WO1995025172A1 - Recombinant antibody fragments which are synthesized and biotinylated in e. coli, their use in immunoassays and immunopurification techniques - Google Patents

Recombinant antibody fragments which are synthesized and biotinylated in e. coli, their use in immunoassays and immunopurification techniques Download PDF

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WO1995025172A1
WO1995025172A1 PCT/FR1995/000293 FR9500293W WO9525172A1 WO 1995025172 A1 WO1995025172 A1 WO 1995025172A1 FR 9500293 W FR9500293 W FR 9500293W WO 9525172 A1 WO9525172 A1 WO 9525172A1
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bccp
antigen
biotin
fab fragments
biotinylated
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PCT/FR1995/000293
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French (fr)
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Etienne Weiss
Jean Chatellier
Georges Orfanoudakis
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Universite Louis Pasteur
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Priority to AU19539/95A priority Critical patent/AU1953995A/en
Publication of WO1995025172A1 publication Critical patent/WO1995025172A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/241—Tumor Necrosis Factors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/90—Fusion polypeptide containing a motif for post-translational modification

Definitions

  • the present invention relates to obtaining fragments of recombinant antibodies, Fab, expressed in E. coli, the Fd part of which is biotinylated in vivo, following the fusion of the coding region of the antibody with that of a receptor for biotin.
  • the invention also relates to the exploitation of the bifunctional nature of said biotinylated antibody fragments, on the one hand for carrying out immunoassays, on the other hand for the development of methods of immunopurification of antigens.
  • biotinylated molecules in immunology is widespread because of their ease of detection which is based on the strong affinity of biotin for avidin and streptavidin.
  • the latter are used in form complexed with an enzyme which gives rise to a colored and quantifiable reaction, in the presence of its substrate.
  • streptavidin coupled with alkaline phosphatase is commonly used.
  • biotinylated antibodies ensures very high sensitivity.
  • biotinylation of the antibodies is carried out chemically and the biotin binds at random, which provides a heterogeneous conjugate.
  • Biotin is a small coenzyme which is present, in vivo, in the form linked to intracellular proteins whose enzymatic activities participate in carboxylation reactions essential for cellular metabolism.
  • E. coli contains only one biotinylated protein which is a subunit of acetyl-CoA-carboxylase, the protein carrying carboxy-biotin, which will be designated hereinafter by BCCP (for " biotin carboxyl carrier protein "- according to Fall and Vagel'os, 1973, J. Biol. Chem. 248, 2078-2088-).
  • BCCP The BCCP gene was cloned by Li and Cronan (J. Biol. Chem. 267, 1992, 855-863) who also demonstrated that the expression in E. coli of a fusion protein comprising the sequence of the BCCP biotin acceptor (ie that is to say the sequence comprising from 84 to 110 amino acids from the C-terminal end) allows an efficient fixation of biotin on said protein. This truncated and sufficient sequence will be designated BCCP *, below.
  • the Applicant has taken advantage of this discovery to design genetic constructs using this DNA sequence and coding sequences of antibody Fab fragments to develop an in vivo biotinylation system for antibodies.
  • the present invention relates to a cloning and expression vector carrying a bi-cistronic expression cassette allowing the synthesis in E. coli of Fab fragments of antibodies of defined specificity and comprising a functional antigen binding site (similar to the model vector described by Orfanoudakis, Karim, Bourel and Weiss, 1993, Mol. Immunol. 30, 1519-1528), into which a DNA sequence coding for the C-terminal end of the subunit has been inserted.
  • BCCP of E. coli in phase, downstream of the coding sequence of the Fd fragment of said antibody.
  • this construction allows the synthesis of an Fd-BCCP * fusion protein and then its binding to the light chain fragment of the same antibody, encoded by the same vector, to form the Fab- BCCP * which comprises said fusion protein.
  • the construction according to the present invention makes it possible to recover the DNA fragment coding for BCCP * with the restriction enzymes which have been used for its insertion and then to transfer it to another vector in which. selected fragments of any antibody have been cloned and which has the same junction zone with the necessary restriction sites.
  • the present invention also relates to Fab fragments of antibodies synthesized and secreted by E. coli bacteria transformed by the vector described above. It is another object of the invention to demonstrate that the Fab fragments of antibodies thus obtained are bifunctional, the N-terminal end constituting the binding site of the antigen and the C-terminal end constituting the site of biotin binding, and that this dual functionality can be exploited to develop new immunoassay tests and new methods of immunopurification.
  • the fusion of the BCCP * sequence at the end of the Fd fragment of the antibody results in the biotinylation of the latter, in vivo, in the bacterium.
  • the rate of biotin uptake can be optimized by the addition of biotin at a concentration of about 0.1 mM in the culture medium of the bacteria.
  • the biotin thus linked to the antibody fragments has retained its affinity for avidin and streptavidin, and this property is the basis for the following applications of the antibody fragments obtained.
  • Biotin is revealed by the addition of avidin or streptavidin, coupled to an enzyme label giving a colored reaction in the presence of its substrate, such as alkaline phosphatase, or to a radioactive isotope.
  • the invention thus makes it possible to carry out all of the conventional immunodetection tests of the ELISA, RIA, etc. type, direct or indirect.
  • the invention can also be applied to the field of medical imaging, the antibodies labeled with a radioactive isotope being able to be detected, in vivo, by immunoscintigraphy. It is also another object of the present invention to use the fragments of biotinylated antibodies in vivo to purify antigens by immunoaffinity.
  • the bifunctionality of the Fab-BCCP * fusion protein is used to fix the antibody by its biotinylated end on an inert support, for example of agarose bead type or of synthetic polymer, coated with avidin or streptavidin.
  • the affinity of the latter for biotin leads to the binding of the fusion protein by its C-terminal end and the presentation, towards the exterior, of the antigen recognition site, located at the N-terminal.
  • This free site allows the retention of the antigen from any crude solution in which it is present (culture medium or biological fluid, etc.). It is thus possible to carry out immunopurification in a batch ("batch") or on a column of immobilized beads, the adsorbed antigen then being eluted under appropriate conditions.
  • FIG. 1 schematically represents the expression vector pHL4-bio which is described in example 1 and which derives from pHL4 by the insertion of the coding sequence BCCP *.
  • the vector pHL4 is described in the publication by Orfanoudakis et al. (cited above) and includes the coding sequences V ⁇ -CH1 (Fd) of the heavy chain and VL-CK of the light chain of anti-TNF monoclonal antibody No. 4. These 2 coding sequences constitute a bicistronic operon placed under the control of the Lac promoter and of the "leader" sequence and of the signal peptide of the bacterial pectate lyase.
  • ori origin of replication of ColEl Lac Z: gene for ⁇ -galactosidase fl IG: intergenic region of phage fl AmpR: gene for resistance to ampicillin.
  • FIG. 2 represents the oligonucleotides designed as primers for recovering the BCCP * coding sequence by PCR.
  • the Spel and EcoRI restriction sites are underlined.
  • Example 1 l.A. construction of the expression vector.
  • the coding sequence for BCCP has been published by Li and Cronan (see above). It was used to design primers (which are represented in FIG. 2) allowing the amplification by PCR (polymerase chain reaction) of the sequence corresponding to the 101 amino acids of the C-terminal end of the BCCP of the strain of. ⁇ . ' coli 71-18 BMH (Yanish-Perron et al., 1985,
  • the primer for the 5 'end contains a Spe I restriction site and the primer for the 3' end, an Eco RI site.
  • E. coli 71-18 BMH bacteria from an overnight culture in M9 medium were placed in the presence of a PCR reaction mixture containing the 2 primers and were subjected to 25 amplification cycles, in an automatic device ( Perkin-Elmer / Cetus®).
  • Amplification products one size about 350 bp, were purified by agarose gel electrophoresis to be inserted into the vector pHL4 digested with Spe I / Eco RI.
  • This vector already described by Orfanoudakis et al. (see above) and represented diagrammatically in FIG. 1, comprises the coding sequences VH ⁇ CH1 (OR Fd) and V -CK of the Fab fragment of a chosen murine monoclonal antibody, raised against TNF. These sequences are aligned in the form of a bi-cistronic operon placed under the control of the Lac promoter. They are separated by 21 junction nucleotides which allow the insertion of a foreign coding sequence, downstream of the Fd sequence.
  • the BCCP * sequence is inserted between the Spe I and Eco RI sites, which places it in phase, downstream of the Fd sequence.
  • the construction is checked by sequencing.
  • the resulting vector was called pHL4-bio (because it codes for the biotin acceptor).
  • Isolated colonies of E. coli 71-18 BMH transformed by pHL4 or by pHL4-bio were cultured in LB medium at 30 ° C. overnight.
  • the proteins secreted into the culture medium after induction of the Lac promoter by 1 mM IPTG (isopropyl- ⁇ -D-thiogalactoside) were recovered after centrifugation of the cell pellet and concentrated by precipitation with ammonium sulphate. They were analyzed by electrophoresis on polyacrylamide-SDS gel and immunoblotting on nitrocellulose filters.
  • biotinylated goat antibodies are used, directed against the kappa chains of mouse antibodies, and streptavidin labeled with alkaline phosphatase.
  • streptavidin labeled with alkaline phosphatase is used.
  • Anti-kappa antibodies recognize a compound approximately 80 kDa and 50 kDa in the proteins secreted by the bacteria transformed respectively by pHL4-bio and by pHL. When aliquots of the same proteins are incubated in the presence of streptavidin labeled with alkaline phosphatase, this binds strongly to the 80 kDa compound identified by the anti-kappa, whereas it does not react with the 50 kDa proteins.
  • Example 2 Demonstration of the binding of the antigen and of the biotin on the fusion protein.
  • the ability to bind the Fab4-BCCP - antigen was evaluated with different amounts of TNF immobilized on nitrocellulose filters.
  • the TNF used is recombinant human TNF0.
  • Fab4-BCCP * protein binds to TNF with the same efficiency as Fab4, when its presence is revealed by the addition of anti-kappa antibodies.
  • the Fab4-BCCP * / TNF interaction was followed in a competition test in ELISA (see example 4) in the presence of an excess of antigen.
  • a defined amount of Fab4 or Fab4-BCCP * and varying amounts of TNF were mixed in wells of ELISA microplates coated with antigen. After washing, the fixed molecules are revealed by the addition of streptavidin labeled with alkaline phosphatase.
  • the concentration of free TNF necessary to cause 50% inhibition of the binding of Fab4-BCCP * is 0.2 ⁇ g / ml which corresponds to a relative binding constant of 1.2 10 ⁇ 8 M, that is ie of the same order as that of 1.5 10 ⁇ 8 M which was found for Fab4 (publication by Orfanoudakis et al.).
  • the percentage of Fab4-BCCP * fusion proteins labeled with biotin was evaluated using streptavidin immobilized on agarose beads. Unsaturated amounts of E. coli culture supernatant were incubated with the beads and then the fixed proteins were analyzed by immunoblotting with anti-kappa. By comparison with the quantity of supernatant incubated, it is measured that 3% of the molecules are biotinylated.
  • the bacterial cultures were carried out in the presence of different d-biotin concentrations (0.01 to 1 mM). This is added when the culture has an OD of 1, at the same time as the IPTG.
  • the Fab4-BCCP * fusion protein concentrate can be used for direct or indirect ELISA testing.
  • Microplates for ELISA assay were incubated overnight at 4 ° C with variable amounts of recombinant TNF ⁇ (from 0.01 to 5 ⁇ g / ml in bicarbonate buffer
  • the graphical representation of the results shows that the Fab4-BCCP * proteins specifically bind TNF, saturable way.
  • the reactivity is strictly dependent on the concentration of Fab4-BCCP * used, which confirms the bifunctionality of the fusion protein since all the molecules which fix streptavidin are capable of reacting with TNF.
  • This property makes it possible to use the fusion protein in any type of assay by im unodetection.
  • Example 5 Use of the fragments of biotinylated antibodies for the purification of antigens by immunoaffinity.
  • the bifunctionality of the biotinylated Fab-BCCP * proteins makes it possible to adsorb them onto beads covered with streptavidin and then to use the latter to purify, by immunoaffinity, the antigen corresponding to the Fab.
  • a 0.25 ml suspension of agarose beads coated with streptavidin was incubated in the presence of concentrated Fab4-BCCP * (4 ml) in 10 ml of 20 mM Tris-HCl buffer, pH8, 50 mM NaCl and bovine serum albumin 1 % (buffer A), for 1 hour at room temperature, with gentle stirring. After washing the beads with a 20 mM Tris-HCl buffer, pH 8, 0.5 M NaCl, 0.1% NP40. then in Tris-HCl buffer, pH 8, 50 mM NaCl, the beads are resuspended in buffer A.

Abstract

Production of recombinant antibody fragments Fab expressed in E. Coli, wherein the Fd portion is biotinylated in vivo following the fusion of the coding region of the antibody with that of a biotine receptor. The invention also concerns the utilisation of the bifunctional character of said biotinylated antibody fragments for the production of immunoassays and for developing antigen immunopurification methods.

Description

Fragments d'anticorps recombinants synthétisés et biotinylés dans E.coli, leur utilisation en immunodosages et en purification par immunoaffinité .Fragments of recombinant antibodies synthesized and biotinylated in E. coli, their use in immunoassays and in purification by immunoaffinity.
La présente invention concerne l'obtention de fragments d'anticorps recombinants, Fab, exprimés dans E. coli, dont la partie Fd est biotinylée in vivo, suite à la fusion de la région codante de l'anticorps à celle d'un récepteur de biotine. L'invention concerne également l'exploitation du caractère bifonctionnel desdits fragments d'anticorps biotinylés, d'une part pour la réalisation de dosages immunologiques, d'autre part pour le développement de méthodes d' immunopurification d'antigènes.The present invention relates to obtaining fragments of recombinant antibodies, Fab, expressed in E. coli, the Fd part of which is biotinylated in vivo, following the fusion of the coding region of the antibody with that of a receptor for biotin. The invention also relates to the exploitation of the bifunctional nature of said biotinylated antibody fragments, on the one hand for carrying out immunoassays, on the other hand for the development of methods of immunopurification of antigens.
L'utilisation de molécules biotinylées en immunologie est largement répandue à cause de leur facilité de détection qui repose sur la forte affinité de la biotine pour l'avidine et la streptavidine . Ces dernières sont utilisées sous forme complexée à une enzyme qui donne lieu à une réaction colorée et quantifiable, en présence de son substrat . Ainsi la streptavidine couplée à la phosphatase alcaline est couramment utilisée.The use of biotinylated molecules in immunology is widespread because of their ease of detection which is based on the strong affinity of biotin for avidin and streptavidin. The latter are used in form complexed with an enzyme which gives rise to a colored and quantifiable reaction, in the presence of its substrate. Thus streptavidin coupled with alkaline phosphatase is commonly used.
Dans les diverses techniques immunologiques de détection, de dosage, de marquage ou de purification, l'utilisation d'anticorps biotinylés assure une très grande sensibilité. D'une manière générale, la biotinylation des anticorps est effectuée chimiquement et la biotine se lie au hasard, ce qui fournit un conjugué hétérogène.In the various immunological detection, assay, labeling or purification techniques, the use of biotinylated antibodies ensures very high sensitivity. Generally speaking, the biotinylation of the antibodies is carried out chemically and the biotin binds at random, which provides a heterogeneous conjugate.
La biotine est un petit coenzyme qui est présent, in vivo, sous forme liée à des protéines intracellulaires dont les activités enzymatiques participent à des réactions de carboxylation essentielles au métabolisme cellulaire.Biotin is a small coenzyme which is present, in vivo, in the form linked to intracellular proteins whose enzymatic activities participate in carboxylation reactions essential for cellular metabolism.
Contrairement aux cellules eucaryotes, E. coli ne contient qu'une seule protéine biotinylée qui est une sous- unité de 1 'acétyl-CoA-carboxylase, la protéine porteuse de carboxy-biotine, qui sera désignée ci-après par BCCP (pour "biotin carboxyl carrier protein" - selon Fall et Vagel'os, 1973, J. Biol. Chem. 248, 2078-2088-) .Unlike eukaryotic cells, E. coli contains only one biotinylated protein which is a subunit of acetyl-CoA-carboxylase, the protein carrying carboxy-biotin, which will be designated hereinafter by BCCP (for " biotin carboxyl carrier protein "- according to Fall and Vagel'os, 1973, J. Biol. Chem. 248, 2078-2088-).
Le gène de la BCCP a été clone par Li et Cronan (J. Biol. Chem. 267, 1992, 855-863) qui ont aussi démontré que l'expression dans E. coli d'une protéine de fusion comprenant la séquence de l'accepteur de biotine de la BCCP (c'est-à-dire la séquence comprenant de 84 à 110 acides aminés de l'extrémité C-terminale) permet une fixation efficace de la biotine sur ladite protéine. Cette séquence tronquée et suffisante sera désignée BCCP*, ci-après.The BCCP gene was cloned by Li and Cronan (J. Biol. Chem. 267, 1992, 855-863) who also demonstrated that the expression in E. coli of a fusion protein comprising the sequence of the BCCP biotin acceptor (ie that is to say the sequence comprising from 84 to 110 amino acids from the C-terminal end) allows an efficient fixation of biotin on said protein. This truncated and sufficient sequence will be designated BCCP *, below.
La Demanderesse a tiré avantage de cette découverte pour concevoir des constructions génétiques utilisant cette séquence d'ADN et des séquences codantes de fragments Fab d'anticorps pour mettre au point un système de biotinylation in vivo des anticorps .The Applicant has taken advantage of this discovery to design genetic constructs using this DNA sequence and coding sequences of antibody Fab fragments to develop an in vivo biotinylation system for antibodies.
Ainsi la présente invention concerne un vecteur de clonage et d'expression portant une cassette d'expression bi-cistronique permettant la synthèse dans E. coli de fragments Fab d'anticorps de spécificité définie et comprenant un site fonctionnel de liaison de l'antigène (semblable au vecteur modèle décrit par Orfanoudakis, Karim, Bourel et Weiss, 1993, Mol. Immunol . 30, 1519-1528), dans lequel on a inséré une séquence d'ADN codant pour l'extrémité C-terminale de la sous-unité BCCP de E. coli, en phase, en aval de la séquence codante du fragment Fd dudit anticorps. Ainsi, dans les conditions appropriées d'induction, cette construction permet la synthèse d'une protéine de fusion Fd-BCCP* et ensuite sa liaison au fragment de chaîne légère du même -anticorps, codée par le même vecteur, pour former le Fab-BCCP* qui comprend ladite protéine de fusion.Thus, the present invention relates to a cloning and expression vector carrying a bi-cistronic expression cassette allowing the synthesis in E. coli of Fab fragments of antibodies of defined specificity and comprising a functional antigen binding site ( similar to the model vector described by Orfanoudakis, Karim, Bourel and Weiss, 1993, Mol. Immunol. 30, 1519-1528), into which a DNA sequence coding for the C-terminal end of the subunit has been inserted. BCCP of E. coli, in phase, downstream of the coding sequence of the Fd fragment of said antibody. Thus, under the appropriate induction conditions, this construction allows the synthesis of an Fd-BCCP * fusion protein and then its binding to the light chain fragment of the same antibody, encoded by the same vector, to form the Fab- BCCP * which comprises said fusion protein.
La construction selon la présente invention permet de récupérer le fragment d'ADN codant pour la BCCP* avec les enzymes de restriction qui ont été utilisés pour son insertion et ensuite de le transférer dans un autre vecteur dans lequel des. fragments choisis d'un anticorps quelconque ont été clones et qui présente une même zone de jonction avec les sites de restriction nécessaires.The construction according to the present invention makes it possible to recover the DNA fragment coding for BCCP * with the restriction enzymes which have been used for its insertion and then to transfer it to another vector in which. selected fragments of any antibody have been cloned and which has the same junction zone with the necessary restriction sites.
La présente invention concerne également les fragments Fab d'anticorps synthétisés et sécrétés par des bactéries E. coli transformées par le vecteur décrit plus haut. C'est un autre objet de l'invention de démontrer que les fragments Fab d'anticorps ainsi obtenus sont bifonctionnels, l'extrémité N-terminale constituant le site de liaison de l'antigène et l'extrémité C-terminale constituant le site de liaison de la biotine, et que cette bifonctionnalité peut être exploitée pour mettre au point de nouveaux tests d' immunodosage et de nouvelles méthodes d' i munopurification. La fusion de la séquence BCCP* à l'extrémité du fragment Fd de l'anticorps entraîne la biotinylation de celui-ci, in vivo, dans la bactérie. Le taux de fixation de la biotine peut être optimisé par l'addition de biotine à une concentration d'environ 0,1 mM dans le milieu de culture des bactéries. La biotine ainsi liée aux fragments d'anticorps a gardé son affinité pour l'avidine et la streptavidine, et cette propriété est la base des applications suivantes des fragments d'anticorps obtenus.The present invention also relates to Fab fragments of antibodies synthesized and secreted by E. coli bacteria transformed by the vector described above. It is another object of the invention to demonstrate that the Fab fragments of antibodies thus obtained are bifunctional, the N-terminal end constituting the binding site of the antigen and the C-terminal end constituting the site of biotin binding, and that this dual functionality can be exploited to develop new immunoassay tests and new methods of immunopurification. The fusion of the BCCP * sequence at the end of the Fd fragment of the antibody results in the biotinylation of the latter, in vivo, in the bacterium. The rate of biotin uptake can be optimized by the addition of biotin at a concentration of about 0.1 mM in the culture medium of the bacteria. The biotin thus linked to the antibody fragments has retained its affinity for avidin and streptavidin, and this property is the basis for the following applications of the antibody fragments obtained.
C'est un autre objet de la présente invention d'utiliser les fragments d'anticorps biotinylés in vivo pour la réalisation de dosages immunologiques dans lesquels la bi-fonctionnalité de la protéine de fusion Fab-BCCP* est exploitée pour fixer l'antigène à l'extrémité N-terminaleIt is another object of the present invention to use the fragments of biotinylated antibodies in vivo for carrying out immunoassays in which the bi-functionality of the fusion protein Fab-BCCP * is exploited to fix the antigen to the N-terminal end
(qui correspond au site naturel de fixation de l'antigène sur l'anticorps choisi) et pour détecter et quantifier la liaison antigène-anticorps par la révélation de la biotine liée à l'extrémité C-terminale de la protéine de fusion. La biotine est révélée par l'addition d'avidine ou de streptavidine, couplée à un marqueur enzymatique donnant une réaction colorée en présence de son substrat, comme la phosphatase alcaline, ou à un isotope radioactif. L'invention permet ainsi de réaliser tous les tests classiques d' immunodétection de type ELISA, RIA, etc..., directs ou indirects. L'invention peut également être appliquée au domaine de l'imagerie médicale, les anticorps marqués par un isotope radioactif pouvant être détectés, in vivo, par immunoscintigraphie . C'est aussi un autre objet de la présente invention d'utiliser les fragments d'anticorps biotinylés in vivo pour purifier des antigènes par immunoaffinité . Dans ce cas, la bifonctionnalité de la protéine de fusion Fab-BCCP* est exploitée pour fixer l'anticorps par son extrémité biotinylée sur un support inerte, par exemple de type bille d'agarose ou de polymère de synthèse, recouvert d'avidine ou de streptavidine. L'affinité de ces dernières pour la biotine entraîne la fixation de la protéine de fusion par son extrémité C-terminale et la présentation, vers l'extérieur, du site de reconnaissance de l'antigène, situé en N-terminal. Ce site, libre, permet la rétention de l'antigène à partir de toute solution brute dans laquelle il est présent (milieu de culture ou fluide biologique...) . On peut ainsi réaliser 1 'immunopurification en lot ("batch") ou sur colonne de billes immobilisées, l'antigène adsorbé étant ensuite élue dans des conditions appropriées .(which corresponds to the natural site for fixing the antigen on the chosen antibody) and for detecting and quantifying the antigen-antibody bond by revealing the biotin linked to the C-terminal end of the fusion protein. Biotin is revealed by the addition of avidin or streptavidin, coupled to an enzyme label giving a colored reaction in the presence of its substrate, such as alkaline phosphatase, or to a radioactive isotope. The invention thus makes it possible to carry out all of the conventional immunodetection tests of the ELISA, RIA, etc. type, direct or indirect. The invention can also be applied to the field of medical imaging, the antibodies labeled with a radioactive isotope being able to be detected, in vivo, by immunoscintigraphy. It is also another object of the present invention to use the fragments of biotinylated antibodies in vivo to purify antigens by immunoaffinity. In this case, the bifunctionality of the Fab-BCCP * fusion protein is used to fix the antibody by its biotinylated end on an inert support, for example of agarose bead type or of synthetic polymer, coated with avidin or streptavidin. The affinity of the latter for biotin leads to the binding of the fusion protein by its C-terminal end and the presentation, towards the exterior, of the antigen recognition site, located at the N-terminal. This free site allows the retention of the antigen from any crude solution in which it is present (culture medium or biological fluid, etc.). It is thus possible to carry out immunopurification in a batch ("batch") or on a column of immobilized beads, the adsorbed antigen then being eluted under appropriate conditions.
Les exemples suivants illustrent la mise en oeuvre de la présente invention dans un modèle choisi, comprenant un anticorps monoclonal choisi, dirigé contre le facteur de nécrose tumorale (TNF) humain, dont le clonage et l'expression d'un fragment Fab fonctionnel ont déjà été décrits. (Orf noudakis, Karim, Bourel et eiss, 1993, Mol. Immunol. 30, 1519-1528) . II est évident que l'invention ne se limite pas à ce modèle et que des constructions semblables peuvent être réalisées avec tout fragment Fab d'un anticorps choisi et clone dans, le même vecteur, par la fusion avec la même séquence codante BCCP* et que les mêmes applications des protéines de fusion peuvent être développées.The following examples illustrate the implementation of the present invention in a chosen model, comprising a chosen monoclonal antibody, directed against human tumor necrosis factor (TNF), the cloning and expression of a functional Fab fragment of which have already been described. (Orf noudakis, Karim, Bourel and eiss, 1993, Mol. Immunol. 30, 1519-1528). It is obvious that the invention is not limited to this model and that similar constructions can be carried out with any Fab fragment of a chosen antibody and cloned in, the same vector, by fusion with the same coding sequence BCCP * and that the same applications of fusion proteins can be developed.
La figure 1 représente schématiquement le vecteur d'expression pHL4-bio qui est décrit dans l'exemple 1 et qui dérive du pHL4 par l'insertion de la séquence codante BCCP*. Le vecteur pHL4 est décrit dans la publication de Orfanoudakis et al. (citée plus haut) et comprend les séquences codantes VΗ-CH1 (Fd) de la chaîne lourde et VL-CK de la chaîne légère de l'anticorps monoclonal n° 4 anti-TNF. Ces 2 séquences codantes constituent un opéron bi- cistronique mis sous le contrôle du promoteur Lac et de la séquence "leader" et du peptide signal de la lyase de pectate bactérienne.FIG. 1 schematically represents the expression vector pHL4-bio which is described in example 1 and which derives from pHL4 by the insertion of the coding sequence BCCP *. The vector pHL4 is described in the publication by Orfanoudakis et al. (cited above) and includes the coding sequences VΗ-CH1 (Fd) of the heavy chain and VL-CK of the light chain of anti-TNF monoclonal antibody No. 4. These 2 coding sequences constitute a bicistronic operon placed under the control of the Lac promoter and of the "leader" sequence and of the signal peptide of the bacterial pectate lyase.
Les sites de restriction utilisés pour la construction sont indiqués .The restriction sites used for construction are indicated.
Les abréviations suivantes sont utilisées : ori : origine de réplication de ColEl Lac Z : gène de la β-galactosidase fl IG : région intergénique du phage fl AmpR : gène de résistance à 1 'ampicilline .The following abbreviations are used: ori: origin of replication of ColEl Lac Z: gene for β-galactosidase fl IG: intergenic region of phage fl AmpR: gene for resistance to ampicillin.
La figure 2 représente les oligonucléotides conçus comme amorces pour récupérer la séquence codante BCCP* par PCR. Les sites de restriction Spel et EcoRI sont soulignés.FIG. 2 represents the oligonucleotides designed as primers for recovering the BCCP * coding sequence by PCR. The Spel and EcoRI restriction sites are underlined.
Exemple 1. l.A. construction du vecteur d'expression.Example 1. l.A. construction of the expression vector.
La séquence codante de la BCCP a été publiée par Li et Cronan (voir plus haut) . Elle a été utilisée pour concevoir des amorces (qui sont représentées sur la figure 2) permettant l'amplification par PCR (réaction de polymérase en chaîne) de la séquence correspondant aux 101 acides aminés de l'extrémité C-terminale de la BCCP de la souche de. Ë.' coli 71-18 BMH (Yanish-Perron et al., 1985,
Figure imgf000007_0001
The coding sequence for BCCP has been published by Li and Cronan (see above). It was used to design primers (which are represented in FIG. 2) allowing the amplification by PCR (polymerase chain reaction) of the sequence corresponding to the 101 amino acids of the C-terminal end of the BCCP of the strain of. Ë. ' coli 71-18 BMH (Yanish-Perron et al., 1985,
Figure imgf000007_0001
L'amorce pour l'extrémité 5' contient un site de restriction Spe I et l'amorce pour l'extrémité 3', un site Eco RI .The primer for the 5 'end contains a Spe I restriction site and the primer for the 3' end, an Eco RI site.
Des bactéries E. coli 71-18 BMH provenant d'une culture de nuit en milieu M9 ont été mises en présence d'un mélange réactionnel pour PCR contenant les 2 amorces et ont été soumises à 25 cycles d'amplification, en appareil automatique (Perkin-Elmer/Cetus®) .E. coli 71-18 BMH bacteria from an overnight culture in M9 medium were placed in the presence of a PCR reaction mixture containing the 2 primers and were subjected to 25 amplification cycles, in an automatic device ( Perkin-Elmer / Cetus®).
Les produits de l'amplification, d'une taille d'environ 350 pb, ont été purifiés par électrophorèse sur gel d'agarose pour être insérés dans le vecteur pHL4 digéré par Spe I/Eco RI. Ce vecteur, déjà décrit par Orfanoudakis et al. (voir plus haut) et représenté schématiquement sur la figure 1, comporte les séquences codantes VH~CH1 (OU Fd) et V -CK du fragment Fab d'un anticorps monoclonal murin choisi, dressé contre le TNF . Ces séquences sont alignées sous forme d'un opéron bi-cistronique placé sous le contrôle du promoteur Lac. Elles sont séparées par 21 nucléotides de jonction qui permettent l'insertion d'une séquence codante étrangère, en aval de la séquence Fd.Amplification products, one size about 350 bp, were purified by agarose gel electrophoresis to be inserted into the vector pHL4 digested with Spe I / Eco RI. This vector, already described by Orfanoudakis et al. (see above) and represented diagrammatically in FIG. 1, comprises the coding sequences VH ~ CH1 (OR Fd) and V -CK of the Fab fragment of a chosen murine monoclonal antibody, raised against TNF. These sequences are aligned in the form of a bi-cistronic operon placed under the control of the Lac promoter. They are separated by 21 junction nucleotides which allow the insertion of a foreign coding sequence, downstream of the Fd sequence.
Dans le présent exemple, la séquence BCCP* est insérée entre les sites Spe I et Eco RI ce qui la place en phase, en aval de la séquence Fd. La construction est vérifiée par séquençage. Le vecteur résultant a été appelé pHL4-bio (parce qu'il code pour l'accepteur de biotine) .In the present example, the BCCP * sequence is inserted between the Spe I and Eco RI sites, which places it in phase, downstream of the Fd sequence. The construction is checked by sequencing. The resulting vector was called pHL4-bio (because it codes for the biotin acceptor).
l.B. Caractérisation des protéines exprimées par les bactéries transformées par pHL4-bio.l.B. Characterization of proteins expressed by bacteria transformed by pHL4-bio.
Des colonies isolées de E. coli 71-18 BMH transformées par pHL4 ou par pHL4-bio ont été mises en culture en milieu LB à 30°C pendant une nuit. Les protéines sécrétées dans le milieu de culture après induction du promoteur Lac par 1 ' IPTG 1 mM (isopropy1-β-D- thiogalactoside) ont été récupérées après centrifugation du culot cellulaire et concentrées par précipitation au sulfate d'ammonium. Elles ont été analysées par électrophorèse sur gel de polyacrylamide-SDS et immunoempreintes sur filtres de nitrocellulose .Isolated colonies of E. coli 71-18 BMH transformed by pHL4 or by pHL4-bio were cultured in LB medium at 30 ° C. overnight. The proteins secreted into the culture medium after induction of the Lac promoter by 1 mM IPTG (isopropyl-β-D-thiogalactoside) were recovered after centrifugation of the cell pellet and concentrated by precipitation with ammonium sulphate. They were analyzed by electrophoresis on polyacrylamide-SDS gel and immunoblotting on nitrocellulose filters.
Pour réaliser les immunoempreintes on utilise des anticorps de chèvre biotinylés, dirigés contre les chaînes kappa d'anticorps de souris, et de la streptavidine marquée à la phosphatase alcaline. Pour détecter les protéines biotinylées on utilise de la streptavidine marquée à la phosphatase alcaline.To carry out the immunoblotting, biotinylated goat antibodies are used, directed against the kappa chains of mouse antibodies, and streptavidin labeled with alkaline phosphatase. To detect biotinylated proteins, streptavidin labeled with alkaline phosphatase is used.
Les anticorps anti-kappa reconnaissent un composé d'environ 80 kDa et de 50 kDa dans les protéines sécrétées par les bactéries transformées respectivement par pHL4-bio et par pHL . Quand des aliquots des mêmes protéines sont incubés en présence de streptavidine marquée à la phosphatase alcaline, celle-ci se lie fortement au composé de 80 kDa identifié par les anti-kappa, alors qu'elle ne réagit pas avec les protéines de 50 kDa.Anti-kappa antibodies recognize a compound approximately 80 kDa and 50 kDa in the proteins secreted by the bacteria transformed respectively by pHL4-bio and by pHL. When aliquots of the same proteins are incubated in the presence of streptavidin labeled with alkaline phosphatase, this binds strongly to the 80 kDa compound identified by the anti-kappa, whereas it does not react with the 50 kDa proteins.
(On détecte aussi une faible bande à 22 kDa qui correspond à la BCCP produite naturellement par la bactérie- hôte) .(We also detect a weak band at 22 kDa which corresponds to the BCCP produced naturally by the host bacteria).
La même analyse a été réalisée en présence de β- mercaptoéthanol. Dans ces conditions, avec la streptavidine marquée, on ne détecte aucune protéine dans le surnageant de pHL4 et une protéine de 40 kDa dans le surnageant de pHL4- bio (40 kDa correspondant à la mεεse approximative calculée pour la protéine de fusion Fd-BCCP*) .The same analysis was carried out in the presence of β-mercaptoethanol. Under these conditions, with the streptavidin labeled, no protein is detected in the supernatant of pHL4 and a protein of 40 kDa in the supernatant of pHL4- bio (40 kDa corresponding to the approximate mass calculated for the fusion protein Fd-BCCP * ).
Des quantités dentiques de fragment:s de chaînes L sont détectées par les ε - - i-kappa dans les 2 surnageants.Dental quantities of fragment: s of L chains are detected by the ε - - i-kappa in the 2 supernatants.
L'ensemble de ces résultats indique que les bactéries transformées par pHL4-bio produisent une protéine complexe comprenant le fragment Fd-BCCP* lié par un pont disulfure à la chaîne légère (VL) et que cette protéine a été efficacement biotinylée in vivo. Cette protéine sera appelée Fab4-BCCP*.All of these results indicate that the bacteria transformed by pHL4-bio produce a complex protein comprising the Fd-BCCP * fragment linked by a light chain disulfide bridge (VL) and that this protein has been effectively biotinylated in vivo. This protein will be called Fab4-BCCP *.
Exemple 2. Mise en évidence de la liaison de l'antigène et de la biotine sur la protéine de fusion.Example 2. Demonstration of the binding of the antigen and of the biotin on the fusion protein.
T.a capacité de fixer l'antigène de la protéine Fab4-BCCP - a été évaluée avec différentes quantités de TNF immobilisé sur filtres de nitrocellulose . (Le TNF utilisé est du TNF0 humain recombinant.)The ability to bind the Fab4-BCCP - antigen was evaluated with different amounts of TNF immobilized on nitrocellulose filters. (The TNF used is recombinant human TNF0.)
On obs.erve que la protéine Fab4-BCCP* se lie au TNF avec la même efficacité que la Fab4, quand on révèle sa présence par l'addition d'anticorps anti-kappa.It is observed that the Fab4-BCCP * protein binds to TNF with the same efficiency as Fab4, when its presence is revealed by the addition of anti-kappa antibodies.
D'autre part quand les mêmes filtres sont révélés en présence de streptavidine marquée à la phosphatase alcaline, seule la Fab4-BCCP* donne un signal positif.On the other hand when the same filters are revealed in the presence of streptavidin labeled with phosphatase alkaline, only Fab4-BCCP * gives a positive signal.
Cette expérience montre donc que les 2 domaines de la protéine de fusion Fab4-BCCP* sont fonctionnels : le site spécifique de l'anticorps reconnaît son antigène et l'extrémité BCCP* fixe la biotine qui lie la streptavidine.This experiment therefore shows that the 2 domains of the Fab4-BCCP * fusion protein are functional: the specific site of the antibody recognizes its antigen and the BCCP * end fixes the biotin which binds streptavidin.
Pour démontrer que la présence de l'extrémité BCCP* n'interfère pas avec la capacité de fixation de l'antigène par comparaison avec le Fab4, l'interaction Fab4-BCCP*/TNF a été suivie dans un test de compétition en ELISA (voir exemple 4) en présence d'un excès d'antigène. Une quantité définie de Fab4 ou de Fab4-BCCP* et des quantités variables de TNF ont été mélangées dans des puits de microplaques à ELISA recouverts d'antigène. Après lavage, les molécules fixées sont révélées par l'addition de streptavidine marquée à la phosphatase alcaline.To demonstrate that the presence of the BCCP * end does not interfere with the binding capacity of the antigen by comparison with Fab4, the Fab4-BCCP * / TNF interaction was followed in a competition test in ELISA ( see example 4) in the presence of an excess of antigen. A defined amount of Fab4 or Fab4-BCCP * and varying amounts of TNF were mixed in wells of ELISA microplates coated with antigen. After washing, the fixed molecules are revealed by the addition of streptavidin labeled with alkaline phosphatase.
La concentration de TNF libre nécessaire pour provoquer 50 % d'inhibition de la fixation de Fab4-BCCP* est de 0,2 μg/ml ce qui correspond à une constante de liaison relative de 1,2 10~8 M, c'est-à-dire du même ordre que celle de 1,5 10~8 M qui a été trouvée pour Fab4 (publication de Orfanoudakis et al . ) .The concentration of free TNF necessary to cause 50% inhibition of the binding of Fab4-BCCP * is 0.2 μg / ml which corresponds to a relative binding constant of 1.2 10 ~ 8 M, that is ie of the same order as that of 1.5 10 ~ 8 M which was found for Fab4 (publication by Orfanoudakis et al.).
La fusion de l'extrémité BCCP* n'affecte donc pas la capacité de liaison de l'antigène du Fab4.The fusion of the BCCP * end therefore does not affect the binding capacity of the Fab4 antigen.
Exemple 3. Optimisation de la biotinylation des molécules de Fab4-BCCP* sécrétées par E. coli.Example 3. Optimization of the biotinylation of the Fab4-BCCP * molecules secreted by E. coli.
Le pourcentage de protéines de fusion Fab4-BCCP* marquées par la biotine a été évalué à l'aide de streptavidine immobilisée sur des billes d'agarose. Des quantités non saturantes de surnageant de culture de E. coli ont été incubées avec les billes puis les protéines fixées ont été analysées par immunoempreinte avec de 1 'anti-kappa. Par comparaison avec la quantité de surnageant incubé on mesure que 3 % des molécules sont biotinylées.The percentage of Fab4-BCCP * fusion proteins labeled with biotin was evaluated using streptavidin immobilized on agarose beads. Unsaturated amounts of E. coli culture supernatant were incubated with the beads and then the fixed proteins were analyzed by immunoblotting with anti-kappa. By comparison with the quantity of supernatant incubated, it is measured that 3% of the molecules are biotinylated.
Pour augmenter ce rendement, les cultures bactériennes ont été réalisées en présence de différentes concentrations de d-biotine (de 0,01 à 1 mM) . Celle-ci est ajoutée quand la culture a une D.O. de 1, en même temps que l'IPTG.To increase this yield, the bacterial cultures were carried out in the presence of different d-biotin concentrations (0.01 to 1 mM). This is added when the culture has an OD of 1, at the same time as the IPTG.
Dans ces conditions on obtient un rendement 5 fois plus élevé de biotinylation, avec un plateau à partir d'une concentration de 0,1 mM de d-biotine. On obtient ainsi 15 % de Fab-BCCP* biotinylé.Under these conditions, a 5 times higher biotinylation yield is obtained, with a plateau from a concentration of 0.1 mM of d-biotin. 15% of biotinylated Fab-BCCP * are thus obtained.
Dans les conditions habituelles de production on obtient 1 mg de Fab-BCCP* par litre de culture bactérienne, dont 0,15 mg de molécule biotinylée.Under the usual production conditions, 1 mg of Fab-BCCP * is obtained per liter of bacterial culture, of which 0.15 mg of biotinylated molecule.
Exemple 4. Utilisation des fragments d'anticorps biotinylés dans des dosages immunologiques.Example 4. Use of biotinylated antibody fragments in immunoassays.
Le concentré de protéines de fusion Fab4-BCCP* peut être utilisé pour réaliser des tests ELISA directs ou indirects .The Fab4-BCCP * fusion protein concentrate can be used for direct or indirect ELISA testing.
Des microplaques pour dosage ELISA ont été incubées pendant une nuit à 4°C avec des quantités variables de TNFα recombinant (de 0,01 à 5 μg/ml en tampon bicarbonateMicroplates for ELISA assay were incubated overnight at 4 ° C with variable amounts of recombinant TNFα (from 0.01 to 5 μg / ml in bicarbonate buffer
0,1 M) . Après lavage, les plaques ont été incubées pendant 1 heure à température ordinaire en présence de sérumalbumine bovine pour bloquer les sites résiduels de liaison de protéines . Après lavage, des dilutions en série du concentré de Fab4-BCCP* ont été distribuées dans les puits. Après 1 heure d'incubation à '37°C les protéines de fusion qui se sont adsorbées sur l'antigène sont détectées par une incubation de 1 heure à 37°C en présence de streptavidine marquée à la phosphatase alcaline. Celle-ci est visualisée par l'addition de para-nitrophényl-phosphate (1 mg/ml - Sigma®) en tampon Tris-HCl 0,1 M pH 9, NaCl 0,1 M, MgCl2 5 mM et hydrolyse pendant 1 heure. La densité optique des puits est mesurée à 405 nm, par exemple à l'aide d'un lecteur automatique d'ELISA (Molecular Devices®) .0.1 M). After washing, the plates were incubated for 1 hour at room temperature in the presence of bovine serum albumin to block the residual protein binding sites. After washing, serial dilutions of the Fab4-BCCP * concentrate were distributed to the wells. After 1 hour incubation at '37 ° C the fusion proteins which are adsorbed to the antigen are detected by incubation for 1 hour at 37 ° C in the presence of streptavidin labeled with alkaline phosphatase. This is visualized by the addition of para-nitrophenyl phosphate (1 mg / ml - Sigma®) in 0.1 M Tris-HCl buffer pH 9, 0.1 M NaCl, 5 mM MgCl2 and hydrolysis for 1 hour . The optical density of the wells is measured at 405 nm, for example using an automatic ELISA reader (Molecular Devices®).
La représentation graphique des résultats montre que les protéines Fab4-BCCP* lient spécifiquement le TNF, de manière saturable. La réactivité est strictement dépendante de la concentration en Fab4-BCCP* utilisée, ce qui confirme la bifonctionnalité de la protéine de fusion puisque toutes les molécules qui fixent la streptavidine sont capables de réagir avec le TNF.The graphical representation of the results shows that the Fab4-BCCP * proteins specifically bind TNF, saturable way. The reactivity is strictly dependent on the concentration of Fab4-BCCP * used, which confirms the bifunctionality of the fusion protein since all the molecules which fix streptavidin are capable of reacting with TNF.
Cette propriété permet d'utiliser la protéine de fusion dans n'importe quel type de dosage par im unodétection.This property makes it possible to use the fusion protein in any type of assay by im unodetection.
Exemple 5. Utilisation des fragments d'anticorps biotinylés pour la purification d'antigènes par immuno¬ affinité.Example 5. Use of the fragments of biotinylated antibodies for the purification of antigens by immunoaffinity.
La bifonctionnalité des protéines Fab-BCCP* biotinylées permet de les adsorber sur des billes couvertes de streptavidine et ensuite, d'utiliser celle-ci pour purifier, par immunoaffinité, l'antigène correspondant au Fab.The bifunctionality of the biotinylated Fab-BCCP * proteins makes it possible to adsorb them onto beads covered with streptavidin and then to use the latter to purify, by immunoaffinity, the antigen corresponding to the Fab.
Une suspension de 0,25 ml de billes d'agarose recouvertes de streptavidine a été incubée en présence de Fab4-BCCP* concentrés (4 ml) dans 10 ml de tampon Tris-HCl 20 mM, pH8, NaCl 50 mM et sérumalbumine bovine 1 % (tampon A) , pendant 1 heure à température ordinaire, sous agitation douce. Après lavage des billes en .tampon Tris-HCl 20 mM, pH 8, NaCl 0,5 M, NP40 0,1 %. puis en tampon Tris-HCl, pH 8, NaCl 50 mM, les billes sont remises en suspension dans le tampon A.A 0.25 ml suspension of agarose beads coated with streptavidin was incubated in the presence of concentrated Fab4-BCCP * (4 ml) in 10 ml of 20 mM Tris-HCl buffer, pH8, 50 mM NaCl and bovine serum albumin 1 % (buffer A), for 1 hour at room temperature, with gentle stirring. After washing the beads with a 20 mM Tris-HCl buffer, pH 8, 0.5 M NaCl, 0.1% NP40. then in Tris-HCl buffer, pH 8, 50 mM NaCl, the beads are resuspended in buffer A.
La préparation de billes ainsi recouvertes de streptavidine-biotine-BCCP*-Fab (9,8 ml) a été mise en contact avec un lysat bactérien (0,2 ml) clarifié d'une souche transformée par un vecteur d'expression d'une protéine de fusion comportant le TNF. Après une incubation de 2 heures à 4°C sous agitation douce, puis lavage dans les conditions décrites plus haut, les protéines du lysat bactérien qui se sont adsorbées sur les billes ont été éluées avec du tampon Tris-HCl 50 mM, pH 8, NaCl 0,50 M, éthylène glycol 50 % . Ces protéines ont été analysées par électrophorese sur gel de polyacrylamide-SDS et coloration au bleu de Coomassie. Elles correspondent bien à la protéine de fusion attendue, Les molécules de Fab-BCCP*-bio sont donc utilisables pour purifier, en une seule étape, un antigène présent dans un extrait brut de bactérie. The preparation of beads thus coated with streptavidin-biotin-BCCP * -Fab (9.8 ml) was brought into contact with a clarified bacterial lysate (0.2 ml) of a strain transformed with an expression vector of a fusion protein comprising TNF. After a 2 hour incubation at 4 ° C. with gentle shaking, then washing under the conditions described above, the proteins of the bacterial lysate which are adsorbed on the beads were eluted with 50 mM Tris-HCl buffer, pH 8, 0.50 M NaCl, ethylene glycol 50%. These proteins were analyzed by electrophoresis on polyacrylamide-SDS gel and staining with Coomassie blue. They correspond well to the expected fusion protein. The Fab-BCCP * -bio molecules can therefore be used to purify, in a single step, an antigen present in a crude extract of bacteria.

Claims

REVENDICATIONS
1.- Vecteur de clonage et d'expression portant une cassette d'expression bi-cistronique permettant la synthèse dans E. coli de fragments Fab d'anticorps de spécificité définie et comprenant un site fonctionnel de liaison de l'antigène, caractérisé en ce qu'il comprend une séquence d'ADN codant pour l'extrémité C-terminale de la sous-unité porteuse de carboxy-biotine (BCCP) de l'acétyl-1.- Cloning and expression vector carrying a bi-cistronic expression cassette allowing the synthesis in E. coli of Fab fragments of antibodies of defined specificity and comprising a functional antigen binding site, characterized in that that it comprises a DNA sequence coding for the C-terminal end of the carboxy-biotin-carrying subunit (BCCP) of acetyl-
CoA carboxylase de E. Coli, insérée en phase en aval de la séquence codante du fragment Fd desdits fragments Fab d'anticorps, ladite séquence d'ADN permettant, dans les conditions appropriées d'induction, la synthèse d'une protéine de fusion Fd-BCCP* et du Fab-BCCP* qui comprend ladite protéine de fusion. CoA carboxylase from E. Coli, inserted in phase downstream of the coding sequence of the Fd fragment of said antibody Fab fragments, said DNA sequence allowing, under appropriate induction conditions, the synthesis of an Fd fusion protein -BCCP * and Fab-BCCP * which comprises said fusion protein.
2.- Fragments Fab d'anticorps caractérisés en ce qu'ils sont synthétisés et sécrétés par des bactéries E. coli transformées par un vecteur selon la revendication 1.2. Fab fragments of antibodies characterized in that they are synthesized and secreted by E. coli bacteria transformed by a vector according to claim 1.
3.- Fragments Fab d'anticorps selon la revendication 2, caractérisés en ce qu'ils sont bifonctionnels, l'extrémité N-terminale constituant le site de liaison de l'antigène et l'extrémité C-terminale constituant le site de liaison de la biotine.3.- Fab fragments of antibodies according to claim 2, characterized in that they are bifunctional, the N-terminal end constituting the binding site of the antigen and the C-terminal end constituting the binding site of biotin.
4.- Fragments Fab d'anticorps selon la revendication 2 ou 3, caractérisés en ce qu'ils ont été biotinylés in vivo par la bactérie qui les a synthétisés.4.- Fab fragments of antibodies according to claim 2 or 3, characterized in that they have been biotinylated in vivo by the bacterium which has synthesized them.
5.- Procédé d'obtention de fragments Fab d'anticorps selon la revendication 4, caractérisé en ce qu'on optimise le taux de fixation de biotine par addition de biotine à une concentration d'environ 0,1 mM dans le milieu de culture des bactéries.5. A process for obtaining Fab fragments of antibodies according to claim 4, characterized in that the rate of biotin fixation is optimized by adding biotin to a concentration of approximately 0.1 mM in the culture medium. bacteria.
6.- Utilisation des fragments Fab d'anticorps selon l'une quelconque des revendications 2 à 5 pour la réalisation de dosages immunologiques dans lesquels la bi¬ fonctionnalité de la protéine de fusion Fab-BCCP* est exploitée pour fixer l'antigène à l'extrémité N-terminale et détecter et quantifier la liaison antigène-anticorps par la révélation de la biotine liée à l'extrémité C-terminale, en présence d'avidine ou de streptavidine couplée à un marqueur de type phosphatase alcaline ou à un isotope radioactif.6.- Use of Fab fragments of antibodies according to any one of claims 2 to 5 for carrying out immunological assays in which the bi¬ functionality of the Fab-BCCP * fusion protein is used to fix the antigen to the N-terminus and detect and quantify the antigen-antibody binding by revealing the biotin linked to the C-terminus, in presence of avidin or streptavidin coupled with an alkaline phosphatase type marker or a radioactive isotope.
7.- Utilisation des fragments Fab d'anticorps selon l'une quelconque des revendications 2 à 5 pour la purification d'antigènes par immunoaffinité à l'aide de supports inertes recouverts d'avidine ou de streptavidine, sur lesquels la protéine de fusion Fab-BCCP* est liée par son extrémité biotinylés, le site de reconnaissance de l'antigène, en N-terminal de ladite protéine, étant libre et permettant la rétention de l'antigène à partir de toute solution brute dans laquelle il est présent, et ensuite, son élution dans des conditions appropriées. 7. Use of the Fab fragments of antibodies according to any one of claims 2 to 5 for the purification of antigens by immunoaffinity using inert supports coated with avidin or streptavidin, on which the Fab fusion protein -BCCP * is linked by its biotinylated end, the antigen recognition site, at the N-terminal of said protein, being free and allowing the retention of the antigen from any crude solution in which it is present, and then, its elution under appropriate conditions.
PCT/FR1995/000293 1994-03-17 1995-03-13 Recombinant antibody fragments which are synthesized and biotinylated in e. coli, their use in immunoassays and immunopurification techniques WO1995025172A1 (en)

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WO1996021156A1 (en) * 1995-01-04 1996-07-11 Abbott Laboratories Method for preparing scintillation proximity assay targets
US6451311B2 (en) 1999-12-22 2002-09-17 Dade Behring Marburg Gmbh Anti-procalcitonin antibodies and the preparation and use thereof
US6562946B2 (en) 1999-12-22 2003-05-13 Dade Behring Marburg Gmbh Human procalcitonin and the preparation and use thereof
US6905687B2 (en) 1999-12-22 2005-06-14 Dade Behring Marburg Gmbh Human procalcitonin and the preparation and use thereof
US7338770B2 (en) 1999-12-22 2008-03-04 Dade Behring Marburg Gmbh Human procalcitonin and the preparation and use thereof
FR2816943A1 (en) * 2000-11-22 2002-05-24 Inst Nat Sante Rech Med Biotin derivatives useful as diagnostic agents and as vectors to introduce specific compounds into target cells to regulate cell proliferation and to treat atheroma and cancers
WO2002042311A2 (en) * 2000-11-22 2002-05-30 Institut National De La Sante Et De La Recherche Medicale (I.N.S.E.R.M) Biotin derivatives, methods for making same and uses thereof as vectors
WO2002042311A3 (en) * 2000-11-22 2002-09-19 Inst Nat Sante Rech Med Biotin derivatives, methods for making same and uses thereof as vectors
US7867781B2 (en) 2000-12-22 2011-01-11 Siemens Healthcare Diagnostics Products Gmbh Detection methods
WO2003064656A1 (en) * 2002-01-29 2003-08-07 Sense Proteomic Limited Protein tag comprising a biotinylation domain and method for increasing solubility and determining folding state
AU2003238441B2 (en) * 2002-01-29 2008-10-30 Sengenics Corporation Pte Ltd Protein tag comprising a biotinylation domain and method for increasing solubility and determining folding state
US8999897B2 (en) 2002-01-29 2015-04-07 Sense Proteomic Limited Protein tag comprising a biotinylation domain and method for increasing solubility and determining folding state
US8628933B2 (en) 2003-12-01 2014-01-14 Siemens Healthcare Diagnostics Products Gmbh Homogeneous detection method
US8399209B2 (en) 2003-12-01 2013-03-19 Siemens Healthcare Diagnostics Products Gmbh Homogeneous detection method
US7951910B2 (en) 2004-08-24 2011-05-31 Siemens Healthcare Diagnostics Products Gmbh Peptides with the marburg I polymorphism of factor VII-activating protease (FSAP) and their preparation and uses
US7622558B2 (en) 2004-08-24 2009-11-24 Siemens Healthcare Diagnostics Products Gmbh Antibodies which are directed against the Marburg I polymorphism of factor VII-activating protease (FSAP), and their preparation and use
EP2399933A1 (en) 2005-05-09 2011-12-28 Siemens Healthcare Diagnostics Products GmbH Binding partner of the placental growth factor, in particular antibodies opposed to the placental growth factor, production and use of same
EP2818479A1 (en) 2005-05-09 2014-12-31 Siemens Healthcare Diagnostics Products GmbH Binding partner of the placental growth factor, in particular antibodies opposed to the placental growth factor, production and use of same
US9453071B2 (en) 2005-05-09 2016-09-27 Siemens Healthcare Diagnostics Products Gmbh Binding partners of the placental growth factor, especially antibodies directed against the placental growth factor, and production and use thereof
US9469687B2 (en) 2005-05-09 2016-10-18 Siemens Healthcare Diagnostics Products Gmbh Binding partners of the placental growth factor, especially antibodies directed against the placental growth factor, and production and use thereof

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