Direct Immunoassay for Free Pregnancy-Associated Plasma Protein A (PAPP-A).

BACKGROUND: Pregnancy-associated plasma protein A (PAPP-A), especially in its noncomplexed form (fPAPP-A), is linked to vulnerable atherosclerotic plaques and risk of cardiac events. An assay for sensitive detection of fPAPP-A has been lacking. Our aim was to develop and validate a direct fPAPP-A assay to meet this need. METHODS: Monoclonal antibodies binding exclusively fPAPP-A were produced by immunizing mice with recombinant PAPP-A. In the optimized immunoassay, we used an fPAPP-A-specific capture antibody together with a lanthanide-chelate-labeled monoclonal antibody recognizing all PAPP-A forms. The assay was evaluated with CLSI guidelines and compared to a 2-assay subtractive fPAPP-A approach. Clinical performance was assessed with acute coronary syndrome patients. RESULTS: The limits of detection and quantitation were 0.4 mIU/L and 1.3 mIU/L, respectively, and the assay was linear up to 1000 mIU/L (R2 = 0.999). Both serum and heparin plasma were suitable matrices, and the complexed form of PAPP-A caused no significant interference. Correlation between the developed assay and the 2-assay approach was fair (Pearson's r = 0.819). Median concentration in healthy individuals was 1.0 mIU/L. fPAPP-A concentration was higher in patients who had myocardial infarction or died during the 1-year follow-up period than in those who did not (1.13 mIU/L vs 0.82 mIU/L, P = 0.008, model adjusted with age and sex). fPAPP-A measured with this direct assay predicted this end point as well as (follow-up 1 year) or better (30 days) than the 2-assay fPAPP-A alone or in combination with cTnI. CONCLUSIONS: The new assay enables sensitive and reliable measurement of low cardiac-related fPAPP-A concentrations from blood samples.

Novel homogenous time-resolved fluorometric RT-PCR assays for quantification of PSA and hK2 mRNAs in blood.

OBJECTIVES: The purpose of this study was to design, validate, and optimize internally standardized real-time quantitative RT-PCR assays and to identify and avoid problems with assay reliability and examine the impact of an exogenous internal standard. DESIGN AND METHODS: The model system consisted of internally standardized quantitative real-time RT-PCR assays specific for PSA and hK2 mRNA based on time-resolved fluorometric detection of lanthanide chelates. RESULTS: Reproducibility was best when large copy numbers (>5000 per milliliter blood) were analyzed. Addition of an exogenous target-mimicking internal standard had no significant effect on the reproducibility of the method, but increased the calculated copy numbers by an average of 2-fold. CONCLUSIONS: We developed an internally standardized, specific and reproducible real-time RT-PCR analysis method for PSA and hK2 mRNA in circulating cells in the bloodstream. Both PSA and hK2 assays are sufficiently sensitive to detect two LNCaP cells per milliliter whole blood.