Detection of Clostridium botulinum neurotoxin type A using immuno-PCR

Aims: An immuno-polymerase chain reaction (immuno-PCR) has been developed f or the sensitive detection of antigens, which greatly extends the detection limits of immunoassays. In the current study, the method was applied to th e detection of Clostridium botulinum neurotoxin type A (BTx-A). Methods and Results: Anti-BTx-A antibody-DNA conjugates were synthesized us ing a heterobifunctional cross-linker reagent to covalently link the report er DNA and the antibodies. The antibody-DNA conjugates with antigens were a mplified by PCR, and dose-dependent relationships for each analyte were dem onstrated. Detection limits of immuno-PCR for BTx-A (3.33 x 10(-17) mol) ex ceeded the conventional enzyme-linked immunosorbent assay (3.33 x 10(-14) m ol) by a 1000-fold enhancement in detection sensitivity. Conclusions: Detection of BTx-A antigens by immuno-PCR demonstrated 100% se nsitivity and 100% specificity in 100-fold magnitude below the detection li mit of ELISA. Significance and Impact of the Study: It is concluded that the immuno-PCR m ethod could be used to detect a very low level of BTx-A for clinical diagno sis.