Development and characterization of anti-G12V mutated K-Ras monoclonal antibodies using hybridoma technology

Abstract

Background. Kirsten rat sarcoma oncogene, KRAS, is a gene that encodes for K-Ras protein, a membrane-anchored protein which is involved in intracellular signal transduction. Cells harbouring KRAS mutations have been reported to progress toward cancer development and several studies have suggested the importance of KRAS mutational screening prior to cancer treatment. However, currently available diagnostic methods are costly, time consuming and laborious. Thus, monoclonal antibodies are proposed as an alternative in K-Ras mutational testing. Objective. This study aims to generate and characterise anti-K-Ras monoclonal antibodies based on their specificity and sensitivity towards G12V-mutated K-Ras proteins, which is one of the most commonly mutated KRAS isoforms. Methods. Mice were first immunised with G12V-mutated K-Ras peptides and spleens were harvested. Hybridoma cells which secreted K-Ras-specific antibodies were generated by fusing splenocytes with X63-Ag 8.6539 myeloma cells. Hybridoma polyclonal wells secreting G12V-mutated K-Ras antibodies were subcloned into a single clone producing monoclonal antibodies. The specificity and sensitivity of monoclonal antibodies were evaluated by ELISA and the reactivity of monoclonal antibodies were tested using immunoblotting and immunocytochemistry. Results. The cross-reactivity results indicated that anti-G12V monoclonal antibodies developed in this study is highly specific to G12V mimotopes with a cross-reactivity of 4.2-16.7% towards wild-type and other mutated K-Ras isoforms. The limit of detection of this monoclonal antibody was determined as 3.28 μg/mL. It was also found to be reactive in immunocytochemical assays and native G12V-mutated K-Ras in immunoblotting. Conclusion. A highly specific and sensitive monoclonal antibody was successfully developed, characterised and applied to several assays such as indirect ELISA, Western Blot and immunocytochemistry. These data indicate the potential for this anti-G12V KRAS monoclonal antibody to be further developed for use in various research, diagnostic and therapeutic applications.