Establishment and primary application of a highly-sensitive orexin-A radioimmunoassay

Orexin-A was labeled by 125I using the chloramine-T method, and was purified with a Sephadex G-25 chromatographic column. The reaction between antigen and antibody was carried out by a one-step balance method and was incubated at 4 degrees C for 24 hours, then bonded and free antigen were separated by PR reagent. The detection range of this RIA is 21-2000 pg/mL; the lowest detection level is 21 pg/mL. The intra-assay and inter-assay variations were 7.8% and 9.7%, respectively. Plasma orexin-A levels of 30 normal individuals and 30 patients with hyperlipidemia (serum triglyceride > 1.7 mmol/L and serum total cholesterol > 5.7 mmol/L) were detected by this RIA, while orexin-A levels of plasma and hypothalamus in rat intestinal ischemia-reperfusion injury model were also measured. Plasma orexin-A levels of normal individuals was 338.48 +/- 20.24 pg/mL, while those of patients with hyperlipidemia were 343.51 +/- 15.49 pg/mL; there were no significant differences between these two groups t = -0.1976; P = 0.8441. We also found that orexin-A levels of rat plasma and hypothalamus did not express a significant change during the early stages of intestinal ischemia-reperfusion injury. These results have shown that this orexin-A radioimmunoassay is stable, simple, and specific, being sensitive enough to test orexin-A levels in human plasma, rat plasma, and hypothalamus.