METHOD FOR SELECTING HYBRIDOMA CELLS FROM A PLURALITY OF HYBRIDOMA CELLS BY MEANS OF A BIRA EXPRESSION VECTOR

Abstract

The invention relates to an improved method for the selection of hybridoma cells from a plurality of hybridoma cells for the generation of monoclonal antibodies as well as hybridoma cells together with suitable expression vectors by means of intracellular biotinylation and the use thereof.

Claims

1. A hybridoma cell containing at least one polynucleotide encoding a biotin protein ligase stably integrated into the genome after its transformation.

2. The hybridoma cell of claim 1, comprising at least one polynucleotide stably integrated into the genome after its transformation encoding a surface protein comprising a biotinylation peptide.

3. The hybridoma cell according to claim 1, comprising a surface protein containing a biotinylation peptide, in particular selected from the group SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3.

4. The hybridoma cell according to claim 1, wherein the integrated polynucleotide encoding biotin protein ligase is contained in an expression vector.

5. The hybridoma cell according to claim 1, wherein the integrated polynucleotide encoding biotin-protein ligase and surface protein containing biotinylation peptide is contained in an expression vector and controlled by a promoter.

6. The hybridoma cell according to claim 1, wherein the biotin-protein ligase is released intracellularly, and the biotinylation peptide is biotinylated intracellularly.

7. The hybridoma cell according to claim 1, wherein the integrated polynucleotide encoding biotin protein ligase is contained in an expression vector comprising at least one sequence of SEQ ID No. 7, SEQ ID No. 9 and SEQ ID No. 13; SEQ ID No. 15; SEQ ID No. 16; or SEQ ID No. 18.

8. The hybridoma cell according to claim 1, wherein a fusion cell line DSM ACC 3343 or DSM ACC 3344 is used.

9. A method for producing a hybridoma cell containing at least one polynucleotide encoding biotin-protein ligase stably integrated into the genome after its transformation, wherein the polynucleotide encoding biotin-protein ligase is used in an expression vector.

10. The method for producing a hybridoma cell according to claim 9 comprising at least one polynucleotide coding for biotin-protein ligase and for a surface protein comprising a biotinylation peptide stably integrated into the genome after its transformation, wherein the polynucleotide coding for biotin-protein ligase and for a surface protein comprising a biotinylation peptide is used in an expression vector.

11. A method of using a hybridoma cell containing at least one polynucleotide coding for biotin-protein ligase stably integrated into the genome after its transformation according to claim 1 for carrying out a selection of secreted monoclonal antibodies.

12. A DSM ACC 3343 or DSM ACC 3344 fusion cell line.

13. The fusion cell line DSM ACC 3343 or DSM ACC 3344 according to claim 12 comprising a polynucleotide encoding biotin protein ligase and a surface protein comprising a biotinylation peptide, which is contained in an expression vector and controlled by a promoter.

14. A DSM 32960 cell line containing SEQ ID No. 18.

15. A kit comprising a fusion cell line DSM ACC 3343 and/or DSM ACC 3344 according to claim 12 comprising a polynucleotide encoding biotin protein ligase in an expression vector.

Description

EXAMPLE 1

[0053] Description of the production of the hybridoma cell in the presence of the antigen (hybridoma/antigen relationship), so that the antigen is later represented in the construct with (strept)avidin:

[0054] Hybridoma cell production is performed to protocol, published in: Holzlöhner P, Hanack K (2017) “Generation of Murine Monoclonal Antibodies by Hybridoma Technology.” J Vis Exp. January 2; (119). doi: 10.3791/54832.

[0055] After successful immunization with the antigen, the spleen cells are removed from the mouse and fused with myeloma cells to hybridomas. For this purpose, the deposited fusion cell lines (supra) are used. Prior to fusion, quality control is performed to determine if the modified cell lines have the surface construct. The following protocol is used for this purpose:

[0056] To verify the presence of the surface construct, the transgenic myeloma cells (5×10.sup.6) are harvested, washed with PBS and incubated with a murine anti-HA antibody (1 μg per 1×10.sup.6 cells) for 30 min at 4° C. Cells are then washed twice again and incubated with 10 μL of an anti-murine IgG microbead solution for 15 min at 4° C. After washing twice, the cells are sorted using a magnetic column. The obtained cell pellet is taken up in full medium. The cells are cultured in the full medium for 2 days. This naturally contains biotin, so the cells are uniformly and completely biotinylated during this incubation. Successful biotinylation is verified by the addition of PE-labeled streptavidin and incubation for 20 min at 4° C. After confirmation of successful biotinylation of the cells, they are used for fusion with B lymphocytes.

[0057] After fusion, conventional HAT selection is performed. Afterwards, the cells are placed on normal full medium.

[0058] After 10 days of HAT selection, the cells are placed on full medium. Cells are then harvested and washed for sorting. They are then incubated with either the antibody capture matrix (ZAMAK-IgG-Avidin) or the avidin-coupled antigen (e.g. ovalbumin) for 3 hours at 37° C. After another washing step, the cells are incubated either with fluorescently labeled antigen or with a fluorescently labeled secondary antibody for 20 min at 4° C. The cells are washed again and the pellet is taken up in 500 μL buffer. This is followed by flow cytometric analysis and sorting of the cells.

[0059] Possibility (Option) A (FIG. 1) is the preparation of an antibody capture matrix in which a subclass-specific antibody, in this case a goat anti-mouse IgG antibody (ZAMAK-IgG) is coupled to avidin. The coupling is performed according to a standard protocol. For this, 1 mg of the ZAMAK-IgG is mixed with 0.5 mg of avidin and 0.25% glutaraldehyde, made up to 1 mL with 1×PBS and incubated for 2 hours at 4° C. This is followed by the addition of sodium borohydride (stock solution 100 mg/mL) and another incubation of 2 hours at 4° C. The mixture is then centrifuged at 13000×g and the supernatant dialyzed against 1×PBS. The coupling conjugate can then be sterile filtered and stored.

[0060] Possibility (Option) B (FIG. 1) is an embodiment example for the case that the antigen is ovalbumin. The coupling procedure is the same as described under option A. However, for ovalbumin, 3 mg of the substance is mixed with 2 mg avidin and 0.25% glutaraldehyde, made up to 1 mL with PBS and further treated as described above.

[0061] Then ELISA is used to check whether the matrix is positive, i.e. antigen/antibody and avidin are present.