EP1109871A1 - Revetement de surfaces solides au moyen de polyhydroxypolymeres actives - Google Patents

Revetement de surfaces solides au moyen de polyhydroxypolymeres actives

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Publication number
EP1109871A1
EP1109871A1 EP99932688A EP99932688A EP1109871A1 EP 1109871 A1 EP1109871 A1 EP 1109871A1 EP 99932688 A EP99932688 A EP 99932688A EP 99932688 A EP99932688 A EP 99932688A EP 1109871 A1 EP1109871 A1 EP 1109871A1
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EP
European Patent Office
Prior art keywords
polyhydroxypolymer
activated
range
groups
solid surface
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP99932688A
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German (de)
English (en)
Inventor
Klaus Gregorius Nielsen
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Affitech AS
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Affitech AS
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Publication of EP1109871A1 publication Critical patent/EP1109871A1/fr
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
    • C09D105/00—Coating compositions based on polysaccharides or on their derivatives, not provided for in groups C09D101/00 or C09D103/00
    • C09D105/02—Dextran; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
    • C09D129/00—Coating compositions based on homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by an alcohol, ether, aldehydo, ketonic, acetal, or ketal radical; Coating compositions based on hydrolysed polymers of esters of unsaturated alcohols with saturated carboxylic acids; Coating compositions based on derivatives of such polymers
    • C09D129/02—Homopolymers or copolymers of unsaturated alcohols
    • C09D129/04—Polyvinyl alcohol; Partially hydrolysed homopolymers or copolymers of esters of unsaturated alcohols with saturated carboxylic acids

Definitions

  • the present invention relates to a convenient method for coating activated polyhydroxypolymers, e.g. tresyl or maleimido activated dextran, onto solid surfaces.
  • activated polyhydroxypolymers e.g. tresyl or maleimido activated dextran
  • WO 94/03530 describes modification of the hydrophilic properties of solid surfaces by treatment thereof with an activated polysaccharide.
  • modification process it is required that the solid surface is carrying nucleophilic groups, e.g. amino groups or thiol group, in order to facilitate immobilisation of, e.g., periodate oxidised dextran or tresyl activated dextran thereto.
  • EP 59631 5 A2 describes a method for coating solid surfaces with dialdehyde starch comprising a contacting step and a heating step without an intermediate rinsing step. It is stated that the dialdehyde starch is nearly irreversibly attached to some polymers after the moderate heating step (50°C to 100°C). It is however also mentioned that the dialdehyde starch can be rinsed off quite easily if the heating step is omitted.
  • WO 91 /0581 7 and WO 90/06954 describe the immobilisation of polysaccharides to surfaces carrying adsorbed polyamines and WO 91 /09877 describes the immobilisation of, e.g., a periodate oxidised cellulose ester to a surface carrying amino groups.
  • WO 92/07706 describes the immobilisation of conjugates of biopolymers and polyimines to a solid surface carrying anionic groups capable of reacting with the amino groups of the polyimine.
  • WO 92/03732 describes the immobilisation of various water-soluble compounds onto solid surfaces, where the water-soluble compounds carry hydrophobic groups so as to facilitate the adsorption.
  • coating of solid surfaces with polyhydroxypolymers can be accomplished by very simple means, i.e. without a mandatory prior acti- vation of the surfaces to be coated and without inclusion of, e.g., aldehyde groups, amino group or hydrophobic group in the polyhydroxypolymer. It has in particular been found that the coating of microtitre plates (e.g. polystyrene microtitre plates) with activated polyhydroxypolymers (e.g.
  • activated polysaccharides such as tresyl activated dextran (TAD) or maleimido activated dextran (MAD)
  • TAD tresyl activated dextran
  • MAD maleimido activated dextran
  • the present invention provides a method for coating a solid surface with a water- soluble activated polyhydroxypolymer, where the solid surface comprises substantially no amino groups, imino groups or thiol groups, the method comprising the step of:
  • the present invention also relates to post-treatment of the coated surface so as to convert the functional groups to other functional groups. Furthermore, the present invention relates to the solid surfaces obtained, their use in immobilisation of biomolecules, as well as the thus obtained solid surfaces comprising immobilised biomolecules.
  • Binding of a peptide to polystyrene microtitre wells coated with TAD at different pH values Binding of a peptide to polystyrene microtitre wells coated with TAD at different pH values.
  • the wells coated with TAD at acidic pH bind more peptide than wells coated at basic pH. It is not clear if this effect is due to improved adsorption of the TAD at low pH or due to hydrolysis of the tresyl groups at high pH.
  • a B-cell epitope scan using overlapping peptides performed on a TAD coated microtitre plate (A) and a conventional microtitre plate (B, MAXISorp) .
  • TNF ⁇ antiserum was added.
  • Peptides containing B-cell epitopes were then expected to be recognized by the antiserum. It is obvious that more peptides were recognized on the TAD coated surface than on the conventional plate.
  • TAD coated on surfaces other than organic polymers A glass test tube and a nickel spatula were treated with TAD as described in Experimental and tested for ability to bind the peptide biotin-MP9.
  • the controls (- TAD coating) were a glass test tube and a nickel spatula respectively, treated with the coating solvent only. For comparison an OD 4 9o/cm 2 value was calculated. Obviously, only the TAD coated surfaces bind the peptide.
  • Figure 5 The effect of NaCI when coating MAD on a polystyrene microtitre plate (MAXISorp) .
  • the presence of NaCI increased the binding of cysteine by approx. 25%.
  • the presence of cysteine binding was detected by adding biotin-NHS.
  • the signal from the lysine as well as the signal from the control with buffer only was very low. This documents that the binding to the MAD surface is thiol specific.
  • MAXISorp polystyrene microtitre plate
  • FIG. 8 Generation of a surface with functional carboxylic acid groups.
  • a TAD surface prepared as described in Example 1 was reacted with 6-amino hexanoic acid under various conditions.
  • a TAD surface prepared as described in Example 1 was reacted with 2,2'dithio-bis(ethylamine). Subsequent reaction of the TAD surface and the thiol-modified TAD surface with biotin-maleimide and biotin- MP9 showed that the TAD surface was specific for amines and the thiol-modified surface was specific for maleimide without any significant cross-specificity.
  • the present invention relates to a method for coating surfaces with activated polyhydroxypolymers such as activated polysaccharides.
  • Polyhydroxypolymers include naturally occurring polyhydroxy compounds such as polysaccharides and synthetic polyhydroxy compounds such as synthetic organic polymers e.g. polyvinylalcohol and poly(hydroxymethylmethacrylate). An important common feature of such compounds is that they are relatively hydrophilic, which is reflected in a good water solubility.
  • Illustrative examples of naturally occurring polyhydroxy compounds are polysaccharides, gum xanthan, etc.
  • Illustrative examples of synthetic organic polymers are polyvinylalcohol, poly(hydroxymethylmethacrylate), poly(hydroxyethylmethacrylate), poly(hydroxypropylmethacrylate), etc. as well as the corresponding copolymers.
  • polysaccharide is intended to be used with its normal meaning, i.e. "a combination of nine or more monosaccharides, linked together by glycosidic bonds", cf. Hawley's Condensed Chemical Dictionary, 1 1 th ed.. Sax and Lewis, eds., Van Nostrand Reinhold Co., New York, 1 987.
  • Examples of such polysaccharides are dextran (e.g. Dex- tran 40, Dextran 70, Dextran 75), agarose, cellulose and starch.
  • the present invention is considered especially applicable for polysaccharides and polyvinylalcohol, in particular polysaccharides such as dextran.
  • the (weight) average molecular weight of the native polyhydroxypolymer in question is typically at least 1 ,000, such as at least 2,000, preferably in the range of 2,500-2,000,000, more preferably in the range of 3,000-1 ,000,000, in particular in the range of 5,000-500,000. It has been shown in the examples that polyhydroxypolymers having an average molecular weight in the range of 10,000-200,000 are particularly advantageous.
  • the polyhydroxypolymer is preferably wa- ter soluble to an extent of at least 10 mg/ml, preferably at least 25 mg/ml, such as at least 50 mg/ml, in particular at least 100 mg/ml, such as at least 1 50 mg/ml. It is known that dextran, even when activated as described herein, fulfils the requirements with respect to water solubility.
  • the ratio between C (carbon a- toms) and OH groups (hydroxy groups) of the unactivated polyhydroxypolymers is in the range of 1 .3 to 2.5, such as 1 .5- 2.3, preferably 1 .6-2.1 , in particular 1 .85-2.05.
  • a C/OH ratio of the unactivated polyhydroxypolymer represents a highly advantageous level of hydrophilicity.
  • Polyvinylalcohol and polysaccharides are examples of polyhydroxypolymers which fulfil this requirement. It is believed that the above-mentioned ratio should be roughly the same for the activated polyhydroxypolymer as the activation ratio should be rather low.
  • native polyhydroxypolymer and similar terms are intended to mean the polyhydroxypolymer before chemical modification. Thus, in a native polysaccharide substantially all monosaccharide units are intact and recognisable.
  • the polyhydroxypolymers carry functional groups (activation groups), which facilitates the anchoring of secondary molecules (e.g. peptides, proteins, antibodies, antigens, nucleic acids, etc. (see below)) to the solid surface.
  • functional groups e.g. tresyl (trifluoroethylsulphonyl), maleimido, cyanogenbromide, tosyl (p-toluenesulfonyl), triflyl (trifluoromethanesulfonyl), pentafluorobenzenesulfonyl, and vinyl sulphone groups.
  • Preferred examples of functional groups within the present invention are tresyl, maleimido, tosyl, triflyl, pentafluorobenzenesulfonyl, and vinylsulphone groups, among which tresyl, maleimido, and tosyl groups are particularly relevant.
  • the functional groups of the activated polyhydroxypolymers according to the invention are attached to the polyhydroxypolymer via a fraction of the hydroxy groups of the native polyhydroxypolymer.
  • the skeleton of the native polyhydroxypolymer is substantially unaffected by the activation.
  • aldehyde functionalities e.g. arising from periodate oxidation of polysaccharides, may be disadvantageous as functional groups of the activated polyhydroxypolymer as oxidation of a diol to two aldehyde groups markedly reduces the hydrophilicity of the polyhydroxypolymer.
  • substantially no aldehyde groups are included in the polyhydroxypolymer other than any (normally masked) aldehyde functionalities of a native polysaccharide.
  • Functional groups should in particular not be aldehyde groups arising from treatment of a polysaccharide with excessive amounts (i.e. more than 1 mole per mole hydroxy groups in the polysaccharide) of periodate.
  • the functional groups are not polymers in themselves as the method according to the invention is simpler as known methods where e.g. poly-L-lysine and other (poly)amines/(poly)imines are used as "activation groups" for immobilising a polysaccharide to a solid surface. It should in par- ticular be understood that the functional groups are not polyimines such as polyethylene imine or polyamines such as poly-L-lysine.
  • substantially no amino (primary, secondary and tertiary aliphatic and aromatic amines), imino, ammonium (aliphatic and aromatic ammonium groups such as pyridinium groups), and thiol groups should be included in the polyhydroxypolymers when used within the present invention.
  • hydrophobic ligands e.g. phenyl, naphthyl, pyridyl and pyridone groups
  • activation groups substantially no groups of this character should be included in the polyhydroxypolymer.
  • the functional groups are some-how involved in the adsorption of the polyhydroxypolymer to the solid surface. This is i.a. illustrated by the difference in the optimal conditions for coating of dextran carrying different functional groups (tresyl and maleimido).
  • the polyhydroxypolymers are generally prepared by methods known to the person skilled in the art.
  • Tresyl activated polyhydroxypolymers can be prepared using tresyl chloride as described for activation of dextran in Example 1 or as described in Gregorius et al., J. Immunol. Meth. 181 ( 1 995) 65-73.
  • Maleimido activated polyhydroxypolymers can be prepared using p-maleimidophenyl iso- cyanate as described for activation of dextran in Example 3.
  • maleimido groups could be introduced to a polyhydroxypolymer, such as dextran, by derivatisation of a tresyl activated polyhydroxypolymer (such as tresyl activated dextran (TAD)) with a diamine compound (generally H 2 N-CnH2n-NH2, where n is 1 -20, preferably 1 -8), e.g.
  • SMCC succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 -carboxylate
  • maleimide activated polyhydroxypolymers Although the different reagents and routes for activation formally results in slightly different maleimide activated products with respect to the linkage between the maleimide functionality and the remainder of the parent hydroxy group on which activation is performed, all and every are considered as "maleimide activated polyhydroxypolymers" .
  • Tosyl activated polyhydroxypolymers can be prepared using tosyl chloride as described for activation of dextran in Example 2. Triflyl and pentafluorobenzenesulfonyl activated polyhydroxypolymers are prepared as the tosyl or tresyl activated analogues, e.g. by u- sing the corresponding acid chlorides.
  • Cyanogenbromide activated polyhydroxypolymer can be prepared by reacting the polyhy- droxypolymer with cyanogenbromide using conventional methods.
  • the resulting functional groups are normally cyanate esters with two hydroxy groups of the polyhydroxypolymer.
  • the degree of activation can be expressed as the ratio between the free hydroxy groups and the activation groups (i.e. functionalised hydroxy groups) . It is believed that a ratio between the free hydroxy groups of the polyhydroxypolymer and the activation groups should be between 250: 1 and 4: 1 in order to obtain an advantageous balance between the hydrophilicity and the reactivity of the polyhydroxypolymer. Preferably the ratio is between 100: 1 and 6: 1 , more preferably between 60: 1 and 8: 1 , in particular between 40: 1 and 10: 1 .
  • Especially interesting activated polyhydroxypolymers for use in the method according to the invention are tresyl, tosyl and maleimido activated polysaccharides, especially tresyl activated dextran (TAD), tosyl activated dextran (TosAD), and maleimido activated dex- tran (MAD).
  • TAD tresyl activated dextran
  • TosAD tosyl activated dextran
  • MAD maleimido activated dex- tran
  • the solid surface to which the polyhydroxypolymer is attached can be selected from a wide variety of solid surfaces used in the analytical and diagnostic fields, however the solid surfaces are generally characterised in the lack of chemical functionalities (e.g. amines, imines and thiols) which are believed to facilitate the coating of surfaces with activated polyhydroxypolymers in conventional methods.
  • chemical functionalities e.g. amines, imines and thiols
  • the most important types of solid surfaces are those of organic polymers, glasses, ceramics and metals.
  • polystyrene, polycarbonate, polypropylene, polyethylene, polyethyleneglycol terephthalate, polyvinylacetate, polymethylpentene, polyvinylpyrrolidi- none, polyacrylonitrile, polymethylmethacrylate and polyvinylchloride are illustrative examples, where polystyrene and polycarbonate are especially interesting examples.
  • borosilicate glass Pyrex glass
  • soda-lime glass are especially relevant examples, e.g. in the form of specimen tubes, vials, and slides for microscopy.
  • the surface of the glass may be treated with acid prior to coating.
  • nickel, iron, copper gold, silver, aluminium and zinc are the most rele- vant illustrative examples. Such surfaces are normally cleaned before coating in order to remove any metal oxides.
  • the solid surface is the surface of a polystyrene body, a polycarbonate body, a borosilicate glass body, or a soda-lime glass body.
  • the body in itself may have a form or may be designed and shaped for the particular desired use.
  • the body may be in the form of a sheet, a film, a bead, a pellet, a disc, a plate, a ring, a rod, a net, a filter, a tray, a microtitre plate, a stick, or a multi-bladed stick.
  • microtitre plates e.g. polystyrene microtitre plates, sticks and beads.
  • the surface of the body in question is not already chemically modified by coating with a compound before use in the present method.
  • the sur- face of the body is not carrying amino, imino or thiol groups.
  • the surface may however be irradiated so as to modify the chemical and/or physical properties of the surface (typically an oxidative process). It has been shown that irradiation is irrelevant in one case (tresyl activated dextran) and slightly advantageous in another case (maleimido activated dextran).
  • solid surfaces are the surfaces of polystyrene microtitre plates, polystyrene beads, polystyrene sticks, polycarbonate microtitre plates, glass beads, and glass plates.
  • the method according to the present invention includes a number of steps, i.e. the contacting step, the rinse step, and the optional drying step. These steps will be described in detail in the following:
  • the coating solution comprising the activated polyhydroxypolymers is preferably an aqueous solution.
  • an aqueous solution comprises a pH adjusting agent and/or a chaotropic agent, and optional one or more auxiliary components.
  • the solvent i.a. for environmental reasons, economic reasons and because many organic polymer materials, such as polystyrene, are damaged by various organic solvents such as dimethyl formamide and acetone. Furthermore, organic solvents often interfere with physical, non-covalent adsorption to solid phases. It is thus preferred that the solvent comprises less than 5% of organic solvents constituents, more preferably no organic solvent constituents are included.
  • the concentration of activated polyhydroxypolymer in the coating solution is typically in the range of 0.001 mg/ml to 5 mg/ml, typically in the range of 0.01 mg/ml to 1 mg/ml, and preferably in the range of 0.1 mg/ml to 0.5 mg/ml.
  • the coating solution which is contacted with the solid surface has a pH in the range of 1 .5-10 and/or a ion strength in the range of 0.1 to 8.
  • pH adjusting agents may be acetic acid, (e.g. 0.5% acetic acid pH 2.6), a citrate/phosphate buffer (e.g. 0.035 M citrate, 0.075 M phosphate, pH 5.0), phosphate buffered saline (PBS) (e.g. 0.01 M phosphate, 0.1 5 M NaCI, pH 7.2) or a carbonate buffer (e.g. 0.1 M carbonate, pH 9.6).
  • PBS phosphate buffered saline
  • carbonate buffer e.g. 0.1 M carbonate, pH 9.6
  • acetic acid and HCI are very convenient as "left over" will be removed by evaporation be evaporation in the drying process.
  • the pH of the coating solution is typically in the range of 1 .5-1 0, preferably in the range of 2.0-7.5, more preferably in the range of 2.0-5.5. It has been shown for a tresyl activated polyhydroxypolymer that equally advantageous products are obtained within the pH range of 2.0-5.5.
  • the desired ionic strength is obtained by using a chaotropic agent in the solution of the activated polyhydroxypolymer.
  • the chaotropic agent must not contain groups (e.g. thiols or amines) that react with the reactive sites of the activated polyhydroxypolymer or have any adverse effect on adsorption of the activated polyhydroxypolymer to the solid phase (as can e.g. be experienced with some detergents).
  • NaCI from 0.5 M to 4 M in water has been used with satisfactory result.
  • guanidinium chloride, sodium thio- sulphate, and sodium thiocyanate may be used. Lower concentration can be used but there is a tendency to increased variability of the coating with decreasing concentration of the chaotropic agent.
  • the ionic strength of the solution of the polyhydroxypolymer is typically 0.1 -8, preferably 0.5-6, more preferably 0.8-5, in particular 1 .2-4.
  • a pH adjusting agent may be used in conjunction with a chaotropic agent so as to fulfil both the pH and the ionic strength recommendations.
  • Auxiliary agents may also be included in the coating solution, however, preferably no other constituents are included as it is preferred that all ingredients are efficiently removed at least in the drying step.
  • the coating time can be from 1 min to over night, e.g. from 3 hours to over night, but the coating time appears to be uncritical. In large scale production over night coating is often very convenient.
  • the coating can be performed at temperature ranging from 4°C to 56°C with equally good results.
  • room temperature is very convenient in order to reduce temperature caused edge effects in e.g. microtitre plates which otherwise could lead to variability in performance between a well in the centre on the microtitre plate and a well close to the edge of the microtitre plate.
  • evaporation during incubation is less or virtually absent at room temperature compared to elevated temperature.
  • Preferred conditions are coating over night at room temperature. These conditions have proven advantageous for tresyl activated dextran (TAD) as well as for maleimido activated dextran (MAD).
  • the coating solution is removed from the solid surface or the solid surface is removed from the coating solution, whatever is most convenient.
  • the coating solution is normally decanted or pipetted off. It should be understood that the coating solution is normally not allowed to completely evaporate, as the performance of the coating is thereby beyond control in that a fraction of the polyhydroxypolymer will be attached to the solid surface by weak passive adsorption and may not be efficiently rinsed off in the subsequent step.
  • the rinse solution is typically an aqueous solution or simply water.
  • an acidic solution is advantageously used in the rinse step in order to avoid that unspecific binding of remaining polyhydroxypolymer or other components takes place due to a change in pH.
  • the rinse solution should contain no components that could react with the reactive sites of the activated polyhydroxy- polymer (e.g. amino or thiol groups) or interfere with the coating and should be easy to remove in the drying process.
  • the rinse solution contains only little salts or other non-volatile components.
  • Acetic acid e.g. 0.5% in water
  • Acetic acid is very suitable as the rinsing solution in situation where acidic conditions have been used in the contacting step as it has a low pH value (approx. 2.6) and is easily removed by evaporation in the drying process.
  • Water is suitably used where the contacting step has been performed under chaotropic conditions.
  • the surface is dried in order to remove the rinsing solution and other volatile components, e.g. acetic acid. Drying is important especially for storage of the coated surface and can be performed at a temperature in the range 20°C-56°C, preferably in the range of 20°C-45°C, with good results. Drying at around 37°C ensures that a relatively fast evaporation of residual rinsing solution takes place and is often more ad- vantageous for large scale production of e.g. coated microtitre plates than drying at 56°C. The drying time can be further reduced under reduced pressure.
  • the coated surface can be stored for later use, or may be used shortly after.
  • the drying step may even be omitted if the rinse solution is compatible with the solutions used in the desired application.
  • coated surfaces prepared according to the present invention have an excellent storage stability expressed as a shelf-life of more than 2-3 years.
  • the stability of the coated surface is so that the difference in absorbance for the most absorbing amino acid in a test for the amino acid side chain specificity (as described for TAD in example 6) when tested on an uncoated solid surface and on a similar solid surface coated with the activated polyhydroxypolymer in question has decreased with at the most 25%, preferably at the most 1 5%, more preferably a the most 10%, in particular at the most 5%, after storage at 37°C for one year. Storage is effected at ambient conditions with respect to atmospheric pressure and atmospheric composition. It is believed that especially pre- ferred coated surfaces obtained according to the method according to the invention fulfil these requirements even when stored at 50°C for one year under the same conditions.
  • the polyhydroxypolymer is a polysaccharide, in particular dextran.
  • Especially important functional groups in connection with polysaccharides are tresyl, tosyl and maleimido.
  • the method comprises a) contacting a solution of a tresyl activated polysaccharide in an aqueous medium having a pH in the range of 1 .5-7.5 with a polystyrene surface; b) rinsing the polystyrene surface with a rinse solution; and c) drying the polystyrene surface coated with the tresyl activated polysaccharide.
  • the method comprises a) contacting a solution of a maleimido activated polysaccharide in an aqueous medium having a ionic strength in the range of 0.5-6 with a polystyrene surface; b) rinsing the polystyrene surface with a rinse solution; and c) drying the polystyrene surface coated with the maleimido activated polysaccharide.
  • the method comprises a) contacting a solution of a tosyl activated polysaccharide in an aqueous medium having a pH in the range of 1 .5-7.5 with a polystyrene surface; b) rinsing the polystyrene surface with a rinse solution; and c) drying the polystyrene surface coated with the tosyl activated polysaccharide.
  • the coated solid surfaces carrying an activated polyhydroxypolymer may be further functionalised before their final use or some or all of the functional groups of the activated polyhydroxypolymer may be reacted so as to form other functional groups.
  • functional groups with the ability to facilitate the coating of a solid surface with a polyhydroxypolymer may be chosen in the initial process (step a) to c)) and these functional groups may afterwards be converted to other functional groups.
  • the tresyl and tosyl groups are good examples of such functional groups for the initial process.
  • a tresyl activated polyhydroxypolymer may be reacted with an ⁇ -amino carboxylic acid (generally H ⁇ N-CnH ⁇ n-COOH, where n is 1 -20, preferably 1 -8) so as to form an immobilised carboxylic acid functionalised polyhydroxypolymer.
  • an ⁇ -amino carboxylic acid generally H ⁇ N-CnH ⁇ n-COOH, where n is 1 -20, preferably 1 -8
  • a tresyl activated polyhydroxypolymer may be reacted with an ⁇ , ⁇ - diamino-alkane (generally H 2 N-CnH 2 n-NH2, where n is 1 -20, preferably 1 -8) so as to form an immobilised amino functionalised polyhydroxypolymer.
  • an ⁇ , ⁇ - diamino-alkane generally H 2 N-CnH 2 n-NH2, where n is 1 -20, preferably 1 -8
  • a tresyl activated polyhydroxypolymer may be reacted with a cystamine or an analogue (generally H2N-CnH2n-S-S-CnH2n-NH2, where n is 1 -10, prefer- ably 1 -4) and subsequently reduced (e.g. with sodium dithionite) so as to form an immobilised thiol functionalised polyhydroxypolymer.
  • a cystamine or an analogue generally H2N-CnH2n-S-S-CnH2n-NH2, where n is 1 -10, prefer- ably 1 -4
  • the formation of the maleimido functionalised polyhydroxypolymer via the tosyl activated polyhydroxypolymer described above may also be accomplished after coating of the solid surface by reacting an immobilised amino functional polyhydroxypolymer with a maleimide reagent, e.g.
  • a reagent selected from succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 -carboxylate (SMCC), sulfo-succinimidyl 4-(N- maleimidomethyl)cyclohexane-1 -carboxylate (sulfo-SMCC), succinimidyl 4- ⁇ p-maleimido- phenyDbutyrate (SMPB), sulfo-succinimidyl 4-(p-maleimidophenyl)butyrate (sulfo-SMPB), N- ⁇ -maleimidobutyryloxy-succinimide ester (GMBS) and N- ⁇ -maleimidobutyryloxy-sulfo- succinimide ester.
  • SMCC succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 -carboxylate
  • sulfo-SMCC succinimidyl 4- ⁇ p-maleimido-
  • the method further comprises the subsequent step (step d)) of converting an amino reactive functionality of the solid surface coated with the thus activated polyhydroxypolymer to another functionality (e.g. selected from carboxylic acid, amino, thiol, and maleimido) by reacting the amino reactive functionality with a reagent which comprises an amino group, preferably a primary amino group.
  • the amino reactive functionalities may be functionalities selected from tresyl, tosyl, cyanogenbromide, triflyl, pentafluorobenzenesulphonyl and vinyl sul- phone.
  • the reagents for such an additional step are exemplified above and the conditions (e.g. in an aqueous buffered solution) will be known for the person skilled in the art.
  • the present invention also relates to solid surfaces coated with activated polyhydroxypolymers.
  • Such solid surface may advantageously be prepared according to the method of the present invention, but alternative methods may also apply.
  • the solid surfaces prepared according to the invention are particularly useful for immobilising molecules of various origins.
  • a particularly interesting group of molecules is biomolecules such as amino acids, oligo- and polypeptides (a special example is PNA), proteins, immunoglobulins, haptens, enzymes, antibodies (monoclonal and polyclonal), antigenes, polysaccharides, oligo- and polynucleotides (nucleic acids such as RNA and DNA), micro-organisms, procaryotic cells, eucaryotic cells, etc.
  • biomolecules such as amino acids, oligo- and polypeptides (a special example is PNA), proteins, immunoglobulins, haptens, enzymes, antibodies (monoclonal and polyclonal), antigenes, polysaccharides, oligo- and polynucleotides (nucleic acids such as RNA and DNA), micro-organisms, procaryotic cells, eucaryotic cells
  • tresyl activated polyhydroxypolymers are especially suitable for the immobilisation of relatively short peptides and nucleic acids such as peptides consisting of 1 -50, or 1 -30, amino acids and nucleic acids consisting of 1 -30, or 1 -20, nucleotides.
  • the present invention also provides solid surfaces coated with an activated polyhydroxypolymer as described herein, where one or more biomolecules have been immobilised to said polyhydroxypolymer via at least a fraction of the activation groups.
  • the bio- molecules are typically selected from amino acids, oligo- and polypeptides, proteins, immunoglobulins, haptens, enzymes, antibodies, antigenes, polysaccharides, oligo- and polynucleotides, micro-organisms, procaryotic cells, eucaryotic cells.
  • the polyhydroxypolymer is a polysaccharide (in particular dextran) and the biomolecule is selected from peptides (including PNA) consisting of 1 -30 amino acids and nucleic acids consisting of 1 -20 nucleotides.
  • PNA peptides
  • the tresyl group as activation group on the polyhydroxypolymer is especially relevant in these instances.
  • the present invention also relates to the use the solid surfaces obtained or obtain- able according to the method of the present invention for immobilisation of biomolecules.
  • the resulting solid surface carry two types of activated polyhydroxypolymer so as to be able to immobilise a broader range of biomolecules.
  • the polyhydroxy- polymer may carry more than one type of functional groups thereby acting as a di-acti- vated polyhydroxypolymer. This can be accomplished either by using two different activated polyhydroxypolymers in the contacting step or by only partial conversion of the functional groups of the polyhydroxypolymer already coated onto the solid surface.
  • the peptide biotin-MP9 (biotin-FAQKEPAFLKEYHLL) was dissolved (0.01 mg/ml) in 0.1 M carbonate buffer, pH 9.6, and 100 ⁇ l was added to the wells to be tested. After 60 min incubation the wells were washed with washing buffer (PBS including 0.5 M NaCI and 1 % Triton x-100 (Sigma)). Residual binding sites were blocked using carbonate buffer including 1 % bovine serum albumin (BSA, Sigma), 1 5% polyethylene glycol (PEG) 8000 (Sigma), and 10 mM ethanol amine.
  • BSA bovine serum albumin
  • PEG polyethylene glycol
  • the immobilized peptide was detected via the biotin group using a streptavidin-horse radish peroxidase (streptavidin-HRP, Amersham) conjugate in diluting buffer (washing buffer including 1 % BSA) and o-phenyl- enediamine dihydrochloride (OPD, Sigma), 1 mg/ml in substrate buffer (citrate phosphate buffer, pH 5.0) as the chromogenic substrate.
  • streptavidin-HRP streptavidin-horse radish peroxidase
  • OPD o-phenyl- enediamine dihydrochloride
  • Dextran (Sigma, mW 70000, freeze dried from water to remove water bound to the dex- tran), 4.5 g was dissolved in dry N-methyl-pyrrolidinone (NMP, 225 ml) at 90-92°C with magnetic stirring. After cooling to 40°C, 2, 2, 2-trifluoroethanesulfonyl chloride (tresyl chloride), 2764 ⁇ l, was added. After 1 5 min 2020 ⁇ l dry pyridine was added and the heating was removed. After 60 min stirring at room temperature (RT) the TAD was precipitated in 1 200 ml cold ethanol.
  • NMP N-methyl-pyrrolidinone
  • the precipitate was dissolved in 200 ml 0.5% acetic acid and dialyzed against 3 times 5 I 0.5% acetic acid in a dialysis membrane with a molecular cut off on 1 2000-14000 Da. After dialysis the TAD was freeze dried.
  • Dextran (Sigma, mW 70000, freeze dried from water to remove water bound to the dex- tran), 0.8 g was dissolved in dry N-methyl-pyrrolidinone (NMP, 40 ml) at 90-92°C with magnetic stirring. After cooling to 60°C p-toluenesulfonyl (tosyl) chloride, 2.8 g dissolved in dry NMP was added. After 1 min 2 ml dry pyridine was added. After 60 min the precipitate is harvested by decanting the supernatant and precipitate is washed using 10 ml NMP and subsequently using 10 ml ethanol (99.9%).
  • NMP N-methyl-pyrrolidinone
  • the precipitate is dissolved in 5 ml water and precipitated using 30 ml ethanol (99.9%) . Subsequently the precipitate was dissolved in 5 ml water and freeze dried. The introduction of tosyl to the dextran could be detected by UV at 280 nm.
  • Dextran (Sigma, mW 70000, freeze dried from water to remove water bound to the dex- tran) 100 mg, was dissolved in dry 1 -methyl-2-pyrrolidinone (5 ml) at 90-92°C with magnetic stirring. After cooling to room temperature p-maleimidophenyl isocyanate (PMPI, Bioaffinity Systems, Roscoe, II, USA), 50 mg dissolved in 1 ml dry dimethyl sulf- oxide, was added. After over night incubation the product was precipitated with 20 ml ethanol (99.9%), dissolved in 5 ml water and freeze dried.
  • PMPI p-maleimidophenyl isocyanate
  • Example 4 The effect of pH on coating TAD onto a solid phase
  • TAD activated polyhydroxypolymers
  • TAD 0.5 mg/ml in 0.5% acetic acid
  • different materials such as a nickel spatula, a glass test tube, a polycarbonate microtitre plate and a polystyrene microtitre plate and incubated over night at RT.
  • the TAD coated materials were tested as described in "A general method to test the peptide binding capacity".
  • the peptide binding test was performed on TAD coated as well as on not coated samples of each material and the results is shown in Fig 4. It is obvious the TAD coating makes the materials tested in this experiment capable of binding much more peptide than the uncoated materials.
  • Example 6 Coating with maleimido activated dextran (MAD) and identification of the chemical specificity of the MAD coated surface
  • MAD Binding of cysteine to a microtitre plate coated with different amounts of MAD.
  • MAD was added in serial dilutions, starting at 1 mg/ml, to a microtitre plate (MAXISorp) in 4 M NaCI in water and incubated 3 hours at RT. After washing with water the plate was dried over night at 37°C. Cysteine and lysine was added, 0.01 mg/ml in PBS, pH 7.2. After 1 hour incubation the plate was washed and biotin-NHS was added, 0.05 mg/ml in PBS including 0.1 % Tween 20, pH 7.2.
  • the specificity was also examined in a similar experiment using cysteine, lysine, glutamic acid and glycine (Fig 7).
  • the amino acids were added to the MAD coated microtitre plate in PBS, 0.01 mg/ml, pH 7.2, and incubated 1 hour at RT.
  • the detection of immobilized amino acids were the preformed using biotin-NHS as described above.
  • Example 7 The affect of high salt ionic strength when coating MAD on a microtitre plate
  • MAD was diluted from a stock solution (20 mg/ml in water) to 0.5 mg/ml in solutions of NaCI ranging from 0 to 4 M NaCI and added to a microtitre plate (MAXISorp). After 3 hours incubation the plates were washed with water and dried over night at 37°C. Cysteine and lysine, 0.01 mg/ml, (or buffer alone as control) were added dissolved in PBS and incubated 1 hour at RT. After washing, immobilization of cysteine or lysine was detected using biotin-NHS as described in example 6. The results are shown in Fig 5 and indicate that increased ionic strength when coating MAD results in increased binding of a thiol containing molecule, exemplified in this experiment by cysteine.
  • Example 8 Testing the amino acid side chain specificity of a TAD coated surface
  • Example 9 Identification of B-cell epitopes using peptides covalently bound to a TAD coated microtitre plate Peptides covering then entire sequence of murine tumor necrosis factor ⁇ (mTNFa) were synthesised as 1 5-mer peptides with a 5 amino acid overlap. These peptides were immobilised (0.05 mg/ml in carbonate buffer, 2 hours at RT) on a TAD coated microtitre plate and on a conventional microtitre plate of the high binder type (MAXISorp). Subsequently, after washing and blocking (as described in the "A general method to test the peptide binding capacity"), anti-TNF ⁇ anti-serum was added.
  • mTNFa murine tumor necrosis factor ⁇
  • Fig 3 shows the result of this B- cell epitope identification assay. More of the covalently immobilised peptides (A) were recognised than of the non-covalently immobilised peptides (B) .
  • Example 10 Generation of a surface with functional carboxylic groups using a TAD coated surface as platform
  • EDC and NHS were either added together, alone or only water was added and incubated for 30 min at RT. After washing with water the biotinylated peptide (biotin- FAQKEPAFLKEYHLL) was added, 0.01 mg/ml, in PBS including 0.2% Tween 20. The result is seen in Fig 8.
  • EDC alone also generates a reactive ester with a carboxylic group but it is very unstable in water. NHS alone cannot form a reactive pro- duct but if EDC and NHS is present at the same time, the EDC ester reacts rapidly with NHS and forms a stable NHS ester.
  • Example 1 1 Generation of a surface with functional thiol groups using a TAD coated surface as platform
  • a TAD coated microtitre plate 2,2'-dithio-bis(ethylamine) (cystamine, Sigma), 1 mg/ml in carbonate buffer, was added and incubated 2 hours at RT. After washing with water, sodium dithionite (Sigma), 2 mg/ml in water, was added and incubated for 3 hours at RT. The plate was washed with water and the generated thiol groups were detected by adding biotin-maleimide (Sigma, 0.05 mg/ml in PBS) which specifically reacts with thiol groups, and incubate 1 hour at RT.
  • biotin-maleimide Sigma, 0.05 mg/ml in PBS
  • the peptide biotin-MP9 (biotin- FAQKEPAFLKEYHLL) was added, 0.01 mg/ml in carbonate buffer, pH 9.6.
  • the immobilized biotin groups (from either biotin-MP9 or biotin-maleimide) were detected using a streptavidin-horse radish peroxidase (strept- avidin-HRP, Amersham) conjugate in diluting buffer (washing buffer including 1 % BSA) and o-phenylenediamine dihydrochloride (OPD, Sigma), 1 mg/ml in substrate buffer (citrate phosphate buffer, pH 5.0) as the chromogenic substrate.
  • substrate buffer citrate phosphate buffer, pH 5.0
  • Fig 9 shows how the TAD coated surface bound the biotin-maleimide (TAD/biotin-mal) or the peptide biotin-MP9 (TAD/biotin-MP9) in the two first bars.
  • the third bar is binding of the maleimide group to the thiol enriched surface (Thio/biotin-mal) and the last bar is binding of biotin-MP9 (Thio/biotin-MP9) to the thiol enriched surface.
  • the TAD coated surface by treatment with cystamine and sodium dithionite lost its ability to bind the peptide biotin- MP9 but gained the ability to bind a thiol specific reagent as biotin-maleimide.

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  • Organic Chemistry (AREA)
  • Solid-Sorbent Or Filter-Aiding Compositions (AREA)
  • Other Resins Obtained By Reactions Not Involving Carbon-To-Carbon Unsaturated Bonds (AREA)
  • Coating Of Shaped Articles Made Of Macromolecular Substances (AREA)
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  • Medicinal Preparation (AREA)

Abstract

Cette invention a trait à un nouveau procédé, simplifié, de préparation de surfaces solides revêtues de polyhydroxypolymères hydrosolubles activés. Ce procédé consiste à revêtir une surface solide sensiblement exempte de groupes amino, imino ou thiols, d'une solution d'enduisage comprenant un polyhydroxypolymère activé, de manière à provoquer le liaisonnement du polyhydroxypolymère à la surface solide, puis à rincer cette surface et, éventuellement, à la sécher. La réaction de contact doit se faire dans un milieu aqueux d'un pH compris entre 1,5 et 10 et/ou ayant une résistance ionique comprise entre 0,1 et 8. Les polyhydroxypolymères préférés sont des polymères d'origine naturelle, des polymères exempts de groupe aldéhyde tels que les polysaccharides, notamment le dextrane, mais également la cellulose, l'agarose et l'amidon. Des polymères synthétiques, notamment l'alcool polyvinylique, figurent également parmi les polymères préférés. La solubilité dans l'eau du polymère est, de préférence, d'au moins 10 mg/ml et le poids moléculaire est, de préférence, d'au moins 1 000. L'activation du polymère se fait, de préférence, au moyen de groupes fonctionnels choisis dans le groupe constitué par des groupes trésyle, maléimido, bromure de cyanogène, tosyle, triflyle, pentafluorobenzène-sulfonyle et vinyl-sulfone. Idéalement, ces polyhydroxypolymères activés sont des dextranes activés trésylés, tosylés ou maléimido. La surface solide est faite, de préférence, d'un polymère organique (un polystyrène, par exemple) ou est en verre, en céramique ou en métal. Cette invention, qui concerne également les surfaces solides pouvant être obtenues par la mise en oeuvre de ce procédé, porte, en outre, sur l'utilisation qui en est faite aux fins de l'immobilisation de biomolécules.
EP99932688A 1998-07-21 1999-07-16 Revetement de surfaces solides au moyen de polyhydroxypolymeres actives Withdrawn EP1109871A1 (fr)

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DK96398 1998-07-21
DKPA199800963 1998-07-21
US9455898P 1998-07-29 1998-07-29
US94558P 1998-07-29
PCT/DK1999/000407 WO2000005316A1 (fr) 1998-07-21 1999-07-16 Revetement de surfaces solides au moyen de polyhydroxypolymeres actives

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AU783144B2 (en) 2000-02-21 2005-09-29 H. Lundbeck A/S Novel method for down-regulation of amyloid
US7097837B2 (en) 2001-02-19 2006-08-29 Pharmexa A/S Synthetic vaccine agents
AU2002255699A1 (en) * 2001-03-09 2002-09-24 Apollo Biotechnology, Inc. Conjugate probes and optical detection of analytes
US6715316B2 (en) * 2001-05-08 2004-04-06 Corning Incorporated Water-removable coatings for LCD glass
MY144532A (en) 2001-08-20 2011-09-30 Lundbeck & Co As H Novel method for down-regulation of amyloid
WO2004113916A1 (fr) * 2003-06-24 2004-12-29 Ngk Insulators, Ltd. Article pour essai analytique et procede de production correspondant
US7722733B2 (en) 2004-03-29 2010-05-25 Baxter International Inc. Method for sterile connection of tubing
US8617895B2 (en) * 2004-09-23 2013-12-31 Tripath Imaging, Inc. Polycationic quaternary ammonium polymer coatings for immobilizing biological samples
FR2966248B1 (fr) * 2010-10-18 2020-05-01 Centre National De La Recherche Scientifique (Cnrs) Procede de fonctionnalisation de surfaces pour la detection d'analytes
GB201106409D0 (en) * 2011-04-15 2011-06-01 Revolymer Ltd Novel composite
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US5281660A (en) * 1992-11-05 1994-01-25 Corning Incorporated Method of attaching dialdehyde starch to a surface and products produced by that method
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WO2000005316A1 (fr) 2000-02-03
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