Is Your Monoclonal Antibody Authentic? Confirming Hybridoma Cell Line Identity by IgC cDNA Sequencing
Steven J. Madore, Ph.D., Associate Director for Science, ICBR
Yanping Zhang, Ph.D., Scientific Director, ICBR Gene Expression & Genotyping Core
Angel Sampson, Ph.D., Scientific Director, ICBR Monoclonal Antibody Core
Widespread reports of irreproducibility in health sciences research has prompted the NIH to update new grant application guidelines to include a brief description of methods that will be used to ensure identity and validity of key biological and/or chemical resources used in the proposed study. In this regard there is a significant effort now underway using DNA “fingerprinting” as a way to use microsatellite repeat lengths as a powerful tool for human cell line authentication. This method has been applied successfully to properly confirm the identity of a plethora of human cell lines routinely grown in the laboratory. In fact, the ICBR Gene Expression core now uses a well-established and fully validated PCR-based assay for confirming the identity of human lines grown by UF researchers.
Mouse hybridoma cell lines are generated by the fusion of splenic antibody producing B-cells with immortalized murine myeloma cells and serve as easy to grow, cell culture factories for producing clone-specific or monoclonal antibodies. Since the majority of hybridoma lines originate from inbred mouse strains that possess a very high degree of genetic similarity, DNA fingerprinting approaches are ineffective in establishing uniqueness amongst hybridoma cell lines. While direct protein sequencing of secreted immunoglobulins is doable, this method is expensive and requires extensive expertise in protein analysis. An alternative approach for authenticating monoclonal antibody identity is to use the protein coding mRNA as a surrogate for the unique antibody protein. Using PCR-based cloning methodologies that compensate for the sequence diversity seen in the immunoglobulin gene family, complementary DNA (cDNA) that encodes for the heavy and light chains of the antibody secreted by each unique hybridoma clone can easily be generated directly from IgG mRNA and sequenced. The resulting cDNA sequence is a unique identifier for that antibody and can be used to confirm the identity of the corresponding hybridoma. Thus, when hybridomas are shared across research laboratories, investigators can confirm they have indeed received the “correct” hybridoma by sequencing the cDNA and matching the sequence obtained with the expected sequence record. In addition, obtaining the cDNA sequence enables one to predict the primary amino acid sequence of the heavy and light chain variable regions of the IgG protein. This information can be used to derive 2- and 3-dimensional models of the antigen binding region of the antibody protein.
The ICBR has performed a number of pilot studies using total RNA purified from hybridoma cell lines and degenerate primers designed in house in standard reactions to successfully clone the variable regions of both the heavy and light chain regions of monoclonal antibody cDNAs. This method is quite robust and allows one to also predict protein primary sequence and predicted secondary structure from the corresponding cDNA. The cost for sequencing the heavy and light chain variable regions is $750, and requires the submission of 2.0 µgm of total RNA (RIN>7.0; A260/280 1.8-2.0). Alternatively, a pellet of frozen hybridoma cells (> 5 x 105) can be submitted for RNA extraction at a minimum cost of $50.00. Turnaround time from sample submission to sequence delivery is 2-4 weeks.
If you are interested in this service please contact Dr. Yanping Zhang (firstname.lastname@example.org; 352-273-8043), Dr. Angel Sampson (email@example.com; 352-273-8039), or Dr. Steven Madore (firstname.lastname@example.org; 352-273-8038).