Radioimmunoassay of somatostatin.
Radioimmunoassay of somatostatin.
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生长抑素的放射免疫测定。
DOI：
10.1016/0026-0495(78)90032-x
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发表时间：
1978
期刊：
影响因子：
--
通讯作者：
A. V. Schally
中科院分区：
文献类型：
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作者：
A. Arimura;Gudmar Lundqvist;J. Rothman;Robert C. C. Chang;R. Fernández;Robert Elde;D. H. Coy;C. Meyers;A. V. Schally
合成生长抑素的出现使得产生针对这种下丘脑激素的抗血清成为可能，并使我们能够建立放射免疫分析（RIA）方法并进行免疫细胞学研究。这种方法和特定的抗血清促进了各种解剖学、生理学和生化研究，为生长抑素研究的进展做出了相当大的贡献。当我们获得合成生长抑素后，我们也开始对兔子进行免疫接种以产生抗血清。由于我们的目的是建立 RIA，因此有必要制备一种抗体，使未标记的生长抑素和示踪剂竞争结合。由于生长抑素不含有酪氨酸（Tyr）或组氨酸（His），这两种物质都很容易通过常规的碘化方法进行碘化，因此合成了Tyr’-生长抑素。所产生的抗血清必须与‘251-Tyr’-生长抑素结合，因此最好不识别靠近N末端的部分。为了增强生长抑素的抗原性，我们使用戊二醛将合成生长抑素与人 N 和/J 球蛋白 (Hrq’3G) 结合。选择这种双功能偶联剂的原因如下：(1) 戊二醛与肽的伯游离氨基反应，优先与 N 末端的伯游离氨基反应，尽管第 4 和 9 位赖氨酸 (Lys) 的游离氨基也可能受到影响； (2)产生的抗体通常其识别位点位于远离载体蛋白偶联位点的分子部分，因此产生的抗体会结合“I-Tyr”-生长抑素。幸运的是，用 H&G-生长抑素缀合物免疫的三只兔子中的两只产生了良好滴度的抗血清，即 101 号和 103 号。“生长抑素与各种载体蛋白（例如甲状腺球蛋白和海螺血蓝蛋白）缀合的各种方法都有报道。Vale 等人”使用 Tyr”-生长抑素与人血清白蛋白 (HSA) 缀合，通过双二唑联苯胺。在早期研究中我们使用Tyr'-生长抑素进行标记。它通过乳过氧化物酶方法进行碘化，并在 CM-纤维素柱上使用乙酸铵缓冲液（pH 4.6）以两种不同浓度（0.002 M 和 0.1 M）进行纯化。后来，N-Tyr-生长抑素被标记并用作生长抑素 RIA 中的示踪剂，因为“SI-N-Tyr-生长抑素被发现非常稳定，因此在储存 1.5 年后无需重新纯化即可使用” mo at-5O”C，而“”“I-Tyr”-生长抑素仅稳定 1 周。 ‘ZsI-Tyr’-生长抑素和 I’”-N-Tyr-
T HE AVAILABILITY OF SYNTHETIC SOMATOSTATIN made it possible to generate antiserum against this hypothalamic hormone and enabled us to establish the radioimmunoassay(RIA) method and perform immunocytologic studies. Such a method and a specific antiserum prompted various anatomic, physiologic, and biochemical investigations, which have made a considerable contribution to the progress of somatostatin research. As soon as we obtained synthetic somatostatin, we also started immunizing rabbits to generate antiserum. Since we aimed to establish an RIA, it was necessary to prepare an antibody with which the unlabeled somatostatin and the tracer compete in the binding. Since somatostatin does not have either tyrosine (Tyr) or histidine (His), both of which are readily iodinated by a conventional method of iodination, Tyr’-somatostatin was synthesized. The antiserum to be generated had to bind with ‘251-Tyr ‘-somatostatin and, therefore, should preferably not recognize the portion near the N-terminus. To enhance the antigenicity of somatostatin, we conjugated synthetic somatostatin with human N and/Jglobulins (Hrq’3G) using glutaraldehyde. This bifunctional coupling reagent was chosen for the following reasons:(1) glutaraldehyde reacts with a primary free amino group of the peptide, preferentially that of the N-terminus, although the free amino group of lysine (Lys) at positions 4 and 9 might be affected as well; and (2) the antibody generated usually has its recognition site at the portion of the molecule distal from the site of coupling with the carrier protein, so the antibody to be generated would bind ““I-Tyr’-somatostatin. Fortunately, two of three rabbits immunized with the H&G-somatostatin conjugate yielded the antisera of good titer, No. 101 and No. 103.’Various methods of conjugation of somatostatin with various carrier proteins, such as thyroglobulin and Whelk hemocyanin” were reported. Vale et al.’used Tyr”-somatostatin conjugated with human serum albumin (HSA) by bisdiazolized benzidine.In the early study we used Tyr’-somatostatin for labeling. It was iodinated by the lactoperoxidase method and purified on a CM-cellulose column using ammonium acetate buffer, pH 4.6, at two different concentrations, 0.002 M and 0.1 M.’Later N-Tyr-somatostatin was labeled and used as the tracer in RIA for somatostatin, since ‘“SI-N-Tyr-somatostatin was found to be so stable that it was used without repurification after storage for 1.5 mo at-5O” C, whereas ‘““I-Tyr’-somatostatin was stable for only 1 wk. Both ‘ZsI-Tyr’-somatostatin and I’“-N-Tyr-