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