The EURIA-β-Endorphin kit (EURO-DIAGNOSTICA AB, Sweden) was used for the β-endorphin radioimmunoassay (RIA). This RIA is based on the principle of double-antibody precipitation. The evaporated samples were diluted with 220 μl diluent (0.05 M phosphate, pH 7.4, 0.25% human serum albumin, 0.05% sodium azide, 0.25% EDTA and 500 KIU Trasylol®/ml) and incubated with 100 μl of anti-β-endorphin antiserum for 24 h at 4°C. After incubation, the labeled peptide was added to each sample and incubated for an additional 24 h, at 4°C. Thereafter, the double antibody PEG was added, and the test tubes were incubated for 60 min and then centrifuged for 15 min at 3000 × g at 4°C. Finally, the supernatants were decanted and the radioactivity of the precipitates was counted in a gamma counter.
