Development of a sensitive enzymeimmunoassay for oxytocin determination in bovine plasma

A highly sensitive and specific second antibody enzymeimmunoassay (EIA) on microtiterplates for oxytocin determination in bovine plasma using the biotin-streptavidin amplification system was developed. Biotin was coupled to oxytocin and used to bridge between streptavidin-peroxidase and the immobilized oxytocin antiserum in the competitive assay. The assay was carried out directly in 200 microliters of bovine plasma. Oxytocin standards prepared in hormone-free plasma were used. The sensitivity of the assay was 0.25 pg/well which corresponded to 1.25 pg/ml plasma; the 50% relative binding was seen at 2.8 pg/well. Plasma volumes for the assay ranging from 50 to 200 microliters did not influence the shape of the oxytocin standard curve; however a distinct drop in the OD450 was observed with higher plasma volumes. The oxytocin antiserum used in the assay showed no significant cross-reaction with other octapeptides tested. The assay was compared with a radioimmunoassay (RIA) procedure employing prior solvent extraction of plasma samples. The oxytocin concentrations assayed by EIA and RIA in plasma samples obtained from four cows before, during and after milking were highly correlated and very similar (r = 0.97). Hence the assay developed offers an attractive alternative to the RIA since no prior laborious plasma extraction is needed. Further, the assay has the distinct advantage of being non-radioactive in nature.