Novel ELISA Using Intracellularly Biotinylated Antigen for Detection of Antibody Following DNA Immunization

Abstract

DNA immunization or vaccination, which refers to the injection of DNA encoding the corresponding antigen proteins, has become an attractive method for inducing the production of antibodies (Abs) in animals, since it does not require proteins as antigens. However, a method for detecting Abs produced in response to antigens is still essential for the quantification of Abs in the sera of immunized animals and for the screening of monoclonal antibody (mAb)-producing hybridomas. Here, we report a new system for the evaluation of Abs against antigens that are difficult to purify, by employing intracellular biotinylation of the antigen protein. The antigen tagged with a peptide to be biotinylated (Bio-tag) and codon-optimized bacterial BirA biotin ligase were co-expressed in mammalian cells, and the biotinylated Bio-tagged antigen was captured on a streptavidin-coated plate. Abs against five human nuclear antigens that were difficult to purify as full-length recombinant proteins were detected in the sera of DNA-immunized mice, and IgG mAbs against three of these antigens were selected by ELISA. The results demonstrate that this system employing intracellular biotinylation of the antigen is a powerful technique for stimulating the production of Abs following DNA immunization.