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Clinical Chemistry 46: 1755-1761, 2000;
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(Clinical Chemistry. 2000;46:1755-1761.)
© 2000 American Association for Clinical Chemistry, Inc.

Articles

Miniature Single-Particle Immunoassay for Prostate-specific Antigen in Serum Using Recombinant Fab Fragments

Harri Härmäa,1, Piia Tarkkinen1, Tero Soukka1 and Timo Lövgren1

1 Department of Biotechnology, University of Turku, Tykistökatu 6A, FIN-20520 Turku, Finland.
a Author for correspondence. fax 358-2-3338050; e-mail harri.harma{at}utu.fi

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-µm 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 µg/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 (Sy|x = 0.084 and 0.057 µg/L), with intercepts of 0.013 ± 0.018 and 0.013 ± 0.017 µg/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.




The following articles in journals at HighWire Press have cited this article:


Home page
 Clin. Chem.Home page
P. Tarkkinen, T. Palenius, and T. Lovgren
Ultrarapid, Ultrasensitive One-Step Kinetic Immunoassay for C-Reactive Protein (CRP) in Whole Blood Samples: Measurement of the Entire CRP Concentration Range with a Single Sample Dilution
Clin. Chem., February 1, 2002; 48(2): 269 - 277.
[Abstract] [Full Text] [PDF]


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