Point-of-care time-resolved immunofluorometric assay for human pregnancy-associated plasma protein A: use in first-trimester screening for Down syndrome.

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 beta-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 degrees 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 microL 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 < or = 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); S(y: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 degrees 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.