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.