Development of a sensitive enzymeimmunoassay (EIA) for FSH determination in bovine plasma

A highly sensitive enzymeimmunoassay (EIA) procedure for FSH determination in bovine plasma on microtiterplates using the biotin–streptavidin amplification system and the second antibody coating was developed. Biotin was coupled to FSH and used to bridge between streptavidin–peroxidase and the immobilized antiserum in the competitive assay. The EIA was carried out directly in 50 μl of bovine plasma and compared with an established radioimmunoassay (RIA) employing 100 μl plasma. Same FSH standards and FSH specific antiserum were used in both procedures. FSH standards prepared in hormone free plasma were used. The sensitivity of the EIA procedure was 6.25 pg/well FSH which corresponded to 125 pg/ml plasma; the 50% relative binding sensitivity was seen at 200 pg/well. In comparison to RIA, the EIA was at least four times more sensitive besides requiring 6 times less FSH specific antiserum. Plasma volumes for the EIA ranging from 12.5 to 50 μl did not influence the shape of the standard curve even though a slight drop in the OD450 was seen with higher plasma volumes. When both EIA and RIA methods were used to measure FSH in cows, the levels were detectable only by the EIA procedure. The assay detects high and low plasma FSH levels within the physiological variation as well as changes in plasma FSH after stimulation with a GnRH analog. In conclusion, in addition to being non-radioactive and low cost in nature, the method offers several advantages over the conventional FSH RIA procedure; these are (a) higher sensitivity, (b) less labour and time saving, (c) more economical use of precious FSH antiserum and (d) long shelf-life of the biotinyl–FSH label (in contrast to the short half life of iodinated FSH in RIA).