Adaptation of the cytohistochemical luteinizing hormone assay for measurement in bovine blood

The highly sensitive cytohistochemical assay was adapted for LH measurements in peripheral blood of cattle. The reducing ability of rat theca lutein tissue exposed to bovine LH was estimated by scanning and integrating microphotometry after Prussian blue staining; we achieved a linear correlation between the logarithm of the LH concentration and the average integrated transmission values. Cross-reacting and nonspecific interfering blood serum compounds were grossly removed by means of ion-exchange chromatography on CM-Sephadex-C-50 columns yielding about 77% of LH. Without chromatography there would have been increasing LH recovery with increasing LH concentrations. Also the presence of FSH either in unextracted serum or after apposition, resulted in LH overestimation due to dose related changes in the slope of the standard curve. Dilution curves of chromatographed bovine blood serum and of purified LH, hCG and PMSG were parallel. The recovery of added LH in the assay averaged 103%. The linear LH dose response curve maximally ranged from 10fg-10ng purified bovine pituitary LH standard. The intra assay variation was 18.8±6.9% and the interassay variation was 5.6±4.7% (x̄±SD). The LH pattern in purified bovine serum during the oestrus cycle and after administration of GnRH showed a close correlation whether measured by means of radioimmunoassay or cytohistochemical assay. The surge RIA-LH values were similar to the cytohistochemically measured ones, but the basal cytohistochemical LH levels were consistently lower (35-430 pg/ml compared to 0.8-2.1 ng/ml by RIA). These results give no indication for a different biological and immunological LH activity in bovine blood.