Pandey R N, Davis L E, Anderson B, Hollenberg P F
J Immunol Methods. 1986 Nov 20;94(1-2):237-46. doi: 10.1016/0022-1759(86)90238-3.
Two chemical methods, diazocoupling and reaction with isocyanates, are commonly used to conjugate primary aromatic amines with carrier proteins in order to elicit antibody responses against the aromatic amine haptenic group. Limitations of these conjugation techniques include the requirement for specific functional groups on the carrier protein which generally limits the degree of haptenic substitution obtainable, the many possible side reactions yielding hapten-hapten and carrier-carrier conjugates which waste valuable materials and lower desired hapten-carrier conjugate yields, and, in some cases, conjugation conditions which may denature the carrier protein (e.g., alkaline coupling conditions). We report here a photolabeling approach for conjugating primary aromatic amines to carrier proteins which avoids some of the problems of other conjugation methods and which was used to elicit antibodies against the primary aromatic amine hapten. The method described here is of general application for coupling primary aromatic amines to the carrier proteins and circumvents many of the problems inherent in the isocyanate or diazocoupling methods. 3-Azido-N-ethylcarbazole (ANEC), the azido analog of 3-amino-N-ethylcarbazole, was conjugated to bovine serum albumin (BSA), human transferrin (TR), thyroglobulin (TH), poly-(lysine X tyrosine), and poly-(lysine X phenylalanine) using standard photolabeling procedures. After photolysis, the conjugated proteins or polypeptides were separated from the unbound products of ANEC photolysis on a Sephadex G-10 column. The conjugated proteins were extracted with isobutanol which demonstrated that approximately 20% of the ANEC was covalently coupled to the protein carriers and that the larger portion of the aromatic haptens was non-covalently and hydrophobically bound to the carriers. The ANEC-protein conjugates used for immunization demonstrated a total covalently and non-covalently bound ANEC epitope density of 90 per BSA, 107 per TR and 800 per TH molecule. Rabbits were immunized with the three conjugated proteins and the production of antibody specific for the 3-amino-N-ethylcarbazole hapten was demonstrated by enzyme-linked immunosorbent assay and by inhibition studies using hapten-carrier conjugates of free hapten. The results demonstrate that antibodies against aromatic amine haptens may be raised by immunizing animals with hapten-carrier protein conjugates produced by photolabeling. Since the coupling conditions are very mild and the functional group requirements are so general (requiring only the presence of C-H, N-H, C = O, C = S, or S-H bonds) most carrier proteins should be suitable for use in this method.(ABSTRACT TRUNCATED AT 400 WORDS)
两种化学方法，即重氮偶联法和与异氰酸酯反应法，通常用于将伯芳香胺与载体蛋白偶联，以引发针对芳香胺半抗原基团的抗体反应。这些偶联技术的局限性包括：载体蛋白上需要特定的官能团，这通常限制了可获得的半抗原取代程度；存在许多可能的副反应，会产生半抗原-半抗原和载体-载体偶联物，浪费了宝贵的材料并降低了所需的半抗原-载体偶联物产量；在某些情况下，偶联条件可能会使载体蛋白变性（例如碱性偶联条件）。我们在此报告一种将伯芳香胺与载体蛋白偶联的光标记方法，该方法避免了其他偶联方法的一些问题，并用于引发针对伯芳香胺半抗原的抗体。这里描述的方法普遍适用于将伯芳香胺与载体蛋白偶联，并规避了异氰酸酯或重氮偶联方法中固有的许多问题。使用标准光标记程序，将3-叠氮基-N-乙基咔唑（ANEC），即3-氨基-N-乙基咔唑的叠氮类似物，与牛血清白蛋白（BSA）、人转铁蛋白（TR）、甲状腺球蛋白（TH）、聚（赖氨酸X酪氨酸）和聚（赖氨酸X苯丙氨酸）偶联。光解后，在葡聚糖凝胶G-10柱上从未结合的ANEC光解产物中分离出偶联的蛋白质或多肽。用异丁醇提取偶联的蛋白质，结果表明约20%的ANEC与蛋白质载体共价偶联，且大部分芳香半抗原通过非共价和疏水方式与载体结合。用于免疫的ANEC-蛋白质偶联物显示，每个BSA分子的共价和非共价结合的ANEC表位密度为90，每个TR分子为107，每个TH分子为800。用这三种偶联蛋白对兔子进行免疫，并通过酶联免疫吸附测定以及使用游离半抗原的半抗原-载体偶联物的抑制研究，证明产生了对3-氨基-N-乙基咔唑半抗原特异的抗体。结果表明，通过用光标记产生的半抗原-载体蛋白偶联物免疫动物，可以产生针对芳香胺半抗原的抗体。由于偶联条件非常温和且对官能团的要求非常普遍（仅需要存在C-H、N-H、C = O、C = S或S-H键），大多数载体蛋白应该适用于此方法。（摘要截短为400字）
