Integrated isotyping and CDR identification of mouse monoclonal antibodies using multiplex RT-PCR

The complementarity-determining regions (CDRs) of monoclonal antibodies are essential for antigen recognition and antibody engineering. Accurate determination of CDR sequences typically requires cDNA synthesis from hybridoma-derived mRNA followed by sequencing of the variable regions. However, murine monoclonal antibodies are composed of diverse heavy and light chain isotypes, necessitating prior isotype determination to select appropriate primers for cDNA synthesis. Conventional workflows rely on immunoassays for isotype identification, which adds time and complexity. Here, we developed a streamlined, isotype-independent workflow for the molecular characterization of mouse monoclonal antibodies. A multiplex set of reverse transcription primers (Multiplex-RT) incorporating a universal adaptor sequence was designed to enable cDNA synthesis across major murine isotypes without prior isotype knowledge. Variable regions were subsequently amplified by isotype-specific PCR (Iso-PCR), allowing identification of antibody isotypes, IgG subclasses, and CDR sequences in a single workflow. We applied this method to characterize a murine antibody targeting the astrocytic membrane protein MLC1 and engineered a human-mouse chimeric antibody by grafting murine CDRs onto a human IgG1 backbone. The chimeric antibody retained antigen-binding activity, as demonstrated by immunoprecipitation and immunoblotting. This workflow provides a rapid and reliable strategy for sequencing and isotyping mouse monoclonal antibodies and facilitates downstream applications in antibody discovery, recombinant production, and engineering.