Highly sensitive immunoassay of free prostate-specific antigen in serum using europium(III) nanoparticle label technology

Abstract

Background: Recent proceedings in utilization of europium(III) chelate-dyed polystyrene nanoparticles as labels have combined the advantages of an enhanced monovalent binding affinity and a high specific activity of nanoparticle-antibody bioconjugate. Our objective was to evaluate the performance of the nanoparticle label technology with biological samples in an immunoassay of free prostate-specific antigen (PSA-F) using a standard microtitration well platform.

Methods: Long-lifetime luminescent europium(III)-chelate nanoparticles, 107 nm in diameter, were coated with a PSA-F specific monoclonal antibody. The two-step noncompetitive immunoassay was performed in a microtitration well coated with a second monoclonal antibody. The signal of the surface-bound nanoparticle-antibody bioconjugates was measured directly from the bottom of the well using a standard time-resolved plate fluorometer.

Results: The detection limit (mean + 2SD) of the nanoparticle-based PSA-F assay was 0.21 ng/l using a 20-microl sample volume. The assay response was linear up to 5 microg/l, and the functional sensitivity was approximately 0.5 ng/l. The within-run imprecision for spiked serum samples at concentrations 0.0005-0.5 microg/l was 6.4-21.8%, and the within-run and between-run imprecisions for serum samples at concentrations 0.2-2.5 microg/l were 3.4-7.2% and 4.4-7.6%, respectively. The concentrations obtained from serum samples correlated well with the reference immunoassay; slope = 1.018 +/- 0.018; intercept = 0.012 +/- 0.021 microg/l; S(y/x) = 0.112 microg/l; r = 0.993; n = 51.

Conclusions: The developed method demonstrated acceptable performance characteristics allowing clinical studies utilizing patient samples with extremely low concentrations of PSA-F. The present assay detected PSA-F in most of samples from prostatectomized men and in few samples from healthy women that were nondetectable according to the reference immunoassay.