Hybridoma Sequencing
Why sequences of antibody genes are needed:
Sequences of the antibody variable regions of monoclonal antibodies are required for the following purposes;
- Patent applications,
- The production of humanized antibodies,
- To rescue the antibody gene if hybridoma cell lines are not grown or Ab production is lost when taken out of frozen storage,
- Authentication (Validation) of hybridoma cell lines.
- Alternative antibodies formats
(a) Antibody- enzyme fusion
(b) Bispecific antibodies
The antibody (IgG, IgM, IgA, or IgE) variable regions of the heavy chain (VH) and the light chain (VL) from mouse, rabbit, and human clonal B cells are PCR amplified and sequenced.
Material Requirement
1×105 cells per hybridoma are recommended for cloning and sequencing antibody genes.
Flow chart of methodology used for sequencing of hybridoma or clonal B cells and construction and production recombinant antibody.
Rescue of an antibody gene from hybridoma cells
Sometimes hybridoma cells stop producing an antibody. Sequencing the antibody gene allows production of a recombinant antibody in another expression system.
Monoclonal antibodies to ghrelin were produced in rabbit and mouse. The gene for the rabbit antibody was sequenced and used to make a single chain variable fragment antibody in E. coli. The data shown is an ELISA test for detection of ghrelin “hunger hormone”, a peptide hormone produced by ghrelinergic cells in the gastrointestinal tract. The recombinant antibody has the same binding as the original rabbit antibody.
Data courtesy of Bruce D. Gaylinn, Endocrinology and Metabolism