Tawfik D S, Green B S, Eshhar Z
Department of Chemical Immunology, Weizmann Institute of Science, Rehovot, Israel.
Anal Biochem. 1992 Apr;202(1):35-9. doi: 10.1016/0003-2697(92)90201-h.
Several laboratories have now shown that monoclonal antibodies having enzyme-like properties can be generated. The generation of catalytic antibodies makes use of the same basic procedures that have been used for the generation of binding monoclonal antibodies, yet the process involves an additional crucial step: screening for catalytic activity. In this paper we address the unique problems involved in the detection of inefficient catalytic activity that is accompanied by uncatalyzed background reaction. An analysis that allows optimization of assay conditions and estimation of the minimal antibody concentration required to observe catalysis is presented. The results indicate that the structure of the substrate should be optimized to increase its affinity (i.e., decrease its Km) and reduce its concentration to pseudo-first-order conditions (S(O) much less than Km) so that the signal observed in the presence of a catalytic antibody (delta Pcat) is significantly higher than that of the background (delta P(uncat)). Other factors involved in the screening procedures, e.g., sensitivity of the assay, solubility and reactivity of the substrate, and purity of the antibody preparation, are also discussed. The effect of these assay parameters on the ability to detect catalytic activity is demonstrated with p-nitrophenyl ester-hydrolyzing antibodies.
现在已有几个实验室表明，可以产生具有酶样特性的单克隆抗体。催化抗体的产生利用了与产生结合性单克隆抗体相同的基本程序，但该过程还涉及一个关键步骤：筛选催化活性。在本文中，我们探讨了检测伴有非催化背景反应的低效催化活性所涉及的独特问题。本文提出了一种分析方法，可优化检测条件并估计观察到催化作用所需的最低抗体浓度。结果表明，应优化底物结构以增加其亲和力（即降低其米氏常数），并将其浓度降低至假一级反应条件（底物初始浓度远小于米氏常数），以使在催化抗体存在下观察到的信号（催化反应的吸光度变化）显著高于背景信号（非催化反应的吸光度变化）。还讨论了筛选过程中涉及的其他因素，例如检测的灵敏度、底物的溶解度和反应性以及抗体制剂的纯度。用对硝基苯酯水解抗体证明了这些检测参数对检测催化活性能力的影响。
