Methodological aspects of rat β-endorphin analysis—influence of diurnal variation

Abstract

β-Endorphin radioimmunoassays (RIAs) are widely performed following physical, emotional and environmental challenges in the rat. In the literature, a wide range of techniques have been described, but in the present study, we have focused on methodological aspects of β-endorphin RIAs, investigating various characteristics of human and rat specific antibodies. Initial studies verified that the RIA outcome was not appropriate when using non-species compatible components. Novel rat β-endorphin antibodies, r 4114 and r 4268, were raised in rabbits and characterised in terms of specificity, avidity and titer. Both of the new antisera showed 68.1% cross-reactivity with human β-endorphin. The ED 50 was 50 ± 8 pmol/l, and the mean ED 80 was 17 pmol/l for r 4268 but three-fold higher for r 4114. The intra-assay coefficient of variation (CV) was 7% at 100 pmol/l and the inter-assay CV was 10% at the same level for r 4268 and similar for r 4114. Using this novel rat β-endorphin RIA for analyses of diurnal influence and removal from the Animal House cage, no significant changes were observed in either the hypothalamus or peri-aqueductal grey regions. These results suggest that rat β-endorphin concentrations in these brain areas are not affected by order of removal or diurnal variation.