We provide a high-quality hybridoma sequencing service, also called hybridoma development. Hybridoma sequencing specifically refers to the sequencing of the variable regions of monoclonal antibodies being produced from hybridoma cell lines and will be required for the production of humanized antibodies, or for the authentication of a hybridoma cell line.

Total RNA is extracted from 100,000 hybridoma cells and using PCR methods, and the known non- variable flanking constant region sequences of respective antibody isotypes, the antibody variable regions (both heavy chain, V_H, and light chain, V_L) will be amplified for cloning and sequencing.

Degenerate primers are used to amplify the variable regions of murine or rabbit heavy (V_H) and light (V_L) chain antibody transcripts. Not all hybridomas generate single V_H and V_L chain transcripts. Multiple cloned and sequenced V_H and V_L chains are expressed in full-length antibody plasmid
backbones, and resulting V_H-V_L pairs are examined for binding to the target antigen. Single-chain variable fragments (scFv) are synthesized, incorporating a bacterial pelB secretion signal,a linker between the V_H and V_L domains, and a C-terminal 6xHis purification tag.

Figure 2. scFvs are expressed in E. coli, and binding of the purified scFvs to the target antigen is verified by ELISA

Antigen-antibody binding will subsequently be confirmed by transient expression of either single-chain antibodies (scFv) or whole IgGs in a mammalian expression system. Further testing will be performed to confirm that the recombinant scFv or whole IgG retains antigen binding affinity and specificity to its target molecule.

We have >1600 hybridoma clones are available to generate CAR-T cells.