Hybridoma technology is a technique used for mass-production of monoclonal antibodies, or identical antibodies, to an antigen of interest. Hybridoma cell lines are produced by fusing extracted spleen cells from immunized mice with compatible myeloma cell lines (Sp 2/0 cells). The favorable hybridoma cell lines will then be screened and cryopreserved for your research purposes. Hybridoma sequencing refers to the sequencing of the variable regions (the VH and VL domains) of monoclonal antibodies produced from hybridoma cell lines. This sequencing is required for the production of humanized antibodies, or for the authentication of a hybridoma cell line.
To validate or humanize your hybridoma cell line, ProMab Biotechnologies offers hybridoma sequencing using high-throughput Next Generation Sequencing (NGS) to provide both the variable fragment heavy chain (VH) and light chain (VL) for your desired application. Furthermore, we can help you synthesize the single-chain variable fragment (scFv) from the desired VH and VL domains and verify antigen-binding by ELISA.
Starting from a hybridoma cell line, we offer the following services:
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Validation of the hybridoma cell line in terms of antibody production, antibody isotyping, and antigen-binding specificity;
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RNA extraction and reverse transcription;
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scFv sequencing by PCR amplification and subcloning of the variable domains;
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Full antibody sequencing by subcloning both variable and constant domains.
Our dedicated scientists have over 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. To learn more about our antibody sequencing service, or to request a quote, please click below:
We provide a high-quality hybridoma sequencing service from RNA extraction to scFv production. The first step is to extract the RNA from 100,000 hybridoma cells, and using polymerase-chain reaction (PCR) and the known sequence of the non- variable flanking constant region 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 mice heavy and light chain antibody transcripts. As 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. The 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.
To request our hybridoma sequencing service, please click below. In addition to custom antibody sequencing and production services, ProMab Biotechnologies offers over 1600 hybridoma clones that are available for immediate purchase.
| 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 |