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 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.
Härmä
H
H
Department of Biotechnology, University of Turku, Tykistökatu 6A, FIN-20520 Turku, Finland. harri.harma@utu.fi
Tarkkinen
P
P
Soukka
T
T
Lövgren
T
T
D000911:Antibodies, Monoclonal; D002138:Calibration; D002614:Chelating Agents; D005456:Fluorescent Dyes; D006801:Humans; D007118:Immunoassay; D007140:Immunoglobulin Fab Fragments; D007700:Kinetics; D008674:Metals, Rare Earth; D008832:Microchemistry; D008863:Microspheres; D017430:Prostate-Specific Antigen / Q000097:blood* / Q000276:immunology; D011994:Recombinant Proteins; D012680:Sensitivity and Specificity; D013438:Sulfhydryl Compounds
D000911:Antibodies, Monoclonal; D002614:Chelating Agents; D005456:Fluorescent Dyes; D007140:Immunoglobulin Fab Fragments; D008674:Metals, Rare Earth; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D017430:Prostate-Specific Antigen