WO2012164320A1

WO2012164320A1 - Biological system for the production of specifically biotinylated monoclonal antibodies

Info

Publication number: WO2012164320A1
Application number: PCT/HR2011/000024
Authority: WO
Inventors: Bojan POLIĆ; Stipan JONJIĆ
Original assignee: Medicinski Fakultet U Rijeci
Priority date: 2011-05-30
Filing date: 2011-05-30
Publication date: 2012-12-06

Abstract

This invention relates to the biological system for production of the specifically biotinylated monoclonal antibodies. The system consists of a mutant mouse strain (KAC) that is capable of producing IgK light chain containing the AviTag^TM and a myeloma cell line transfected with the BirA transgene (SP2/0- BirA). Upon the immunization of Igk ^KAC/KAC mice with several antigens and PEG-mediated fusion of their splenocytes with the SP2/0-BirA cell line hybridoma clones producing antigen-specific and specifically biotinylated monoclonal antibodies are generated.

Description

FIELD OF THE INVENTION

This invention relates to a biological system for production of specifically biotinylated monoclonal antibodies.

BACKGROUND OF THE INVENTION

Biotinylation is the process of covalently attaching biotin to a protein, nucleic acid or other molecule. The process is widely used in the various fields of biotechnology due to the fact that it does not perturb the natural function of the molecule due to the small size of biotin and that it binds to streptavidin and avidin with the extremely high affinity and specificity. Moreover, biotin-binding to streptavidin and avidin is resistant to extremes of heat, pH and proteolysis, making capture of biotinylated molecules possible in a wide variety of environments.

Biotinylation of monoclonal antibodies represents one of the most used adaptations to different applications. Biotinylation is usually achieved by a chemical reaction that causes random binding of biotin to all lysine residues, which can, however, bring risks regarding the function of the protein. In contrast, specific biotinylation requires lysine residue as a part of the specific amino acid sequence that is recognized by a biotin protein ligase (BPL). So far, the best characterized BPL is Escherichia coli biotin- ligase - BirA.

The enzyme biotin holoenzyme synthetase (BirA) of E. coli catalyses in vivo the covalent addition of biotin to the ε-lysine side chain in its natural substrate, biotin carboxyl carrier protein (BCCP) (Cronan, J. E. Jr. ef al J. Biol. Chem 265 (1990) 10327-10333).

Monoclonal antibodies represent key reagents in research of proteins and their interactions, and, can be produced by a relatively simple and affordable technology. These reagents are essential for a number of classical methods in protein biochemistry (i.e. Western blot, IP, ELISA, etc.), as well as in many new methods that are necessary for a comprehensive proteome research. Therefore, finding of new ways in monoclonal antibody adaptation for use on platforms for a high-throughput proteome research (i.e. protein microarrays) represents necessity. It was the object of this invention to provide a method for site-specific biotinylation of monoclonal antibodies by making use of a biological system.

SUMMARY OF THE INVENTION

This invention provides a biological system for production of the specifically biotinylated monoclonal antibodies. The system consists of a mutant mouse strain (KAC) that is capable to produce Igi light chain containing the AviTag™, and a myeloma cell line transfected with the BirA transgene (SP2/0- BirA). Upon the immunization of lgif^AC/KAC mice with several antigens and PEG-mediated fusion of their splenocytes with the SP2/0-BirA cell line hybridoma clones producing antigen-specific and specifically biotinylated monoclonal antibodies are generated

The advantages of the system are: a) production of specifically biotinylated monoclonal antibodies by hybridoma cell lines; b) production of the biotinylated monoclonal antibodies that is not restricted to any IgH class; c) specifically biotinylated Fab fragments can be derived from the monoclonal antibodies; d) applicability of monoclonal antibodies in different research, diagnostic and therapeutic approaches; e) the monoclonal antibodies are particularly suitable for use on solid-phase platforms (protein microarrays, SA-coated beads, SA-coated microtitre plates, etc.) considering the defined site of biotin-SA interaction on the monoclonal antibody.

DETAILED DESCRIPTION OF THE INVENTION

Monoclonal antibodies represent key reagents in research of proteins and their interactions, and, can be produced by a relatively simple and affordable technology. These reagents are essential for a number of classical methods in protein biochemistry (i.e. Western blot, IP, ELISA, etc.), as well as in many new methods that are necessary for a comprehensive proteome research. Therefore, finding of new ways in monoclonal antibody adaptation for use on platforms- for a high-throughput proteome research (i.e. protein microarrays) represents necessity and challenge in the present and following era.

Labeling of monoclonal antibodies with different biomarkers (Biotin, FITC, PE, POD, AP, etc.) represents widely used manner of monoclonal antibody adaptation to different applications. One of the most used biomarkers in protein biochemistry is biotin. This relatively small molecule has extremely strong binding affinity (Kd =10^'14 M) to streptavidin (SA) and represents one of the strongest covalent interactions known in the nature at all (Bayer E.A. and Wilchek M., Methods Enzymol. 184:138, 1990). This interaction has been widely used in immobilization of chemically biotinylated proteins on the SA coated surfaces (microtiter plates, beads, microarray platforms and others) and it is one of the often chosen principles in affinity chromatography. The usual method for posttranslational biotinylation is random chemical binding of biotin to ε-amino group of lysine residues (Bayer E.A. and Wilchek M., Methods Enzymol. 184:138, 1990). However, this kind of protein biotinylation can lead to the loss of activity or capability of protein-protein interaction due to random modification of the lysine residues, or/and uncontrolled binding to the SA coated matrices, but also because of the conditions required for the chemical modification that can alter the protein structure (i.e. pH). Lately, considerable attention has been focused to the specific, enzymatically mediated biotinylation of proteins, resulting in their better quality and usefulness. Specific biotinylation of proteins is relatively rare in the nature. For example, E.coli has only one biotinylated protein (biotin-carboxyl carrier protein (BCCP) for acetyl-CoA carboxylase), while in other bacteria one can find one to three of such proteins (Fall R Methods Enzymol. 62:390, 1979). Yeast cells (S. cervisiae) have four or five biotinylated proteins, depending on the growth conditions (Lim P. et al., Arch. Biochem. Biophys. 258:219), while in mammals (Robinson B.H. et al., J.Biol.Chem. 258:6660, 1983.; Chandler C.S. and Ballard F.J., Biochem. J. 251:749, 1988) and plants (Nikolau B.J. et al.₍ Anal. Biochem. 149:488, 1985.) one can find four of such proteins.

Lysine residues in these proteins, that are biotinylated on ε-amino group, are situated in an amino acid sequence (Biotin Accepting Peptide - BAP) that is specifically recognized by biotin protein ligases (BPL). The best characterized BPL so far is bacterial BirA (E.coli). It recognizes specific and well conserved peptide sequence within various biological sources (Samols D. et al., J. Biol. Cem. 263:6461, 1988.; Shenoy B.C. et al., J. Biol.Chem. 267:18407, 1992). Except of natural recognition sequence, BirA recognize also shorter and artificially obtained sequences, such as PinPoint (Promega Inc.) or AviTag™ (Avidity Inc.). This, relatively new system, that requires production of a fusion protein containing BAP sequence and presence of BirA has been already successfully used in several cases of protein biotinylation in vitro (Cognet I. et. al., J. Immunol. Methods 301:53, 2005.), in bacteria (Ashraf S.S. et al., Protein Expression and Purification 33:238, 2004.), in eukaryotic cells (Athavankar S. and Peterson B.R., Chemistry and Biology 10:1245, 2003.) and in vivo in transgenic mice (De Boer E. et al. PNAS 100:7480, 2003.). This invention describes a biological system for the production of specifically biotinylated monoclonal antibodies, which is based on the above described mechanism of enzimatically mediated biotinylation (Figure 1).

This invention describes the development of a mouse mutant with introduced DNA sequence of AviTag into the Ig κ locus by gene targeting (knock-in mutation).

This invention also provides a mouse myeloma cell line (Sp2/0) stably transfected with BirA transgene, as a fusion partner in the hybridoma development and monoclonal antibody production.

The further characteristic of this invention is the mouse mutant (KAC - Ig Kappa AviTaged Constant Region) capable of producing specific and AviTag containing monoclonal antibodies upon the immunization with an appropriate antigen.

This invention further provides a method for fusion of activated B cells isolated from the immunized mutants and BirA-SP2/0 transfectant results in the hybridoma cells producing specific monoclonal antibodies that are specifically biotinylated on the IgK chain.

This invention presents a major advantage over prior art due to the fact that IgK locus was chosen for gene targeting.

This particular choice of IgK locus has following advantages:

1. IgK light chain is dominantly (> 95%) present in immunoglobulin molecules in comparison to light chain;

2. IgK chain is capable to pair with any isotype (IgM, IgD, IgGl, lgG2a, lgG2b, lgG3, IgE and IgA) of IgH (heavy chain), which contributes to a high level of flexibility of the labeling system;

3. the labeling of IgK chain gives a possibility of production of labeled F(ab)₂ fragments, since IgK entirely contributes to the formation of F(ab)₂ fragments;

4. More simple strategy of IgK targeting in comparison to any isotype of IgH

More specifically the specific biotinylation of IgK chain in this system givesthe advantage: in possibility of production of any IgH class of antibodies. Further advantage of the system lies in the fact that it enables use Fab fragments. Another advantage of the system is that is uses natural affinity and availability of active (Ag binding) sites of all monoclonal antibodies.

This inventions has a broad spectrum of industrial applications. More specifically these applications are in the field of molecular biology and biomedicine research and diagnostics such as: ELISA, flow cytometry, confocal microscopy, Western blot, immunoprecipitation, affinity chromatography, etc..

Considering the defined site of biotine -SA interaction on the monoclonal antibody, this invention is particularly suitable for use in the field of high-throughput methods in proteome research that use solid- phase platforms (protein microarrays, SA-labeled spheres, SA-labeled microtiter plates, etc.).

Other possible application is a therapeutic field, for example specific drug or radioactivity delivery.

DESCRIPTION OF THE FIGURES

Figure 1 The biological system for the production of biotinylated monoclonal antibodies.

The system consists of two components: a mouse mutant containing KAC mutation that serves for immunization with antigen and isolation of activated B cells, and a stably transfected myeloma cell line (SP2/0) with BirA transgene, as a fusion partner for the activated B cells. Upon the fusion, hybridoma lines produce specific monoclonal antibodies that are enzymatically biotinylated.

Figure 2 The targeting of IgL CK locus.

The homologous recombination of the targeting vector into the IgL CK locus (a) in C57BL6 ES cells resulted with the introduction of the AviTag sequence CK3 exon (KAC mutation) and of the loxP flanked selection marker (Neo^R) (b). Upon the transient transfection of the ES cells positive for the mutation with the Cre expression plasmid, Cre recombinase mediated loxP recombination and deletion of the Neo^R cassette (c).

Figure 3 Generation of mice with KAC mutation. a) Targeting of Igk locus. Partial restriction map of the constant region of Igk locus (Ck) with restriction sites for BamH I (B), EcoR I (E) and Stu I (S) is shown. Selection markers (HSV-tk and neo), loxP sites (►), external hybridization probes A and B and internal probe C are depicted. The expected sizes of the DNA fragments are indicated above the lines. b) Southern-blot analysis of the targeted ES (Bruce 4) clones. The homologous recombination as well as the introduced mutation (AviTag) in the Ck3 was tested by EcoR I digestion and external probe B. The DNA fragments of 9.7 and 15.7 kb represent targeted and wild type Igk locus, respectively. The targeted allele of the 3A3 clone is larger than expected indicating for miss integration of the AviTag sequence. c) Southern-blot analysis of the homologous recombinants upon the transient expression of Cre. The Cre-mediated deletion of the neo cassette (Dneo) in the targeted ES clones was tested by the Stu I digestion and the external probe B. The DNA fragments of 11.8, 16.5 and 16.6 kb representing neo, WT ^' and Dneo alleles, respectivelly, are shown. d) Germ-line transmission of the KAC mutation. Coat color chimeras were bred with C57BL/6J mice and only black mice were tested for the KAC mutation by PC . The bands of 178 and 235 bp representing the mutated and WT allele are shown.

Figure 4 Phenotypical analysis of mice with KAC mutation.

Analysis of the spleen lymphocyte populations. a) Analysis of CD4+, CD8+, NK1.1+ and CD19+ lymphocyte populations in the spleen of the homozygous mice for KAC mutation in comparison to WT mice are shown. b) B cell development. There is no significant differences in B cell development pattern (B220 vs. IgM) of KAC homozygous mice as compared to WT mice. c) Levels of antibody isotype subclasses. Antibody isotype distribution in homozygous KAC mice is comparable to WT mice, regardless of AviTag insertion.

Figure 5 Generation of BirA expression vector.

Plasmid vector for hBirAL expression, a) Modified E. Coli BirA (hBirALj was inserted in pcDNA3.0 plasmid using standard DNA cloning techniques. The plasmid was used for stabile transfection of SP2/0 myeloma cells, b) Alignment of hBirAL protein sequence with bacterial BirA.

Figure 6 Analysis of hBirAL expression in transfeced SP2/0 cells.

Growing collonies were evaluated for hBirAL expression using RT PCR and Cell ELISA. a) hBirAL mRNA expression in transfected Sp20 cells. hBirAL mRNA expression was quantified by Real- Time PCR (LightCycler -Roche) using PCR-CyberGreen Kit (Roche). Amplified hBirAL cDNA was normalized with the amplified Act cDNA. b) hBirAL protein expression in transfected Sp2/0 cells. ELISA plates were coated with the transfected myeloma cells and hBirAL expression was evaluated using anti FLAG M2 mAb.

Figure 7 Evaluation of the fusion partners expressing hBirAL a) Fusion properties of generated hBirAL SP2/0 transfectants. Three selected fusion partners expressing hBirAL were tested for fusion capabilities and biotinylated mAb production to hlgM. The spleen cells of KAC homozygous mice immunized with hlgM were isolated and fused (PEG) with the indicated fusion partners.

b) Efficient mAb biotinylation in generated hybridoma cell lines. Generated hybridoma lines produce efficiently biotinylated mAbs according to the ELISA results (note 3A4 3G12 and 4E42D12 were IgGl, lambda).

Figure 8 Fusion overview

EXAMPLES

1. Design and construction of the DNA vector for Igic gene targeting in mouse ES cells

IgK locus, like other immunoglobulin loci, comprises more genetic elements (VK, JK and CK) that are involved in the rearrangements of the locus (mediated by RAG1 and RAG2 recombinase), thus contributing to the variability in the locus and generation of a broad spectrum of immunoglobulin specificities. Considering the fact that the variability and specificity of immunoglobulins depends partially (contribution of the variable segment of IgH chain) on the combination between different VK and JK elements, we estimated that the constant segment (CK) of the Igk locus would be the best for targeting and introduction of the AviTag sequence (45 bp). CK comprises 3 exons, and on the very beginning of the 3^rd exon there is a codon for amino acid cystein that is important for the formation of a disulphide bond with an appropriate part of the IgH chain. Therefore, targeting was done in a way that the AviTag was introduced between the codon for cystein and the stop codon (in CK₃) (Figure 2.)· The in frame introduction of the short AviTag sequence in proposed way did not cause conformation difficulties of IgK, and, consequent pairing with IgH chain and specificity of the Ig molecule. The targeting vector comprises: CK₃ with the introduced AviTag sequence and positive selection marker (Neo^R cassette) for ES cells together flanked with the 5' and 3' IgK homologous sequence. On the very end of the 5' homology we introduced a marker gene (HSV-tk) for the negative selection of ES cells (against random integrants). In order to prepare the targeting vector an appropriate genomic clone (BAC) containing Igk locus was obtained from the public available genomic library (BacPac). PCR was used to amplify and subclone both arms of homology as well as to introduce AviTag sequence into CK₃. All these elements were subcloned into the pEasy Ftox vector (M. Alimzhanov) which contains a loxP flanked Neo^R cassette, HSV-Tk gene and appropriate cloning sites. Upon completion of the targeting vector, it was entirely sequenced before the following transfection of ES cells.

2. Transfection and selection of ES cells

Bruce 4 (C57BL/6) mouse ES cells were used for the transfection. Before the transfection, ES cells were grown to the exponential growth phase and in an appropriate number optimal for the transfection (10⁷ cells per transfection). The targeting vector was linearized by endonuclase digestion a day before transfection and purified by phenol/chloroform extraction. For the transfection of 10⁷ Bruce ES cells 30 - 50 μg of the purified and linearized vector was used. The transfection was performed by electroporation (BioRad) in appropriated cuvettes (0.4 cm). Upon the transfection, ES cells were resuspended in the ES cell medium (DMEM + supplements), distributed on EF coated 100 mm TC Petri dishes and cultured without positive selection during the next 24 h. Next day, G418 was added in the minimal dose (determined via pretesting) that slowly killed all non-transfected ES cells (positive selection), and 5 days later Gancyclovir wasadded (during the next 3 days) for negative selection of ES cells (against random integrants). During the culture and selection of ES cells ES medium was changed on a daily basis in order to keep optimal growth conditions for ES cells.

3. Picking and screening of the ES cell clones

Picking of positively selected ES clones was performed between days 9 and 14 after the transfection. This has been done in a sterile hood with horizontal air flow with the aid of binocular microscope. Picked ES clones were transferred into the 96 well plates containing trypsin medium in each well in order to disaggregate the colonies and to get single cell suspension. Afterwards the resuspended ES cells were transferred into 96 well plates (3 replica plates) previously coated with EF cells. Upon 2-3 days of culture, two plates were frozen at -80°C, while one plate was further divided into 3 more plates not coated with EF cells. These plates served for the DNA isolation and Southern blot screening of the ES cell clones. Southern blot screening for the homologous recombinants was performed using appropriate endonuclease digestion and pretested (Bruce 4 DNA) external hybridization probes (5' and 3'). Possible presence of random integrants was tested by an internal hybridization probe. Positive ES clones for the homologous recombination and integration of the KAC mutation were thawed and further expanded for the following transient transfection with Cre and Neo deletion.

4. Deletion of Neo^R cassette in positive ES cell clones

Since the presence of a selection marker in the locus can interfere with the expression of the gene of interest or neighboring genes, it had to be deleted. The Cre/loxP system was used to make recombinatorial deletion of the Neo^R cassette. Since NeoR cassette was flanked with loxP sites, the NeoR deletionwas achieved in the homologous recombinants by Cre mediated recombination of loxP sites. The positive ES clones for KAC mutation and negative for random integrants were transiently transfected with the Cre expression plasmid (pGK Cre) using the same conditions as described above. Upon the transfection, smaller portion of the cells was cultured at low density on 100 mm Petri dishes and without selection (48 h), while the rest of transfected cells was frozen. When the ES cell formed colonies of desired size, they were picked up and distributed in two 96 well plates (replicas). One of them was subjected to the selection by G418, while the other was grown without selection. By observing of colonies within the first 2 - 3 days of selection we were able to discriminate the G418 sensitive (Neo^R deleted) over the resistant clones. The G418 sensitive clones were then expanded from the non-treated plate, checked for the Neo^R deletion by Southern blot analysis and prepared for the microinjection.

5. Set up of mouse breeding of blastocyst donors and acceptors

For blastocyst microinjection with the positive ES cells and their transfer into the pseudopregnant female mice it was necessary to set up mouse breeding of embryo donor and acceptor mice.The albino mice (white coat color, CB20) were used as blastocyst donors, since it was planned to transfect Bruce 4 ES cell line (C57BL/6), in order to easy determine the level of chimerism by coat color of mice born upon the transfer of microinjected blastocysts. An appropriate number of breeding pairs of CB20 mice were set up 4 days before the microinjection. Next day, upon the vaginal plaque check (indicator of fertilization), positive females were transferred in separate cage (0.5 day from the fertilization). Three days later (day 3.5) the blastocysts were isolated by flushing their uteri. For the embryo acceptors (the microinjected blastocysts) we used a colony of Fl (C57BL/6 x BLAB/c) mice. The Fl males were vasectomised 2 month before the experiment and tested for fertility. Sterile males were used for mating with Fl females in order to produce pseudopregnant (foster) females. It was set up 3 days before the microinjection. Next day, plaque positive Fl female were separated and used for the embryotransfer at day 2.5 from the "conception."

6. Microinjection of the ES clones and embryotransfer

On the day of microinjection it was necessary to isolate and flush the uteri of blastocyst donors. After that the embryos were washed several times (M2 medium) and transferred into the small Petri dish containing a drop of M2 medium covered with mineral oil by prepared glass capillary and mouth adaptor. In case that some of embryos were undeveloped (morula stage), they were put into a drop of M16 medium and cultured for several hours in C02 incubator to let them develop in blastocyst stage. In parallel with the blastocyst preparation, it was necessary to prepare ES cells for the microinjection. They were trypsinased to achieve a single cell suspension and plated for 45 min. on the Petri dish of the same surface to get rid of EF cells that adhered earlier than ES cells to the surface. ES cells were harvested and washed once, then resuspended in DMEM + HEPES medium and kept on ice until the microinjection.

The microinjection of blastocysts was performed in a shallow dish (lid of a 30 mm Petri dish) and under the inverted microscope possessing two micromanipulators for the control of the microinjection and holder needle. The device for the piezo-electric control of the microinjection that significantly facilitated the microinjection procedure. The needles for microinjection (home made or commercial) were installed on the micromanipulators and connected to the microinjectors. Upon the microinjection of blastocysts, they were transferred into uteri of pseudopregnant mothers.

7. Generation of the KAC mutants and their characterization

After the embryotransfer we obtained chimeric mice in 17 to 18 days. The chimerism (skin color) was visible already several days after the birth. Later on the chimerisam is becoming more visible, particularly with the growth of hair. It was very variable from 80-90% down to hardly visible. When the chimeras become sexually mature, we set up breeding between them and C57BL/6 mice in order to discriminate the pups according to the origin of the germ cells. Namely, if the germ cells originate from the microinjected ES cells ("black") than the pups are pure black and they were subjected later on to genotyping (50% of probability that they have the desired mutation), all other pups (different color, agouti) that originate from CB20 mice did not carry the mutation. Genotyping of mice was done by PC and Southern blot analysis. KAC positive mice (heterozygous) were further mated together in order to get mice homozygous for the KAC mutation.

8. Production of a stabile transfectant of mouse myeloma cell line with BirA transgene In order to produce a stabile transfectant of the mouse myeloma cell line SP2/0 with BirA an appropriate expression DNA vector was produced. BirA gene from E.coli was isolated by PCR and further subcloned into the expression vector (pcDNA 3.0) under the control of a strong eukaryotic promoter (CMV iel) and Neo selection marker. In the BirA cDNA we also introduced the signal sequence on N- terminus (for directing BirA into ER) as well as 6His tag. Myeloma cell lines were transfected with the linearized vector by electroporation (BioRad). Upon the transfection the cells were cultured in selective medium containing G418 and resistant colonies were collected and further tested for the expression of BirA by RT PCR and flow cytometry. The BirA positive transfectants (BirA-SP2/0) were further tested for their capability as fusion partners.

9. Immunization of KAC mice with selected herpes viral antigens

The homozygous mice for the KAC mutation were immunized with selected antigens (i.e. human IgM). The immunization of the homozygous mice was performed according to the following protocol: 1. subcutaneous immunization with 200 μg of Ag / mouse in complete Freund adjuvant (CFA); 2. three weeks after the first immunization the mice were boosted with subcutaneous injection of 200 μg Ag / mouse in incomplete Freund adjuvant (IFA); 3. Two weeks after the boost, the titer of specific antibodies was determined in sera of immunized mice. If the titer was < 1:5 000 the mice were immunized once more in IFA. This could be repeated 1 -2 times, or until the titer of specific antibodies reaches required level; 4. If the titer of specific serum immunoglobulins was > 1:5 000, the mice were boostered once more by intraperitoneal injection of 100 μg Ag / mouse in PBS. Three days after the last boost the spleen cells were isolated and fused with the previously developed BirA-SP2/0 myeloma cell line.

10. Production and characterization of specifically biotinylated monoclonal antibodies

In the production of monoclonal antibodies the technology well known in the state of the art was used for the production and selection of hybridoma lines (C. Milstein and G. Koeller)., the splenocytes isolated from the immunized mice homozygous for the KAC mutation were fused with the BirA-SP2/0 myeloma cell line by PEG (polyethilenglicol). Upon the fusion, the cells were distributed on ten 96 well plates and subjected to the selection in HAT and G418 containing medium, considering the sensitivity of the non- fused and non-BirA producing myeloma cells to this selection. When the hybridoma colonies were formed (after 6-8 days), all wells were tested for the production of specific monoclonal antibodies. The analysis of monoclonal antibodies was not only focused to the specificity of monoclonal antibodies, but also to the efficiency of the biotinylation measured by ELISA.

Claims

1. A biological system for preparing biotinylated monoclonal antibody that consists of: a) mutant mouse strain KAC (KAC - Ig Kappa AviTaged Constant Region) that is capable to produce IgK light chain containing the AviTag™,

b) myeloma cell line transfected with the BirA transgene (SP2/0- BirA).

2. A biological system according to claim 1, where lgx?^AC/KAC mice are immunized with several antigens and their splenocytes fused with the SP2/0-BirA cell line.

3. A biological system according claims 1 and 2, where hybridoma clones producing antigen- specific and specifically biotinylated monoclonal antibodies are generated.

4. A specifically biotinylated monoclonal antibody produced by making use of biological system according claims 1-3, where specific biotinylation occurs on the IgK chain

5. Use of specifically biotinylated monoclonal antibody according to claim 4, as reagent in the field of molecular biology research and diagnostics such as high-throughput methods in proteome research that are using solid-phase platforms (protein microarrays, SA-labeled spheres, SA- labeled microtiter plates, ELISA, flow cytometry, conofocal microscopy, Western blot, immunoprecipitation, affinity chromatography.

6. Use of specifically biotinylated monoclonal antibodies according to claim 4, in therapy such as drug delivery agent or radioactivity delivery agent.