High-specific-activity 111In-labeled anticarcinoembryonic antigen monoclonal antibody: improved method for the synthesis of diethylenetriaminepentaacetic acid conjugates.
High-specific-activity 111In-labeled anticarcinoembryonic antigen monoclonal antibody: improved method for the synthesis of diethylenetriaminepentaacetic acid conjugates.
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发表时间：
1985-11
期刊：
影响因子：
11.2
通讯作者：
R. Paxton;J. Jakowatz;J. Beatty;B. Beatty;W. G. Vlahos;L. Williams;B. Clark;J. Shively
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文献类型：
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作者：
R. Paxton;J. Jakowatz;J. Beatty;B. Beatty;W. G. Vlahos;L. Williams;B. Clark;J. Shively
本文报道了二乙烯三胺五乙酸（DTPA）的N-羟基丁二酰亚胺活性酯与蛋白质偶联的新方法。DTPA-活性酯的制备使用二异丙基碳二亚胺在一个简单的单步合成。通过将DTPA-活性酯反应混合物加入到pH 7.0缓冲的蛋白质溶液（5 mg/ml）中制备DTPA-缀合的蛋白质，并通过Sephadex G-50色谱法纯化。针对癌胚抗原的单克隆抗体与四种不同量的DTPA活性酯反应。固相酶免疫分析表明，当活性酯与抗体的摩尔比为36：1或72：1时，抗体偶联物的免疫活性没有改变，但当摩尔比为180：1或360：1时，抗体偶联物的免疫活性降低。当通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析时，抗体重链和轻链的电泳迁移率略有降低，这一结果与DTPA与蛋白质的共价连接一致。SephadexG-200柱层析显示天然抗体和缀合抗体大小相同。当DTPA偶联抗体与10、50和100 μ Ci的111 In/微克蛋白质孵育时，获得的比活度为9.8、43.1和56.3 μ Ci/微克。酶免疫分析和放射免疫分析结果表明，111 In标记的抗体保留了完整的免疫活性。111 In标记的抗体的高比活度使其适合于使用低剂量的抗体对携带癌胚抗原的肿瘤进行成像。
A new method has been developed for conjugating diethylenetriaminepentaacetic acid (DTPA) to proteins using the N-hydroxysuccinimide active ester of DTPA. The DTPA-active ester was prepared using diisopropylcarbodiimide in a simple single step synthesis. DTPA-conjugated proteins were prepared by adding the DTPA-active ester reaction mixture to protein solutions (5 mg/ml) buffered at pH 7.0 and purified by Sephadex G-50 chromatography. A monoclonal antibody directed against carcinoembryonic antigen was reacted with four different amounts of the DTPA-active ester. Solid-phase enzyme immunoassay showed that the immunological activity of the antibody conjugate was not altered when the active ester: antibody molar ratio was 36:1 or 72:1; however, it decreased when the ratio was 180:1 or 360:1. The antibody heavy and light chains had slightly decreased electrophoretic mobilities when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a result consistent with the covalent attachment of DTPA to the protein. Sephadex G-200 chromatography showed that the native and conjugated antibodies were the same size. When the DTPA-conjugated antibody was incubated with 10, 50, and 100 microCi of 111In/micrograms of protein, specific activities of 9.8, 43.1, and 56.3 microCi/micrograms were obtained. Enzyme immunoassay and radioimmunoassay of the 111In-labeled antibody showed that it retained its full immunological activity. The high specific activity of the 111In-labeled antibody makes it suitable for imaging carcinoembryonic antigen-bearing tumors using low doses of antibody.