A highly sensitive immuno-polymerase chain reaction assay for Clostridium botulinum neurotoxin type A.

Our goal was to develop a sensitive method for detecting Clostridium botulinum neurotoxin type A (BoNT/A). We were able to detect BoNT/A in the femtogram (10(-15)g) range using an indirect immuno-polymerase chain reaction (immuno-PCR) assay and an indirect sandwich immuno-PCR assay. For the indirect immuno-PCR assay, enzyme-linked immunosorbent assay (ELISA) plates were coated with BoNT/A that was recognized by anti-BoNT/A monoclonal antibody. For the indirect sandwich immuno-PCR assay, the monoclonal antibody was immobilized on ELISA plates for detecting BoNT/A that was recognized by its polyclonal antibodies. Reporter DNA was prepared by PCR amplification using biotinylated 5'-primers, and it was coupled with biotinylated antibodies through streptavidin. In order to increase sensitivity and reduce background noise, the amounts of reporter DNA (ranging from 50 fg to 50 ng) and streptavidin (ranging from 0.125 ng to 8 ng) were optimized. Using the optimized concentration of reporter DNA and streptavidin, both indirect and indirect sandwich immuno-PCR assays detected BoNT/A as low as 50 fg. These results are a 10(5)-fold improvement over conventional indirect ELISA and indirect sandwich ELISA methods. The assays we developed are currently the most sensitive methods for detecting BoNT/A.