Grouselle D, Tixier-Vidal A, Pradelles P
Groupe de Neuroendocrinologie Cellulaire et Moléculaire, CNRS URA 1115, Collége de France, Paris.
Neuropeptides. 1990 Nov;17(3):155-62. doi: 10.1016/0143-4179(90)90079-e.
Enzyme immunoassays (EIAs) for Thyroliberin (TRH) and TRH-elongated peptides were developed. Three haptens less than E-H-P-NH2 (TRH). Less than E-H-P-OH (TRH-OH), and S-K-R-Q-H-P-G-K-R-F (P10) were conjugated by the use of different heterobifunctional cross-linking agents either to sun-flower globulin as carrier or to acetylcholinesterase as tracer. For a same hapten, the same chemical group in the peptide was used to prepare the immunogen and the enzyme conjugate. These EIAs were performed with a second antibody solid phase technique using acetylcholinesterase as label. They permitted the measurement of TRH and TRH-elongated peptides with a sensitivity threshold of 10 fmol/well for TRH and 2 fmol/well for P10. TRH EIA only detected authentic TRH whereas TRH-OH EIA detected TRH and TRH peptides elongated on C terminal part. Anti-P10 serum was specific of TRH peptides elongated both on C and N terminal parts and no cross reactivity was observed with TRH. Using these assays, TRH and TRH-elongated peptides were determined in crude or chromatographed mouse and rat hypothalamus tissular extracts. Several TRH extended forms were identified by P10 EIA, whereas TRH-OH EIA permitted detection of both TRH and TRH-elongated peptides in chromatographed extracts. Authentic TRH was measured by TRH EIA both in crude and chromatographed hypothalamic extracts. These assays can permit the study of the processing and maturation of TRH.
开发了用于促甲状腺素释放激素（TRH）和TRH延伸肽的酶免疫测定法（EIA）。三种半抗原比E-H-P-NH2（TRH）少。比E-H-P-OH（TRH-OH）少，以及S-K-R-Q-H-P-G-K-R-F（P10）通过使用不同的异双功能交联剂与作为载体的向日葵球蛋白或作为示踪剂的乙酰胆碱酯酶偶联。对于同一半抗原，肽中的相同化学基团用于制备免疫原和酶结合物。这些EIA采用以乙酰胆碱酯酶为标记的第二抗体固相技术进行。它们能够测定TRH和TRH延伸肽，TRH的灵敏度阈值为10 fmol/孔，P10为2 fmol/孔。TRH EIA仅检测到真实的TRH，而TRH-OH EIA检测到TRH和C末端延伸的TRH肽。抗P10血清对C末端和N末端均延伸的TRH肽具有特异性，未观察到与TRH的交叉反应。使用这些测定法，在粗制或经色谱分离的小鼠和大鼠下丘脑组织提取物中测定了TRH和TRH延伸肽。通过P10 EIA鉴定了几种TRH延伸形式，而TRH-OH EIA能够检测经色谱分离提取物中的TRH和TRH延伸肽。在粗制和经色谱分离的下丘脑提取物中，通过TRH EIA测定真实的TRH。这些测定法可用于研究TRH的加工和成熟过程。
