Validation of a sensitive enzymeimmunoassay for 13,14-dihydro-15-keto-PGF2α in buffalo plasma and its application for reproductive health status monitoring

A simple, sensitive and direct enzymeimmunoassay (EIA) procedure on microtitre plates using the second antibody coating technique was standardized and validated for the determination of 13,14-dihydro-15-keto PGF2α (PGFM) in unextracted buffalo plasma. The assay was carried out directly in 20 μl of buffalo plasma. PGFM standards prepared in charcoal stripped hormone-free plasma were used. The sensitivity of the assay was 0.4 pg/well, which corresponded to 20 pg/ml plasma. Plasma volumes for the assay ranging from 10 to 50 μl did not influence the PGFM standard curve; however, a slight drop in the OD450 was observed with higher plasma volumes. Biological validation of the assay was carried out in buffalo plasma samples obtained during physiological states of cyclicity, peri-estrus, post-insemination, reproductive tract infection and persistent corpus luteum conditions. A pulsatile pattern of plasma PGFM release was observed prior to estrus when PGFM was determined in blood samples collected at hourly intervals of time. The PGFM pulsatility was not observed when blood sampling frequency of either 4 or 12 h was considered. The PGFM levels stayed high in peripheral circulation of buffaloes with reproductive tract infections and remained low throughout the sampling period in buffaloes having persistent corpus luteum. After an initial increase post-insemination, the plasma PGFM levels showed minor fluctuations. The assay was found to be sufficiently reliable and specific for estimation of PGFM levels in buffaloes. The standardization and validation of PGFM assay in buffalo opens the prospects of using PGFM levels as an indicator for reproductive health status monitoring in this species.