Detection of Clostridium botulinum neurotoxin type A using immuno-PCR

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Abstract
Aims: An immuno-polymerase chain reaction (immuno-PCR) has been developed for the sensitive detection of antigens, which greatly extends the detection limits of immunoassays. In the current study, the method was applied to the detection of Clostridium botulinum neurotoxin type A (BTx-A). Methods and Results: Anti-BTx-A antibody-DNA conjugates were synthesized using a heterobifunctional cross-linker reagent to covalently link the reporter DNA and the antibodies. The antibody-DNA conjugates with antigens were amplified by PCR, and dose-dependent relationships for each analyte were demonstrated. Detection limits of immuno-PCR for BTx-A (3·33 × 10−17 mol) exceeded the conventional enzyme-linked immunosorbent assay (3·33 × 10–14 mol) by a 1000-fold enhancement in detection sensitivity. Conclusions: Detection of BTx-A antigens by immuno-PCR demonstrated 100% sensitivity and 100% specificity in 100-fold magnitude below the detection limit of ELISA. Significance and Impact of the Study: It is concluded that the immuno-PCR method could be used to detect a very low level of BTx-A for clinical diagnosis.