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

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Abstract
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 β-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 ε-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.