Development and validation of a solid phase radioimmunoassay system for the determination of pregnancy-associated glycoproteins in bovine serum

Abstract

The Pregnancy-Associated Glycoproteins (PAGs) belong to aspartic protease family and are synthesized by trophoblast cells of ruminant placenta and secreted into maternal blood and milk. These biomarkers very closely associated to pregnancy have demonstrated their efficiency and reliability in detection of vital embryos or pregnancy loss. In order to measure the concentration of PAGs in bovine serum, a direct radioimmunoassay (RIA) system was developed and validated. The radioiodinated bPAG tracer was prepared by chloramine-T direct method and the solid phase was used in RIA/PAGs as a separation system. The optimization tests allowed to set the assays procedure as working buffers, time and temperature of the assays incubation. Assay sensitivity was 0.63 ng/mL, the intra and inter-assay CVs were 7.7% and 5.3% respectively and the accuracy of the assays was expressed by a good parallelism and recovery tests. The serums bPAG values determinated by this developed system were consistent with the reproduction status and pregnancy stages of cows. The developed RIA/PAG system can provide the laboratory with an efficient method for the accurate detection of pregnancy, which could offer veterinarians and farmers an important tool for the reproductive management in dairy herds. Moreover, this diagnostic tool can also be developed for other livestock species.

Keywords

• bovine pregnancy-associated glycoproteins
• radioimmunoassay
• serum
• solid phase
• validation