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
PGF2alpha (
PGFM) in unextracted buffalo plasma. The assay was carried out directly in 20 microl 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 microl 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.