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Clinical Chemistry 48: 473-483, 2002;
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(Clinical Chemistry. 2002;48:473-483.)
© 2002 American Association for Clinical Chemistry, Inc.

Point-of-Care Time-resolved Immunofluorometric Assay for Human Pregnancy-associated Plasma Protein A: Use in First-Trimester Screening for Down Syndrome

Qiu-Ping Qin1a, Michael Christiansen2 and Kim Pettersson1

1 Department of Biotechnology, University of Turku, Tykistökatu 6, 20520 Turku, Finland.

2 Department of Clinical Biochemistry, Statens Seruminstitut, Artillerivej 5, DK-2300 S Copenhagen, Denmark.

aAuthor for correspondence. Fax 358-2-333-8050; e-mail qiqin{at}utu.fi.

Background: Screening for Down syndrome in the first trimester by a combination of fetal nuchal translucency thickness and maternal serum pregnancy-associated plasma protein A (PAPP-A) and free ß-human chorionic gonadotropin has been shown to be effective and efficient. We aimed to develop a fast point-of-care assay that could be placed in one-stop clinics for the measurement of PAPP-A.

Methods: We developed a two-site, one-step assay that uses two monoclonal antibodies (mAbs) to PAPP-A, based on a dry-reagent, all-in-one immunoassay concept with a stable fluorescent lanthanide chelate and time-resolved fluorometry. One antibody (mAb 10E1) was biotinylated, and the other (mAb 234-5) was europium-labeled, both via the {epsilon}-amino groups of surface lysine residues. The assay was performed on an AIO immunoanalyzer at 36 °C in single, streptavidin-coated microtitration wells that contained the dry reagents. PAPP-A, either in free or complexed form, was detected by the antibodies used.

Results: The assay procedure required 20 min and used 10 µL of sample. The calibration curve was linear from 5 to 10 000 mIU/L. The detection limit was 0.5 mIU/L. Intra- and interassay imprecision (CV) was <=4.3% and 8.3%, respectively, for whole blood, plasma, or serum samples. Recovery was 93–96% for serum, 95–108% for heparin-derived whole blood, and 98–103% for heparin-derived plasma. Parallelism was observed in all three matrices. Results correlated [slope = 0.85 (confidence interval, 0.82–0.87); intercept = -33 (confidence interval, -58 to -9); Sy|x = 85 mIU/L; r = 0.991; n = 100] with those obtained by a Delfia assay. Heparin did not affect the assay, but EDTA markedly reduced PAPP-A values. PAPP-A was stable at 4 °C for at least 18 days in serum and for 8 days in heparin-derived whole blood or plasma.

Conclusions: The present assay appears suited for use in one-stop clinics for screening for Down syndrome in the first trimester, with results available within 1 h.




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