Zeptomole detection sensitivity of prostate-specific antigen in a rapid microtitre plate assay using time-resolved fluorescence.
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.
Härmä
H
H
Department of Biotechnology, University of Turku, Tykistökatu 6, FIN-20520 Turku, Finland.
Soukka
T
T
Lönnberg
S
S
Paukkunen
J
J
Tarkkinen
P
P
Lövgren
T
T
D000911:Antibodies, Monoclonal; D001710:Biotin; D005063:Europium; D015200:Fluoroimmunoassay / Q000379:methods* / Q000706:statistics & numerical data; D006801:Humans; D008297:Male; D008863:Microspheres; D017430:Prostate-Specific Antigen / Q000032:analysis*; D012680:Sensitivity and Specificity; D019809:Streptavidin
D000911:Antibodies, Monoclonal; D005063:Europium; D001710:Biotin; D019809:Streptavidin; D017430:Prostate-Specific Antigen