Hybridoma Sequencing

ProMab Biotechnologies has nearly 2 decades of experience generating highly specific and potent monoclonal antibodies.

We have generated over 5000 hybridoma cell lines for our catalog and custom projects for clients from academic institutions to biopharma.

 

Starting from a hybridoma cell line, we offer the following services:

  • Validation of the hybridoma cell line in terms of antibody production, antibody isotyping, and antigen-binding specificity;
  • RNA extraction and reverse transcription;
  • scFv sequencing by PCR amplification and subcloning of the variable domains;
  • Full antibody sequencing by subcloning both variable and constant domains.

 

Figure 1. The process for sequencing hybridoma cells (1) Extracting RNA from 100,000 cells (2) Amplification for cloning and sequencing VH and VL regions (3) assembly of the transcriptome (4) defining scFv for synthesis.

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, VH, and light chain, VL) will be amplified for cloning and sequencing.

Degenerate primers are used to amplify the variable regions of murine or rabbit heavy (VH) and light (VL) chain antibody transcripts. Not all hybridomas generate single VH and VL chain transcripts. Multiple cloned and sequenced VH and VL chains are expressed in full-length antibody plasmid
backbones, and resulting VH-VL 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 VH and VL 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.

Other Custom Services:

Mouse monoclonal antibodies

 
 

Human monoclonal antibodies

 
 

Hybridoma Sequencing

 
 

 

HYBRIDOMAS THAT PRODUCE ANTIBODY AGAINST CELL-SURFACE ANTIGEN
Catlog # Description Entrez Gene ID
30630 Mouse Monoclonal Antibody to EPCAM 4072
30623 Mouse Monoclonal Antibody to ROR2 4920
30622 Mouse Monoclonal Antibody to MSTN 2660
30594 Mouse Monoclonal Antibody to CSF1R 1436
30590 Mouse Monoclonal Antibody to CD14 929
30585 Mouse Monoclonal Antibody to CD14 929
30578 Mouse Monoclonal Antibody to CD10 4311
30576 Mouse Monoclonal Antibody to CD10 4311
30570 Mouse Monoclonal Antibody to CD93 22918
30568 Mouse Monoclonal Antibody to CD93 22198
30567 Mouse Monoclonal Antibody to ITGAM 3684
30565 Mouse Monoclonal Antibody to IGFBP7 3490
30564 Mouse Monoclonal Antibody to IGFBP7 3490
30560 Mouse Monoclonal Antibody to MCAM 4162
30552 Mouse Monoclonal Antibody to EGFR 1956
30551 Mouse Monoclonal Antibody to EGFR 1956
30550 Mouse Monoclonal Antibody to CD22 933
30549 Mouse Monoclonal Antibody to CD22 933
30528 Mouse Monoclonal Antibody to ALCAM 214
30527 Mouse Monoclonal Antibody to IL3RA 3563
30522 Mouse Monoclonal Antibody to IL2RA 3559
30519 Mouse Monoclonal Antibody to CD24 100133941
30516 Mouse Monoclonal Antibody to IL3RA 3563
30512 Mouse Monoclonal Antibody to ALCAM 241
30507 Mouse Monoclonal Antibody to FLT3 2322
30505  Mouse Monoclonal Antibody to MCAM 4162
30498  Mouse Monoclonal Antibody to CSPG4 1464
30495  Mouse Monoclonal Antibody to FGF4 2249
30494  Mouse Monoclonal Antibody to FGF4 2249
30490  Mouse Monoclonal Antibody to NRCAM 4897
30486P  Mouse Monoclonal Antibody to EGFR mutant 1956
30486A  Mouse Monoclonal Antibody to EGFR mutant 1956
30485P  Mouse Monoclonal Antibody to ITGA2B 3674
30485A  Mouse Monoclonal Antibody to ITGA2B 3674
30480P  Mouse Monoclonal Antibody to CD33 945
30480A  Mouse Monoclonal Antibody to CD33 945
30476A  Mouse Monoclonal Antibody to ITGB4 3691
30449P  Mouse Monoclonal Antibody to TGFBR3 7049

Other Components of the CAR Platform: