Harapanhalli R S, Schafranek W, Ong G L, Mattes M J
Center for Molecular Medicine and Immunology, Garden State Cancer Center, Newark, New Jersey 07103, USA.
Anal Biochem. 1995 Oct 10;231(1):50-6. doi: 10.1006/abio.1995.1502.
Protein radioiodination via iodo-5-([4,6-dichlorotria- zin-2-yl]amino)fluorescein(DTAF), a cyanuric chloride derivative of aminofluorescein, was characterized. Commercially available DTAF was iodinated by the Iodogen reaction and then conjugated to IgC antibodies in a 4-h incubation in borate buffer, pH 9.0. With low amounts of protein, 10 micron, molar ratios of iodine:IgC of nearly 1:1 were obtained. With 25 micron protein, which was used routinely, 15-20% efficiency of (125)I incorporation was obtained. Polylysine was labeled efficiently, and labeling was inhibited by ethanolamine, which is consistent with previous data indicating that conjugation is to amino in the protein. A low level of aggregates, primarily dimers, was generated. This procedure is a simple, inexpensive method to test the advantage of lysine-directed protein iodination, with proteins for which oxidative iodination is unsuitable. Abs labeled with DTAF retained strong antigen-binding activity, and, in the case of one Ab, were much more active than the chloramine T-labeled Ab. Catabolic products of iodo-DTAF conjugates, produced after internalization and degradation of Ab bound to the cell surface, were retained within some but not all human carcinoma cell lines.
通过5-([4,6-二氯三嗪-2-基]氨基)荧光素（DTAF，氨基荧光素的三聚氯氰衍生物）对蛋白质进行放射性碘化的特性进行了研究。市售的DTAF通过碘代反应进行碘化，然后在pH 9.0的硼酸盐缓冲液中孵育4小时与IgC抗体偶联。对于少量蛋白质（10微克），碘与IgC的摩尔比接近1:1。对于常规使用的25微克蛋白质，获得了15%-20%的¹²⁵I掺入效率。聚赖氨酸被有效标记，且标记被乙醇胺抑制，这与先前表明偶联是与蛋白质中的氨基结合的数据一致。产生了低水平的聚集体，主要是二聚体。该方法是一种简单、廉价的方法，用于测试赖氨酸导向的蛋白质碘化的优势，适用于氧化碘化不适用的蛋白质。用DTAF标记的抗体保留了很强的抗原结合活性，并且对于一种抗体而言，其活性比用氯胺T标记的抗体高得多。与细胞表面结合的抗体内化和降解后产生的碘代-DTAF偶联物的分解产物，在一些但不是所有的人癌细胞系中被保留。
