Background: A logical progression of the widely used microtiter plate ELISA is toward a protein array format that allows simultaneous detection of multiple analytes at multiple array addresses within a single well. Here we describe the construction and use of such a multiplex ELISA to measure prostate-specific antigen (PSA), α1-antichymotrypsin-bound PSA (PSA-ACT), and interleukin-6 (IL-6). Methods: We silanized glass plates and printed the appropriate capture antibodies to allow for the construction of “sandwich” ELISA quantification assays. We examined specificity of the assay for appropriate antigen, assembled calibration curves, and obtained PSA concentrations for 14 human serum samples. We compared the serum PSA concentrations derived through the use of our array with values obtained independently using a standard ELISA method. Results: R 2 values generated by our microarray for the PSA and PSA-ACT calibration curves were 0.989 and 0.979, respectively. Analyte concentrations used for the construction of these curves were 0.31–20 μg of protein/L of diluent. IL-6 calibration curve concentrations were 4.9–300 ng of IL-6/L of diluent. The R2 value for the IL-6 calibration curve was 0.983. The 14 human serum samples screened by this micro-ELISA technique for PSA concentrations generated a regression equation (linear) with a slope of 0.83 ± 0.10 and intercept of 0.74 ± 0.70 (R2 = 0.88). Conclusions: Multiplexed ELISA arrays are a feasible option for analyte quantification in complex biologic samples.
The utility of several streptavidin-linked fluorescent detector molecules was evaluated on two protein microarray platforms. Tested detector molecules included: Alexa Fluor 546; R-phycoerythrin (RPE), orange fluospheres; Cy3-containing liposomes (Large Unilamellar Vesicles, LUV) labelled with Cy3; and an RPE-antibody complex. The two array architectures tested consisted of an array of murine Fc-biotin and an array of murine IgG (the murine IgG array was probed with a biotinylated rabbit anti-murine IgG). These platforms allowed for the direct comparison of detector utility by detector recognition of array-bound biotin. All of the fluorescent detectors examined demonstrated utility on each of the array platforms. For the Fc-biotin array, detector signal intensity (background adjusted) was as follows: RPE-antibody complex > fluospheres > RPE > liposomes > Alexa 546: for the IgG array: RPE/antibody complex > RPE > fluospheres > Alexa546 > liposomes. The RPE-antibody complex fluoresced 67% and 150% more intensely than the next closest detector molecule for the Fc-biotin and the murine IgG arrays, respectively. A marked increase in background fluorescence (as compared to RPE alone) did not accompany the increase in signal intensity gained through RPE-antibody complex use (a true increase in signal:noise ratio). These results suggest that the RPE-antibody complex is superior to other molecules for fluorescent detection of analytes on protein microarrays.
Background: High-density microarrays are ideally suited for analyzing thousands of genes against a small number of samples. The next step in the discovery process is to take the resulting genes of interest and rapidly screen them against thousands of patient samples, tissues, or cell lines to further investigate their involvement in disease risk or the response to medication. Methods: We used a microarray technology platform for both single-nucleotide polymorphisms (SNPs) and protein expression. Each microarray contains up to 250 elements that can be customized for each application. Slides contained either a 16- or 96-microarray format (4000–24 000 elements per slide), allowing the corresponding number of samples to be rapidly processed in parallel. Results: Results for SNP genotyping and protein profiling agreed with results of restriction fragment length polymorphism (RFLP) analysis or ELISA, respectively. Genotyping analyses, using the microarray technology, on large sample sets over multiple polymorphisms in the NAT2 gene were in full agreement with traditional methodologies, such as sequencing and RFLP analysis. The multiplexed protein microarray had correlation coefficients of 0.82–0.99 (depending on analyte) compared with ELISAs. Conclusions: The integrated microarray technology platform is adaptable and versatile, while offering the high-throughput capabilities needed for drug development and discovery applications.
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