Aspartic proteinase members secreted by the ruminant placenta: specificity of three radioimmunoassay systems for the measurement of pregnancy-associated glycoproteins.

Pregnancy-associated glycoproteins (PAGs) isolated from the placenta of various ruminant species are enzymatically inactive members of the aspartic proteinase family. The measurement of these proteins in the maternal blood can be a good indicator of the presence of a live embryo. As certain aspartic proteinases are present in biological fluids in physiological and pathological conditions at various concentrations, it was necessary to determine the specificity of three radioimmunoassay (RIA) systems currently used for the detection of PAG molecules. Commercially available members of the aspartic proteinase family like pepsinogen, pepsin, chymosin, rennet, cathepsin D and renin were tested in a wide concentration range (10 ng/ml - 1 mg/ml). Pepsinogen cross-reacted in RIA 1, RIA 2 and RIA 3 over 1 mg/ml, 50 microg/ml and 500 microg/ml concentrations, respectively. In the presence of pepsin, cross-reaction was observed in RIA 1, RIA 2 and RIA 3 over 1 mg/ml, 500 microg/ml and 1 mg/ml concentrations, respectively. Chymosin and rennet could cross-react in RIA 2 and RIA 3, while renin and cathepsin D did not decrease the binding of the tracer to antisera more, than that of the minimal detection limit. As the plasma/serum concentrations of the examined aspartic proteinases reported in the literature were outside the concentration range where cross-reaction was observed, it can be concluded that these RIA systems were specific for the detection of PAGs in biological fluids.

Comparison of the ability of three radioimmunoassay to detect pregnancy-associated glycoproteins in bovine plasma.

CONTENTS: Pregnancy-associated glycoproteins (PAGs) constitute a large family of glycoproteins that are synthesized in the superficial layer of the ruminant placenta according to a spatial and temporal expression pattern. When PAGs are released in the maternal blood they can be used for pregnancy diagnosis, pregnancy follow-up and for the monitoring of the trophoblastic function. Three different radioimmunoassay systems (RIA 1, RIA 2 and RIA 3) using antisera produced against PAG I67 (RIA 1), PAG55+62 (RIA 2) and PAG55+59 (RIA 3) were used in this investigation in order to measure the PAG concentration in plasma samples withdrawn from pregnant cows and heifers during different periods following artificial insemination (AI). These systems were able to detect PAG molecules in the maternal blood as early as 21 days after AI in different concentrations (RIA 1: 0.43 +/- 0.24 ng/ml, mean +/- SD; RIA 2: 0.48 +/- 0.24 ng/ml; RIA 3: 0.64 +/- 0.37 ng/ml). On days 32 and 42 RIA 2 (4.30 +/- 1.32 ng/ml and 5.56 +/- 1.95 ng/ml) and RIA 3 (4.17 +/- 1.15 ng/ml and 5.60 +/- 1.89 ng/ml) presented significantly (p < 0.0001) higher PAG concentrations than those of RIA 1 (2.43 +/- 0.81 ng/ml and 4.01 +/- 1.48 ng/ml), respectively. After day 21, significant correlations (p < 0.0001; r >/= 0.929) were determined between the three systems. Additionally the three individual PAG profiles presented in this study showed that PAG molecules secreted in the maternal blood between 21 and 50 days after AI were better recognized by the RIA 2 and RIA 3 systems. This study clearly indicated that the ability of a RIA test to recognize PAG molecules in the maternal blood can be improved by carefully selecting the antiserum.