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Real-Time Immuno-PCR on the iCycler iQ System, Rev A

Kristina Lind and Mikael Kubista, TATAA Biocenter, Medicinaregatan 7A/B, 405 30 Gteborg, Sweden


Introduction
A technique for antigen detection, called immuno-PCR, was developed by Sano et al. (1992). It combines the molecular recognition of antibodies with the high DNA amplification capability of PCR. The procedure is similar to conventional enzyme-linked immunosorbent assays (ELISA) but allows for more sensitive detection. Instead of an enzyme, a DNA molecule is linked to the detection antibody and serves as a template for PCR (Figure 1). The DNA molecule is amplified and the PCR product is measured by gel electrophoresis. An improvement of this method is to amplify the oligomer in a real-time PCR instrument, thereby eliminating post-PCR analysis (Sims et al. 2000). Further, real-time PCR is extremely accurate and sensitive, which should make it possible to quantitate very low amounts of DNA-coupled detection antibody with high accuracy. Here we present early results on the development of real-time immuno-PCR for prostate specific antigen (PSA) using the iCycler iQ system. PSA is a well-known tumor marker for prostate cancer and is widely used to detect, stage, and monitor the disease.


Methods
Anti-PSA10 and anti-PSA66 from CanAg Diagnostics were used as capture and detection antibodies, respectively, in the sandwich immuno-PCR assay. Anti-PSA66 was biotinylated using biotinamido-caproate-N-hydroxysuccinimide ester. Biotinylated DNA was generated by amplifying a 1,098 bp fragment of the gusA gene (E. coli β-glucuronidase gene) with a 5'-biotinylated forward primer (biotin-AACTATGCCGGAATCCATCG- 3') and unmodified reverse p
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