Development of a new ELISA for measurement of pregnancy-associated glycoprotein.

In the present study we report the development of a new ELISA for PAG measurement (Ref. code E.G.7, CER, Maloie) in ruminant species. Purified boPAG67kDa protein was purified in our laboratory and used as standard. Antiserum raised against caprine PAG 55kDa+62kDa (AS707) was used as capture antibody (1:40,000). Antiserum raised against PAG purified from buffalo placentas (AS859) was used as detection antibody (1:32,000 dilution). Avidin-HRP and TMB completed the ELISA system. Reproducibility was determined by calculating the intra- and inter-assay CV. Accuracy was determined by adding increasing concentrations of purified boPAG67kDa to bovine sera containing low PAG concentrations. Specificity was tested regarding 25 different compounds: carbohydrates, aspartic proteinases, hormones and major plasma proteins. Parallelism was assessed by serially diluting pregnant cow serum containing high PAG concentrations. Sera was collected from pregnant cows (n=4) during the whole duration of gestation. Samples were tested by both RIA and ELISA. Samples giving high PAG concentrations were serially diluted in order to fit with optical density (OD) of the ELISA standard curve. Intra-and inter-assay CV ranged from 5.5 to 9.1%. Accuracy ranged from 92.9 to 105.5%. No cross reaction was observed with almost all tested compounds excepting lactic acid at supra-physiological levels (1 mg/ml). Dilutions of bovine maternal sera were parallel to standard curve. Profiles obtained by ELISA in pregnant cows were very similar to those obtained by RIA. In conclusion, a new PAG-ELISA is available for measurement of PAG concentrations in cattle. This new test was validated in vitro and can be used for pregnancy diagnosis and follow-up of gestation in cattle