Demonstration of an alternative approach to immuno-PCR.

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There have been many reports describing the combination of immunoassay techniques and nucleic acid amplification using antibodies labeled directly with nucleic acids (the “direct” immuno-PCR approach) (1)(2)(3). The rationale behind immuno-PCR is to develop ultrahigh-sensitivity labels by exploiting the extremely high productivity of nucleic acid amplification techniques, coupled with highly sensitive approaches to detect the amplified material. Specific applications in which the use of ultrahigh-sensitivity immuno-PCR techniques have been proposed include prion protein detection, in which there is no nucleic acid associated with the infectious agent, and the detection of viral antigens in blood bank screening applications, in which there can be very little viral nucleic acid present at certain stages of the infection. Although the principle of immuno-PCR has been demonstrated in various research applications, there are continuing concerns over both the technical difficulty of linking nucleic acids directly to antibodies (or “adapter” proteins such as streptavidin) and the substantial backgrounds that can be generated by nonspecific binding of the nucleic acid–antibody conjugates to solid phases (2)(4). We have developed an alternative approach based on an “indirect” immuno-PCR technique that links ELISA techniques and PCR. Because this approach works in a completely different way to traditional immuno-PCR, it may circumvent some of the problems associated with the traditional approach. Here we present the proof of principle of our new approach. We used a double stranded 5′-phosphorylated DNA (dsDNA) substrate for alkaline phosphatase (AP). Shown in Fig. 1A⇓ is a schematic of the principle of the enzyme assay method. The procedure takes advantage of the substrate specificity of λ exonuclease for the 5′-phosphorylated form of dsDNA (5). In an immunometric assay, the AP in the antibody conjugate will remove the 5′ phosphate groups, and the DNA …