In 1992 Sano et al. developed immuno-PCR, which is an immunoassay similar to ELISA but uses PCR for the detection instead of a colorimetric enzyme reaction. They constructed a streptavidin-protein A chimera that was bound to a biotinylated linear plasmid. The protein A-streptavidin-DNA-conjugate bound the detection antibody which was connected to the analyte in the microtiter plate. The DNA was then amplified by PCR. The PCR product was finally detected by gel electrophoresis (Sano et al., 1992). In an alternative setup the amplified PCR product was detected by PCR ELISA (Niemeyer et al., 1997). Here biotin labeled PCR primers and a digoxigenin labeled nucleotide was used. Following PCR the products were immobilized in streptavidin coated microtiter plates and analyzed by ELISA using digoxigenin IgG-alkaline phosphatase conjugate. These post-PCR analysis methods are time consuming and cross-contamination is a major problem. Further drawback is that the PCR is run to completion resulting in loss of linearity. A much easier, faster and more convenient approach is to use real-time PCR for direct quantification of the DNA (Figure 12.1). This was first demonstrated by Sims et al., 2000. Compared to ELISA, real-time immuno-PCR is more sensitive and has much larger dynamic range (Figure 12.2). These advantages are due to the fundamentally different signal generation and detection processes. In ELISA the detection antibody is coupled to an enzymatic system that generates linear signal increase with time, while in real-time immuno-PCR the detection antibody is coupled to a nucleic acid template that generates an exponential signal growth in time
Schematic set-up of real-time immuno-PCR and ELISA.
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Comparison of real-time immuno-PCR (squares) and ELISA (triangles) on standard samples. Reprinted from Journal of Immunological Methods, 304, 107-116, Figure 5 with permission from Elsevier B.V.
when amplified by PCR. Further, ELISA signal is read out as intensity value at the end of the reaction, while in real-time PCR the number of amplification cycles to reach a particular signal level is registered.
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