RESUMEN
Prostate-specific antigen (PSA) was detected in microtitre wells coated with a PSA-specific antibody using biotinylated antibody and streptavidin-coated, highly fluorescent 107 nm nanoparticles, which contained more than 30000 europium ions entrapped by beta-diketones. PSA was monitored directly on the surface of a well without any additional enhancement step. The sensitivity of the assay was 1.6 ng/L, corresponding to 50 fmol/L or 250 zeptomoles (250 x 10(-21) mol/L) of PSA. The high specific activity and low non-specific binding of the streptavidin-coated nanoparticles improved the sensitivity of the PSA assay 100-fold compared to the conventional europium-labelled streptavidin tracer in the same assay format. Additionally, the streptavidin-coated nanoparticle label made very rapid assays possible, due to the high affinity of the streptavidin-biotin complex and a high number of binding sites available for tracing the biotinylated antibody on the surface. Due to the inherent problems of tracing analyte with a complex of biotinylated antibody and streptavidin-coated nanoparticles, the streptavidin-coated nanoparticles reacting with the surface-captured analyte and biotinylated antibody was favoured and factors influencing this are discussed. This universal labelling technology can be applied to detect any biotinylated molecule, either in solution or on a solid phase, in order to improve detection sensitivities in many areas of biochemical analysis, such as cyto- and histochemistry, multianalyte DNA-chip assays and single-particle assays.
Asunto(s)
Fluoroinmunoensayo/métodos , Antígeno Prostático Específico/análisis , Anticuerpos Monoclonales , Biotina , Europio , Fluoroinmunoensayo/estadística & datos numéricos , Humanos , Masculino , Microesferas , Sensibilidad y Especificidad , EstreptavidinaRESUMEN
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 format to decrease reagent consumption, cost, and assay time. We used recombinant Fab fragments to capture and detect free and total prostate-specific antigen (PSA) from serum in a submicroliter volume single-particle immunoassay. METHODS: Genetically engineered thiol-Fab or thiolated monoclonal antibodies (mAbs) were covalently attached onto uniformly sized 60-microm maleimide-activated microparticles. Free and total PSA were detected with europium- or terbium-labeled Fab fragments on a single microparticle using a microfluorometer 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 microg/L, with a total volume of 330 nL per particle. An excellent correlation was found in microparticle and microtiter-well 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 microg/L), with intercepts of 0.013+/-0.018 and 0.013+/-0.017 microg/L (R>0.99). Furthermore, the particle-immobilized Fab fragment had a PSA binding capacity 1.5-fold higher than the intact mAb capacity on a single microparticle. Capacity, 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 quantitatively.