Dual-label time-resolved immunofluorometric assay for simultaneous determination of pregnancy-associated plasma protein A and free β-subunit of human chorionic gonadotrophin

Abstract

Using time-resolved fluorometry, a simple one-step dual-label immunometric assay has been developed, which allows simultaneous determination of pregnancy-associated plasma protein A (PAPP-A) and free β-subunit of human chorionic gonadotrophin (β-hCG) in first-trimester maternal serum samples. Two monoclonal antibodies were biotinylated and immobilized onto the surface of streptavidin-coated microtitration plates, and used to capture PAPP-A and β-hCG, respectively. Europium (Eu) and Samarium (Sm) chelates were conjugated to two additional monoclonal antibodies acting as detection antibodies for PAPP-A and β-hCG. The assay was performed using a 4-h one-step format. The within-run precision with buffer-based calibrators was below 8% over the working range of PAPP-A (40–10 000 mIU/l) and β-hCG (7.3–525 μg/l) and no hook effect was observed. The intra- and inter-assay coefficients of variation were below 7.1% for serum samples. PAPP-A and β-hCG concentrations measured by the dual assay in 39 first-trimester serum samples correlated excellently with those obtained by DELFIA single-label PAPP-A ( r=0.997) and the β-hCG part ( r=0.993) of the DELFIA AFP/βhCG dual-label assay.