Isolation, cloning and analysis of parvovirus-specific canine antibodies from peripheral blood B cells

B-cell cloning methods enable the analysis of antibody responses against target antigens and can be used to reveal the host antibody repertoire, antigenic sites (epitopes), and details of protective immunity against pathogens. Here, we describe improved methods for isolation of canine peripheral blood B cells producing antibodies against canine parvovirus (CPV) capsids by fluorescence-activated cell sorting, followed by cell cloning. We cultured sorted B cells from an immunized dog in vitro and screened for CPV-specific antibody production. Updated canine-specific primer sets were used to amplify and clone the heavy and light chain immunoglobulin sequences directly from the B cells by reverse transcription and PCR. Monoclonal canine IgGs were produced by cloning heavy and light chain sequences into antibody expression vectors, which were screened for CPV binding. Three different canine monoclonal antibodies were analyzed, including two that shared the same heavy chain, and one that had distinct heavy and light chains. The antibodies showed broad binding to CPV variants, and epitopes were mapped to antigenic sites on the capsid. The methods described here are applicable for the isolation of canine B cells and monoclonal antibodies against many antigens.

Automated summary

B-cell cloning methods were used to analyze antibody responses against target antigens, reveal host antibody repertoire and protective immunity against pathogens. Improved methods for isolating canine B cells and producing monoclonal antibodies against canine parvovirus (CPV) capsids were described. The methods included fluorescence-activated cell sorting, in vitro B cell culture, screening for CPV-specific antibody production, and cloning of heavy and light chain sequences. Three different canine monoclonal antibodies were analyzed, showing broad binding to CPV variants and mapping of antigenic sites on the capsid.