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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
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