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Establishment and Primary Application of a Highly‐Sensitive Orexin‐A Radioimmunoassay

Abstract 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°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.