Miniature single-particle immunoassay for prostate-specific antigen in serum using recombinant Fab fragments

Background: Quantitative, miniaturized nucleic acid assays and immunoassays can be developed with single microparticles, microfluorometric detection, and intrinsically fluorescent lanthanide chelates in a multiple assay forma t to decrease reagent consumption, cost, and assay time. We used recombinan t Fab fragments to capture and detect free and total prostate-specific anti gen (PSA) from serum in a submicroliter volume single-particle immunoassay. Methods: Genetically engineered thiol-Fab or thiolated monoclonal antibodie s (mAbs) were covalently attached onto uniformly sized 60-mum maleimide-act ivated microparticles. Free and total PSA were detected with europium- or t erbium-labeled Fab fragments on a single microparticle using a microfluorom eter in a time-resolved mode. Results: The detection limit of the free- and total-PSA assays (mean + 3 SD of zero calibrator) was 0.35 mug/L, with a total volume of 330 nL per part icle. An excellent correlation was found in microparticle and microtiter-we ll assays for 21 serum samples: slopes for free and total PSA were 1.06 +/- 0.03 and 1.03 +/- 0.02, respectively (S-y/x = 0.084 and 0.057 mug/L), with intercepts of 0.013 +/- 0.018 and 0.013 +/- 0.017 mug/L (R > 0.99). Furthe rmore, the particle-immobilized Fab fragment had a PSA binding capacity 1.5 -fold higher than the intact mAb capacity on a single microparticle. Capaci ty, kinetics, and sensitivity of the Fab fragment and intact mAb assays in the microparticle and microtiter well formats are discussed. Conclusions: With site-specific (cysteine tail) covalent attachment of Fab fragments on a microparticle, subattomole amounts of PSA can be detected qu antitatively. (C) 2000 American Association for Clinical Chemistry.