Accelerating Antibody Discovery with the WOLF G2 Cell Sorter

Kirsten rat sarcoma viral oncogene homolog (KRAS) is the most mutated oncogene in human cancers, found in approximately 30 % of tumors. These mutations primarily consist of single-base missense alterations in codon G12. While extensive efforts have focused on developing allele-specific inhibitors for KRAS mutations, mutation-specific antibodies (Abs) remain largely unexplored, with only a few research-use-only catalog Abs available. In this study, we employed the proprietary Epivolve technology to develop site-directed monoclonal Abs (mAbs) that target KRAS oncogenic driver mutation KRAS G12D. These site-directed mAbs demonstrate high binding affinity, with equilibrium dissociation constants (KD) in the nanomolar range, showing over 1,000-fold greater affinity for KRAS G12D compared to wild-type KRAS. Western blot analyses using both purified KRAS protein variants and tumor cell lines harboring G12D mutations confirmed the high specificity of these mAbs. Furthermore, immunocytochemistry analysis revealed co-localization of the site-directed mAbs with endogenously expressed KRAS in cancer cells bearing G12D mutations. The validated high affinity and specificity of these site-directed mAbs highlight their potential for diagnostic applications and therapeutic development targeting KRAS driver mutations.

How the WOLF was used in this study
Li et al., New Biotechnology 2025 demonstrates the use of WOLF sorting during early antibody discovery steps. In this study the authors developed site-directed mAbs targeting the KRAS G12D driver mutation, using flow cytometry to enrich live, marker-defined B cell populations before characterization of binding specificity. The WOLF sorter helped isolate phenotypically defined populations critical for validating their antibody candidates prior to downstream biochemical assays.

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