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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 rimer (5'-ACATATCCAGCCATGCACAC- 3'), and was purified with the QIAquick PCR purification kit (QIAGEN).

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 g/L with phosphate buffer containing BSA and immunoglobulins to reduce nonspecific adsorption (CanAg PSA EIA instructions).

To each well, 25 l of the diluted biotinylated anti-PSA66 was added and the samples were incubated for 1 hr at room temperature. After washing six times with wash buffer 1, the wells were incubated for 30 min at room temperature with 25 l streptavidin solution (0.3 g/ml streptavidin in 10 mM Tris-HCl, pH 7.5, 50 mM KCl, 2.5 mM MgCl2). Thereafter, the samples were washed six times with wash buffer 1 and incubated with 25 l of biotinylated DNA marker (15 pM). The wells were then washed six times with wash buffer 1, followed by four times with wash buffer 2 (0.154 M NaCl, 5 mM Tris, pH 7.75), and then left to stand in wash buffer 2 for 1 hr before removing the buffer. Finally, the PCR mix was added to the wells and real-time PCR was performed in the iCycler iQ system (Figure 2). Approximately 1520 cycles were needed to reach threshold, set at 10,000 relative fluorescence units (RFU), which was substantially above the background but still in the exponential growth phase of the PCR. The CT vs. log (concentration) plot was linear with a large correlation coefficient (r = 0.986), showing that the assay correctly and accurately reflects the amount of PSA in the studied range. However, PCR products that appear in negative control samples limit sensitivity. Analysis revealed two sources of these products. One is formation of primer-dimers, which form independently of the presence of template. There are several ways to suppress their formation through assay optimization techniques. The second source of error is nonspecific adsorption of the detection antibody or DNA. This i s common to standard ELISA and can be reduced by proper blocking of the surface or by binding the capture antibody more tightly to allow for more extensive washing. These approaches are being developed.

The results presented here and also those reported previously (Sims et al. 2000) are promising indications that applications of real-time PCR will expand to protein detection. A challenging future task will then be to quantitate the levels of both mRNA and the corresponding protein in only a few cells, or perhaps even a single cell, to study the correlation between gene and protein expression.


Acknowledgements
We thank CanAg Diagnostics for supplying antibodies, antigens, and buffers. We also thank our colleagues at the TATAA Biocenter for valuable discussions.


References
CanAg PSA EIA Instructions, Product No. 300-10, June 2000. Available from CanAg Diagnostics, www.canag.se

Sano T et al., Immuno-PCR: very sensitive antigen detection by means of specific antibody-DNA conjugates, Science 258, 120122 (1992)

Sims PW et al., Immunopolymerase chain reaction using real-time polymerase chain reaction for detection, Anal Biochem 281, 230232 (2000)


Practice of the patented polymerase chain reaction (PCR) process requires a license. The iCycler iQ system includes a licensed thermal cycler and may be used with PCR licenses available from Applied Biosystems. Its use with authorized reagents also provides a limited PCR license in accordance with the label rights accompanying such reagents. Some applications may require licenses from other parties.

Germall is a trademark of ISP Chemicals, Inc. JumpStar t is a trademark of Sigma-Aldrich Corporation. SYBR is a trademark of Molecular Probes, Inc. Tween is a trademark of ICI Americas, Inc.


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