Yarmush M L, Lu X M, Yarmush D M
Department of Chemical and Biochemical Engineering, Rutgers University, Piscataway, NJ 08855.
J Biochem Biophys Methods. 1992 Dec;25(4):285-97. doi: 10.1016/0165-022x(92)90022-3.
A method to covalently bind antibody fragments, via their carboxyl termini to solid supports, is presented. The strategy involves: (1) reversibly blocking all the accessible carboxyl groups on the antibody molecule with phenylhydrazine, (2) exposing the carboxyl termini of the fragment by enzymatic digestion with pepsin and (3) subsequently coupling the fragment to an appropriate support. Experiments with an anti-bovine serum albumin monoclonal antibody and C-14 phenylhydrazine revealed that the blocking step was nearly completely reversible with a dilute solution of FeCl3. Radioiodinated blocked F(ab')2 fragments were then coupled to an amino-functionalized Sepharose 4B column, and characterized as to their coupling capacity (mass of protein coupled/ml of bead), and antigen-binding activity. The coupling capacity of the blocked fragments was found to be 12%, half the coupling efficiency of unmodified radioiodinated F(ab')2. The antigen-binding capacity (mol antigen bound per mol antibody coupled) for the blocked F(ab')2, on the other hand, was found to be 1.9, which was approx. 3.5-times greater than for the unmodified F(ab')2. Comparisons with other conventional coupling techniques were also made. These preliminary studies suggest that this technique can provide one with the means to obtain more uniform and active populations of immobilized antibody fragments.
本文介绍了一种通过抗体片段的羧基末端将其共价结合到固体支持物上的方法。该策略包括：（1）用苯肼可逆地封闭抗体分子上所有可及的羧基；（2）用胃蛋白酶酶切使片段的羧基末端暴露；（3）随后将片段偶联到合适的支持物上。用抗牛血清白蛋白单克隆抗体和C-14苯肼进行的实验表明，用稀氯化铁溶液可使封闭步骤几乎完全逆转。然后将放射性碘化的封闭F(ab')2片段偶联到氨基功能化的琼脂糖4B柱上，并对其偶联容量（每毫升珠子偶联的蛋白质质量）和抗原结合活性进行表征。发现封闭片段的偶联容量为12%，是未修饰的放射性碘化F(ab')2偶联效率的一半。另一方面，封闭F(ab')2的抗原结合容量（每摩尔偶联抗体结合的摩尔抗原）为1.9，约为未修饰F(ab')2的3.5倍。还与其他传统偶联技术进行了比较。这些初步研究表明，该技术可为人们提供一种获得更均匀且活性更高的固定化抗体片段群体的方法。
