specific (Jackson ImmunoResearch, 115-035-008) secondary antibody diluted 1 :10,000 in PBS-T + 0.5% skim milk powder for 1 h at 22°C.

For Figure 16 the blot was incubated with different dilutions of hybridoma supernatants of clone 6F4-F6 and clone 2D2-F11 in Miniblotter system 28 channels dual blot MN28 units (Immunetics, 168830) over night at 4°C. Dilutions were done with PBS-T + 0.5% w/v skim milk powder. After incubation with the primary antibody membranes were washed three times 5 min with PBS-T at 22°C and incubated with peroxidase-conjugated AffiniPure goat anti-mouse IgG Fey fragment specific (Jackson ImmunoResearch, 115-035-008) secondary antibody diluted 1 :10,000 in PBS-T + 0.5% skim milk powder for 1 h at 22°C. For Figure 15 and 16 membranes were washed three times 10 min with PBS-T at 22°C and bound antibodies were visualized by enhanced chemoluminescence with ECL Western Blotting Detection Reagents (GE Healthcare, RPN2 06) and exposure of Fuji Medical X-ray films (FUJIFILM Corporation, Super HR-E, 47410 08471 ).

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Aoustin, Isabelle

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Claims

89

1. A method for producing an antibody that specifically recognizes a dye which is a portion of a dye/protein complex by selectively eliciting an immune response against the dye, wherein the method comprises the steps of:

(i) immunizing a non-human animal with a first dye/protein complex,

(v) obtaining/isolating said dye-specific antibody, wherein said dye-specific antibody is obtained/isolated by screening using a dye/protein complex that has not been used in the elicitation of the immune response and wherein the protein portion of the dye/protein complex is similar or different than the protein portion of the dye/protein complex used in step (i) and/or (ii) and/or (iii).

2. The method according to claim 1 , wherein the antibody is a monoclonal antibody which is obtained in step (v) by

(i) fusing a B-cell or a plasma-cell or a progenitor cell thereof, from a non- human animal which has been immunized according to steps (ii) to (iv) as defined in claim 1 with myeloma cells in order to obtain hybridoma cells, wherein the non-human animal which has been immunized is a mouse, rabbit or rat 90

(ii) culturing said hybridoma cells;

(iii) identifying a hybridoma cell that produces a dye-specific antibody; and

3. The method according to claim 1 or 2, wherein the B-cell, plasma-cell or the progenitor cell thereof is a cell derived from spleen, lymph nodes or peyer's patches.

4. The method of any one of claims 1 to 3, wherein a step precedes the immunization wherein the dye is coupled to a protein.

5. The method of any one of claims 1 to 4, wherein the dye is a visually detectable dye.

6. The method of any one of claims 1 to 5, wherein the visually detectable dye is a Remazol dye, a Levafix dye, a Procion dye, Sirius dyes, Coomassie Brilliant Blue, Ponceau red, AuroDye, FerriDye, India ink, Amido black or Kongo red.

7. The method of claim 6, wherein the Remazol dye is selected from the group consisting of Remazol Turquoise, Brilliant Red F3B, Brilliant Orange 3R, Golden Yellow RNL, The following Remazol dyes taken from Nilsson et al; Table 3: Remazol Marine Blue GG, Remazol Brilliant Blue BB, Remazol Black B, Remazol Brilliant Green 6B, Remazol Brilliant Blue R spec, Remazol Gold Yellow RNL, Remazol Brilliant Orange FR, Remazol Brilliant Yellow 4GL, Remazol Black GF; The following Remazol dyes taken from page 6 of WO2006/138366: Remazol Brown GR, Remazol Brilliant Red 5BA, Remazol Brilliant Red BB, Remazol Red F3B, Remazol Red 3B, Remazol Brilliant Orange 3R, Remazol Brilliant Yellow GR, Remazol Yellow FG, Remazol 91

Yellow GR, Remazol Brilliant Green 5GA, Remazol Green B, Remazol Brilliant Blue R, Coomassie Blue R-250, Reactive Blue Remazol Turquoise P, Remazol Brilliant Violet 5R, Remazol Red Violet R, Remazol Yellow RR, Remazol Orange RR, Remazol Red RR, Remazol Blue RR, Remazol Brilliant Yellow RGB, Remazol Golden Yellow RGB, Remazol Orange RGB, Remazol Deep Red RGB, Remazol Red RGB, Remazol Navy RGB), Remazol Luminous Yellow FL, Remazol Brilliant Yellow 4GL, Remazol Brilliant Yellow 3GL, Remazol Brilliant Yellow 3GL, Remazol Yellow GL, Remazol Yellow GR, Remazol Golden Yellow RNL, Remazol Yellow 3RS, Remazol Orange BN, Remazol Brilliant Orange 3R spec, Remazol Brilliant Red BB, Red RB, Remazol Red GWF, Remazol Brilliant Red F3B, Remazol Brilliant Red 3BS, Remazol Red 3B, Remazol Brilliant Violet 5R, Remazol Brilliant Blue R spec, Remazol Brilliant Blue RN, Remazol Brilliant Blue BB, Remazol Turquoise Blue G, Remazol Brilliant Green 6B, Remazol Black RL, Remazol Navy Blue GG, Remazol Black B, Remazol Black A, Remazol Black NF and Remazol Deep Black N 150.

8. The method of any one of claimsl to 4, wherein the dye is covalently linked to the protein of the dye/protein complex.

9. The method of any one of claims 1 to 8, with the proviso that the antibody that specifically recognizes a dye which is a portion of a dye/protein complex is not an antibody generated as part of an allergic reaction against textile dyes in human.

10. The method of any one of claims 1 to 9, wherein the dye/protein complex is a dye/protein marker complex.

11. An antibody produced by the method of any one of claims 1 to 10.

12. The antibody of claim 11 , wherein the antibody specifically recognizes a dye which is a portion of a dye/protein complex, wherein said dye is recognized, perceived and/or detected by the human eye as colour (or as black or as 92 white) upon illumination with visible light which ranges from approximately 380 to approximately 800 nm.

13. An antibody that specifically recognizes a dye which is a portion of a dye/protein complex or an antibody obtainable by the method of any one of claims 1 to 10, with the proviso that the antibody that specifically recognizes a dye which is a portion of a dye/protein complex is not an antibody generated as part of an allergic reaction against textile dyes in human.

14. An antibody which specifically recognizes the dye Remazol Brilliant Blue R.

15. A composition comprising two or more antibodies obtainable by the method of any one of claims 1 to 10, wherein each antibody in said composition recognizes a dye of an alternate dye/protein complex.

16. The composition according to claim 15, wherein one of the antibodies is the antibody of claim 14.

17. The antibody of claim 13 or claim 14, wherein said antibody is produced by the hybridoma 2D2-F11 deposited at the Leibnitz-lnstitut DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen) Braunschweig under No: DSM ACC3212.

18. The antibody of any one of claims 11 to 14 and 17, wherein the dye is covalently linked to the protein of the dye/protein complex. 9. The antibody of any one of claims 11 to 14, 17 and 18, wherein the dye/protein complex is a dye/protein marker complex.

20. Use of

an antibody that specifically recognizes a dye which is a portion of a covalently linked dye/protein marker complex or 93 more than one antibodies that specifically recognize a dye which is a portion of a covalently linked dye/protein marker complex wherein each of said more than one antibodies recognizes a dye of an alternate covalently linked dye/protein marker complex

in the detection of one or more dye-prestained protein molecular weight marker proteins.

Use of

an antibody that specifically recognizes a dye which is a portion of a covalently linked dye/protein marker complex or

more than one antibodies that specifically recognize a dye which is a portion of a covalently linked dye/protein marker complex wherein each of said more than one antibodies recognizes a dye of an alternate covalently linked dye/protein marker complex

in the estimation of the size of a sample protein comprising the steps of:

(ii) transferring the separated proteins on a membrane;

(iv) comparing the size of the detected sample protein with the at least one detected dyes which is a portion of a covalently linked dye/protein marker complex. 94

22. Use according to claim 20 or 21 , wherein the antibody that specifically recognizes a dye which is a portion of a covalently linked dye/protein marker complex is an antibody as defined in any one of claims 11 to 14 and 17 to 19.

23. Kit comprising the antibody of any one of claims 11 to 14 and 17 to 19 or the composition of claim 16 or 17.

24. Use of a kit in the detection of one or more protein molecular weight marker proteins prestained with dyes or in the estimation of the size of a sample protein, said kit comprising the antibody of any one of claims 11 to 14 and 17 to 19 or the composition of claim 15 or 16.