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.
Lin
Ji
J
Research Laboratory of Biochemistry, Basic Medical Institute, General Hospital of P.L.A., Beijing, PR China. panny@263.net
Yan
Guang-Tao
GT
Hao
Xiu-Hua
XH
Zhang
Kai
K
Wang
Lu-Huan
LH
Xue
Hui
H
D000818:Animals; D002352:Carrier Proteins / Q000097:blood*; D016022:Case-Control Studies; D006801:Humans; D006949:Hyperlipidemias / Q000097:blood; D007031:Hypothalamus / Q000737:chemistry; D007413:Intestinal Mucosa / Q000098:blood supply / Q000473:pathology; D047908:Intracellular Signaling Peptides and Proteins; D007457:Iodine Radioisotopes; D008297:Male; D009479:Neuropeptides / Q000097:blood*; D000068797:Orexins; D011817:Rabbits; D011863:Radioimmunoassay / Q000379:methods*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012015:Reference Standards; D015427:Reperfusion Injury / Q000097:blood; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D002352:Carrier Proteins; C000596229:HCRT protein, human; D047908:Intracellular Signaling Peptides and Proteins; D007457:Iodine Radioisotopes; D009479:Neuropeptides; D000068797:Orexins