Production and epitope mapping of monoclonal antibodies against the VP2 protein of canine parvovirus and the Hexon protein of canine adenovirus

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Canine parvovirus (CPV) is a non-enveloped ssDNA viruses while canine adenovirus (CAV) is a non-enveloped dsDNA viruses. Previous studies suggested that both the VP2 protein of CPV and the hexon protein of CAV are antigenic surface proteins. In this work, we have prepared monoclonal antibodies that recognize the CPV VP1-VP2∆N225 and CAV hexon (aa 34-348) proteins. The monoclonal antibody (D11) against the CPV VP2 and four monoclonal antibodies (A8, A13, A14 and A15) against the CAV hexon (aa 34-348) were prepared. To further define the location of epitopes on the CPV VP2 and CAV hexon proteins, the full-length and deletion fragments of the CPV VP2 and CAV hexon genes generated by polymerase chain reaction (PCR) were cloned into pET32a expression vectors and the fusion proteins were overexpressed into Escherichia coli BL21 strain. Epitopes mapping using MAbs and E. coil-expressed deletion fragments of the CPV VP2 and CAV hexon. Epitope mapping suggested that the anti-CPV VP2 MAb recognizes aa 466-530, respectively and that four epitopes on the CAV hexon protein were determined by four monoclonal antibodies. Epitopes on the CAV hexon were located at aa 34-205, aa 202-208, aa 206-274 and aa 120-274, respectively. The isotypes of anti-CPV VP1-VP2∆N225 MAb and anti-CAV hexon (aa 34-348) MAbs were all IgG1. The sensitivity of monoclonal antibodies were further determined after purification by protein G agarose and the results confirmed that one anti-CPV VP2 MAb and two anti-CAV hexon (aa 34-348) MAbs recognize the epitopes located on surface of virus particles, which have high sensitivity. Taken all together, these five MAbs can be used as materials for developing immunochromatography test (ICT) strips for rapid detection of CPVs and CAVs.