Real-time PCR was performed in the Bio-Rad iCycler iQ system. The primers used were 5'-GTTAGCCGGGCTGCACTC- 3' and 5'-ACATATCCAGCCATGCACAC-3', which produced a 71 bp product. Reaction volumes were 25 l, containing 1x PCR buffer (Sigma), 4 mM MgCl2 (Sigma), 200 M dNTPs (Sigma), 0.08 g/l BSA (Fermentas), 1 U JumpStart Taq polymerase (Sigma), 300 nM of each primer, and 0.5x SYBR Green I (Molecular Probes, Inc.) Cycling conditions were 95C for 3 min, followed by 40 cycles of 95C for 20 sec, 60C for 20 sec, and 72C for 25 sec. The fluorescence data used for quantitation were collected at the end of each 72C step.
Results and Discussion
The sandwich real-time immuno-PCR system was assembled in a standard PCR plate from Bio-Rad as shown in Figure 1. To each well, 50 l anti-PSA10 (10 g/ml in 0.2 M phosphate buffer) was added and allowed to adsorb to the surface of the PCR plate overnight. The wells were then washed three times with wash buffer 1 (0.154 M NaCl, 5 mM Tris, pH 7.75, 0.005% Tween 20, 0.1% Germall II), and the surface was then blocked from further adsorption by incubating at room temperature overnight with blocking buffer (50 mM Tris, pH 7.0, 6% D-sorbitiol, 0.1% BSA, 0.05 % NaN3). The wells were washed with wash buffer 1, followed by incubation at room temperature with 5 l of PSA standards (containing 2.4 x 106 to 2.8 x 109 molecules) and 20 l Tris-HCl buffered salt solution containing BSA for 1 hr (CanAg PSA EIA instructions). The samples were then washed three times with wash buffer 1. Biotinylated anti-PSA66 was diluted to 0.92