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Abstracts of Oak Ridge Posters |
1 ISEAO Technologies, 2 Royal College Street, London, NW1 0NH, United Kingdom
aauthor for correspondence: fax 44-20-7691-2036, e-mail sharon.banin{at}microsens.co.uk
There have been many reports describing the combination of immunoassay techniques and nucleic acid amplification using antibodies labeled directly with nucleic acids (the "direct" immuno-PCR approach) (1)(2)(3). The rationale behind immuno-PCR is to develop ultrahigh-sensitivity labels by exploiting the extremely high productivity of nucleic acid amplification techniques, coupled with highly sensitive approaches to detect the amplified material. Specific applications in which the use of ultrahigh-sensitivity immuno-PCR techniques have been proposed include prion protein detection, in which there is no nucleic acid associated with the infectious agent, and the detection of viral antigens in blood bank screening applications, in which there can be very little viral nucleic acid present at certain stages of the infection.
Although the principle of immuno-PCR has been demonstrated in various research applications, there are continuing concerns over both the technical difficulty of linking nucleic acids directly to antibodies (or "adapter" proteins such as streptavidin) and the substantial backgrounds that can be generated by nonspecific binding of the nucleic acid–antibody conjugates to solid phases (2)(4).
We have developed an alternative approach based on an "indirect" immuno-PCR technique that links ELISA techniques and PCR. Because this approach works in a completely different way to traditional immuno-PCR, it may circumvent some of the problems associated with the traditional approach. Here we present the proof of principle of our new approach.
We used a double stranded 5'-phosphorylated DNA (dsDNA) substrate for alkaline phosphatase (AP). Shown in Fig. 1A
is a schematic of the principle of the enzyme assay method. The procedure takes advantage of the substrate specificity of 
exonuclease for the 5'-phosphorylated form of dsDNA (5). In an immunometric assay, the AP in the antibody conjugate will remove the 5' phosphate groups, and the DNA is not then degraded by subsequent exonuclease treatment. PCR, or any other dsDNA amplification technique, can then be used to amplify and detect this nondegraded, "protected" dsDNA. In the absence of AP, the DNA is not protected from the exonuclease, is degraded, and therefore is not detected by PCR. Thus, in an ELISA format, protection of the dsDNA and PCR amplification/detection are indicative of the presence of antibody–AP conjugate bound to captured antigen.
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Because each molecule of phosphatase will generate many molecules of detectable dsDNA product, which subsequently enter the PCR process, this indirect immuno-PCR method has effectively two amplification steps, thus giving an increased signal over that of standard direct immuno-PCR.
To prove the principle and assess the sensitivity of our approach for detection of AP, we assayed serial dilutions of unconjugated AP by our method. A 285-bp 5'-phosphorylated dsDNA substrate for the enzyme was first generated from the pUC19 (New England Biolabs) DNA template by a standard PCR process with the 5'-phosphorylated primers (MWG Biotech) PS1 (5'-P-GGCGAAAGGGGGATGTGCTGCAAGG-3') and PAS1 (5'-P-GTGAGCGCAACGCAATTAATGTGAG-3') and Taq polymerase supplied by Qiagen Ltd. We have previously found that the method can be adversely affected by the quality of the DNA substrate used; any nonphosphoryled DNA that contaminates the phosphorylated DNA substrate in the assay cannot be degraded by the exonuclease and will generate a signal even in the absence of AP. To reduce this signal, or "noise", several steps were taken to ensure that the DNA substrate was highly phosphorylated, and therefore degradable by the exonuclease, before use: the DNA substrate was prepared with polyacrylamide-gel-purified oligonucleotide primers to ensure that the primers used to prepare the substrate were highly phosphorylated; and after PCR, the DNA substrate was agarose-gel-purified and treated with T4 polynucleotide kinase (New England Biolabs). These measures decreased the background of noise in the assay, and enabled us to detect AP activity with high sensitivity.
Serial 10-fold dilutions of calf intestinal AP (New England Biolabs) were prepared and incubated with the phosphorylated dsDNA substrate (final concentration in the reaction of 1 ng/µL) for 1 h at 37 °C in Buffer 3 (from the enzyme supplier). We then added 0.5 U of exonuclease (New England Biolabs), and continued the incubation for 1 h. We then amplified 5µL of each reaction mixture in a standard "detection" PCR process using the PS1 and PAS1 primers. The amplification was carried out with an initial denaturation step at 94 °C for 5 min and subsequently 25 thermal cycles of the following profile: 94 °C for 30 s, 62 °C for 30 s, and 72 °C for 30 s.
Shown in Fig. 1B
are the PCR products from the detection step, separated by agarose gel electrophoresis and stained with ethidium bromide. Visual inspection showed that even with our steps to ensure that the DNA substrate was highly phosphorylated, some molecules could not be degraded by the exonuclease and even in the absence of AP generated a background signal or noise. However, the signal generated by dilutions of AP were significantly greater than this background noise and enabled us to detect as few as 10–11 U of AP, which is approximately equivalent to 10 x 10–18 g, or as few as 60 molecules of AP in our assay. This visual interpretation was confirmed by quantification of the PCR products with the dye SYBR Green I (Biogene) and the fluorescence of the dye–DNA complex detected by a fluorescence microplate reader (Molecular Devices Ltd.). As a comparison, the detection limit when we used p-nitrophenylphosphate colorimetric substrate (Sigma) was 10–4 U of AP. These results demonstrate proof of principle of our novel approach and show that the indirect immuno-PCR methodology can be used to detect and quantify AP activity with very high sensitivity.
We also compared the detection limit of our approach with that of the highly sensitive AMPAK system (Dako Ltd.), an ultrasensitive amplification colorimetric system for AP that is comparable in its sensitivity to chemiluminescence, for the detection of AP and found that this indirect immuno-PCR is 100-fold more sensitive.
To calculate the reproducibility and precision of our assay, we used SYBR Green I to quantify the PCR products seen on our gels as described previously. Multiple assays were performed to detect 10–6 U of free AP. The intraassay CV of the signal was 7.4% (n = 8).
We then used the indirect immuno-PCR assay to analyze serial dilutions of commercial AP–antibody conjugates to confirm that the enzyme, in this configuration, was still able to use the 5'-phosphorylated dsDNA substrate. For this study, an anti-mouse IgG–AP conjugate (Sigma) and an anti-Chlamydia AP conjugate (DakoCytomation Ltd.) were used. Our results showed that the indirect immuno-PCR assay had a detection limit more than 100 000-fold lower than the standard colorimetric pNPP substrate and at least 1000 times lower than the AMPAK detection system with these conjugates (data not shown). This detection limit was comparable to that seen for free AP.
We also confirmed that the indirect immuno-PCR method functions well in a complete microplate ELISA procedure for Chlamydia (DakoCytomation Ltd.; data not shown), and optimization of this protocol is ongoing to measure sensitivity.
In summary, we have demonstrated proof of principle of our novel indirect immuno-PCR technique that links the presence of AP conjugate to PCR amplification. Because the technique works in a way completely different from that of traditional immuno-PCR, it offers an alternative approach and may circumvent some of the common problems associated with traditional immuno-PCR. Work is ongoing to fully exploit the potential of this new approach. One exciting development is the adaptation of the indirect immuno-PCR detection method to real-time amplification and detection PCR formats to replace the cumbersome gel electrophoresis procedure. This could allow the direct coupling of fully automated PCR instrumentation to existing immunoassay processing systems, thereby providing an opportunity to combine immunoassay and nucleic acid techniques in one instrument platform.
References
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