Simultaneous Multianalyte ELISA Performed on a Microarray Platform

Abstract

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.

Solid supports for microarray immunoassays

“…Mini-arrays of up to 250 spots were printed onto the bottom of a well (Eggers et al, 1994 (High Throughput Genomics, Inc., Tucson, USA); Mendoza et al, 1999). While most such microspot immunoassays were performed on only few antibodies per well (Moody et al, 2001;Wiese et al, 2001;Tam et al, 2002), there are commercial products with up to 50 antibodies (Eggers et al, 1994). The main problem of this approach lies with the chemiluminescence usually used for detection.…”

“…However, the attainable sensitivity and dynamic range of detection on these microarrays are considerable in comparison to conventional ELISA assays. Mini-arrays of less than 10 elements were used to screen cytokines in different biological samples with a sensitivity of a few pg/ml Moody et al, 2001;Wiese et al, 2001;Tam et al, 2002). Besides the conventional polystyrene surface (Moody et al, 2001), micro-wells were coated with cyanosilane in several studies Wiese et al, 2001;Tam et al, 2002).…”

“…Mini-arrays of less than 10 elements were used to screen cytokines in different biological samples with a sensitivity of a few pg/ml Moody et al, 2001;Wiese et al, 2001;Tam et al, 2002). Besides the conventional polystyrene surface (Moody et al, 2001), micro-wells were coated with cyanosilane in several studies Wiese et al, 2001;Tam et al, 2002). By this means, the antibodies were bound electrostatically via the glycosyl rests of their Fc regions (Falipou et al, 1999).…”

“…The same chemistry was used to coat slides with avidin and then bind randomly biotinylated antibodies (Rowe-Taitt et al, 2000;Wiese et al, 2001;Delehanty and Ligler, 2002) or Fab fragments biotinylated in the hinge region (Rowe et al, 1999). Streptavidin/avidin interaction with biotin, the strongest non-covalent binding process known with a binding affinity constant of about 10 15 l/mol, is often used for immunoassay construction (Schetters, 1999).…”

Simultaneous Multicolor Detection System of the Single‐Molecular Microbial Antigen with Total Internal Reflection Fluorescence Microscopy

“…In general technique, detection was dependent on the secondary antibody that combines biotin or that were the specific antibody to the immunoglobulin of an immunized animal (e.g. ; antihuman IgG), and then finally detected by the colorimetric substrate, the chemiluminescent reagents, or the fluorescence such as Alexa ® fluor or CyChrome ® dyes (26). Enhancement of assay performance has been achieved by several methods.…”