Monoclonal Antibody Sequencing (DNA)
- We can isotype and sequence mouse or rat antibodies from hybridoma cell lines generated at the facility as well as cell lines from outside sources. Knowing the sequence is essential for antibody engineering or for IP protection.
- The antibody sequencing protocol involves isolating the mRNA from hybridoma cells followed by cDNA synthesis and PCR amplification of heavy- and light-chain variable region genes. The genes are then cloned into a vector and sequenced.
- We will provide a sequencing report stating the nucleotide and amino acid sequences of both the heavy-chain variable (VH), and light-chain variable (VL), regions of the monoclonal antibody. This will also include annotated VH and VL amino acid sequences highlighting the complementary determining, and framework regions of the antibody, and the similarity of the sequences to unrearranged germline mouse or rat antibody sequences. These analyses will be conducted using the IMGT/V-Quest program, (The International Immunogenetics Information System; http://www.imgt.org/IMGT_vquest/vquest).
- For sequencing we need a growing culture, a frozen cell pellet containing 10e7 cells or a frozen vial of cells. We do not accept RNA that has been isolated elsewhere. We would prefer these to be tested and free from mycoplasma but for cells of unknown or contaminated status we can isolate the culture then test here. If required we can treat the cells to remove the mycoplasma.