Development and evaluation of three real-time immuno-PCR assemblages for quantification of PSA

Abstract

Real-time PCR is a very sensitive technique to measure DNA concentrations. In real-time immuno-PCR, it is used as the detection system for quantification of proteins. Many ways to perform and assemble real-time immuno-PCR are possible. We have tested three different approaches for the detection of prostate specific antigen (PSA) and compared them with each other and with ELISA. We also demonstrate the applicability of real-time immuno-PCR to classify clinical PSA samples. Assemblage I is performed stepwise attaching the capture antibody to the vessel surface by adsorption and having the DNA-label linked to the detection antibody through biotin and streptavidin. In assemblage II, capture antibody is also adsorbed to the vessel surface but the detection antibody is pre-conjugated to the DNA-label. Assemblage III uses the pre-conjugated detection antibody/DNA-label but binds the capture antibody through biotin to surface immobilized streptavidin. We found assemblage II to be the most sensitive, with a detection limit of 4.8 × 10⁵ PSA molecules. This can be compared to the detection limit of the ELISA, which is 5.7 × 10⁷ molecules. Assemblage III was the most reproducible with a standard deviation (SD) of 0.21 cycles, while assemblage I was the least reproducible (SD = 0.45 cycles). The SD of assemblage II was 0.25 cycles. We conclude that using pre-conjugated detection antibody/DNA-label enhances both the sensitivity and the reproducibility of real-time immuno-PCR. Measurements of PSA in serum samples using real-time immuno-PCR correlated well with measurements performed with ELISA. The real-time immuno-PCR measurements were more sensitive and the dynamic range was larger than with the ELISA.

Introduction

Immunoassays have been used for the quantification of proteins since the 1960s and are the basis of many diagnostic applications. The diversity of antibodies has made it possible to raise antibodies for almost any protein and use them for the specific recognition of antigens in immunoassays. A number of ways to generate and detect signal from analyte bound antibodies have been developed (Yalow and Berson, 1960, Engvall and Perlman, 1971, Chan et al., 1987). During recent years, real-time PCR has become one of the most popular methods to quantify nucleic acids. The very high sensitivity of real-time PCR, which under optimal conditions detects a single molecule, makes it excellent for diagnostic purposes. So far no method developed for protein analysis has reached this sensitivity. In 1992, Sano et al. described a new technique using PCR to detect specific proteins, which they called immuno-PCR. Immuno-PCR combines the molecular specificity of antibodies with the sensitivity of PCR. Antibodies specific for the protein target are immobilized to the surface of a vessel. Sample is added and the targeted proteins are bound by the immobilized antibody molecules. A second specific antibody, coupled to a DNA molecule, is then added. This so-called detection antibody binds to a second epitope on the immobilized protein target. After careful washing to remove all unbound reagents, the DNA is amplified. During the exponential phase of the PCR, the amount of product formed reflects the amount of target protein that was bound by the antibodies.

When the above technique was first developed, the amount of PCR product was assessed either by gel electrophoresis (Sano et al., 1992, Zhou et al., 1993, Furuya et al., 2001) or by PCR-ELISA (Niemeyer et al., 1997). These rather laborious and insensitive post-PCR analytical methods limited the quantification range of immuno-PCR and thereby its applicability. Using real-time PCR instead, the amount of DNA could be quantified with high sensitivity and accuracy over a wide concentration range. This was first shown by Sims et al. (2000), who used real-time immuno-PCR to quantify vascular endothelial growth factor (VEGF). Eliminating the post-PCR step also reduced the assay time and risk of contamination.

Here we use real-time immuno-PCR to quantify prostate specific antigen, PSA. PSA is a well-known tumor marker for prostate cancer and is widely used to detect, stage, and monitor the disease (Ablin, 1997). It is a 32 kDa glycoprotein serine protease that is produced in the prostate gland. PSA levels in the blood are usually low, and elevated serum concentrations indicate disease of the prostate. We have tested the PSA real-time immuno-PCR assay on clinical samples and found that it can be used to classify samples for diagnostic purposes.

There are different ways to assemble immunoassays. Most common are sandwich assays where two specific antibodies are used to identify the protein target. This results in higher specificity than when a single antibody is used. The capture antibody can be adsorbed to the tube surface or attached through biotin to streptavidin coated microtiter plates. The detection antibody can be linked by different means to an enzyme (ELISA) or to DNA (immuno-PCR). In the procedures of Sims et al. (2000) and McKie et al. (2002), a covalent conjugate of detection antibody and DNA-label is used, while the methodologies of Adler et al. (2003), Niemeyer et al. (2003), Gofflot et al. (2004), and Barletta et al. (2004) use biotin–streptavidin coupling to attach the DNA. The Adler and Niemeyer procedures use streptavidin and bis-biotinylated DNA-label together with biotinylated detection antibody to obtain a non-covalent conjugate that is prepurified on a gel filtration column. The Gofflot method premixes streptavidin and biotinylated DNA before addition to the reaction wells. In the Barletta protocol, each assay reagent is added stepwise. In all assays other than Sims et al. (2000), the capture antibody or the antigen is directly adsorbed to the well surface. The Sims procedure uses streptavidin coated tubes to which biotinylated capture antibody is bound.

In this work, we compare three ways to assemble the real-time immuno-PCR detection system for the quantification of PSA. We test the sensitivity, reproducibility, and dynamic range. All three assemblages are sandwich assays. They differ in the way the capture antibody is attached to the surface and how the DNA is linked to the detection antibody. The assemblages are optimized separately. In assemblage I (Fig. 1a), the capture antibody is passively adsorbed to the PCR tube surface and the other assay components are added stepwise, one at a time, with incubation and washing in between. Streptavidin is used to couple biotinylated DNA-label to biotinylated detection antibody. This assemblage requires a total of six washing steps. In assemblage II, the capture antibody is also passively adsorbed to the surface, but here, the DNA is chemically conjugated to the detection antibody and premixed with the protein sample before addition to the well (Fig. 1b). This setup requires three washing steps. Assemblage III makes use of streptavidin coated PCR microtiter plates and a biotinylated capture antibody (Fig. 1c). The DNA is covalently linked to the detection antibody as in assemblage II. Capture antibody, test sample, and detection antibody/DNA-label conjugate are mixed and incubated before addition to the wells. For this assemblage, two washing steps are sufficient. The three real-time immuno-PCR assemblages are compared with each other and also with classical ELISA.