Endocrine Abstracts (2006) 11 P656

Standardisation and validation of a sensitive enzymeimmunoassay procedure to estimate 13,14-dihydro-15-keto-PGF (PGFM) in mithun (Bos frontalis) plasma

DP Mishra, A Dhali, A Mech, M Karunakaran, H Choudhury & C Rajkhowa


National Research Centre on Mithun, Jharnapani, Medziphema, Nagaland - 797106, India.


Mithun (Bos frontalis), a free-range domesticated ungulate, reared primarily for beef and occasionally for milk, is mainly distributed in the South-East Asia. Much scientific reports on reproductive-endocrinology in this species are not available at present. The current study was designed to standardise and validate a simple, sensitive and direct enzymeimmunoassay (EIA) on microtitre plates using second antibody coating technique to determine 13,14-dihydro-15-keto PGF (PGFM) in unextracted mithun plasma. The assay was carried out directly in 20 μl mithun plasma. PGFM standards prepared in hormone-free plasma were used in the assay. It was observed that the different plasma volumes (10, 20, 50 and 100 μl) did not influence the absolute binding sensitivity. However, a slight drop in OD450 was observed with increasing plasma volume. The sensitivity of the assay was found 40 pg/ml plasma. The intra- and inter- assays coefficients of variation that determined using pooled plasma samples containing 97.2±3.1 and 235.6±4.9 pg/ml PGFM were found 5.9 and 8.3 and, 6.5 and 9.7 percent, respectively. Biological validation of the assay was carried out in plasma samples that collected from 10 mithun cows on different days of estrous cycle and during peri-estrus period. In estrous cycle, the plasma PGFM level (P<0.01) attained peak (481.0±43.4 pg/ml) on day 4 prior to estrus and the lowest level (164.3±11.9 pg/ml) was observed on estrus day. A pulsatile PGFM secretion pattern was observed prior to estrus when frequent samples were collected at 1 h intervals. A wide range of plasma PGFM concentration (93 to 612 pg/ml) were successfully detected in cyclic mithuns using the current EIA protocol. In conclusion, the present PGFM-EIA procedure has been found to be sufficiently sensitive and reliable to estimate different physiological levels of PGFM in mithun plasma.

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