Takahashi N, Kakinuma H, Hamada K, Shimazaki K, Takahashi K, Niihata S, Aoki Y, Matsushita H, Nishi Y
Laboratory of Life Science & Biomolecular Engineering, Japan Tobacco, Inc., Yokohama, Kanagawa, Japan.
Eur J Biochem. 1999 Apr;261(1):108-14. doi: 10.1046/j.1432-1327.1999.00235.x.
One of the major obstacles to acquiring catalytic antibodies is that it requires labor-intensive procedures to select catalytic antibodies from huge repertories of antibodies. Here, we selected potential catalytic Abs by utilizing their affinity towards a short transition-state analog which contained only the transition-state structural element, and evaluated in detail its efficiency to enrich catalytic Abs. Hybridoma supernatants elicited against a phosphonate derivative, the TSA1, were screened by a three-step screening process: step 1, ELISA for TSA1-BSA; step 2, ELISA for the short TSA4; and step 3, competitive-inhibition by the short TSA2. Only 22. 8% of positive mAbs from step 1 were found to be catalytic. The rate of catalytic Abs increased to 45.7% using screening steps 1 plus 2, and reached 83.3% using all three screening steps. This clearly suggests that our screening protocol is an efficient method to select potential catalytic Abs. Furthermore, we characterized the properties of both the catalytic Abs and the noncatalytic Abs in detail. The catalytic Abs tended to have lower Kd for TSA1 and the short TSA2 than noncatalytic Abs. It was also observed that catalytic Abs showed clear enantiospecificity toward substrate 6 containing d-phenylalanine while noncatalytic Abs did not. The detailed analysis of kinetic and binding parameters for these antibodies gives us further insight into catalytic antibodies.
获得催化抗体的主要障碍之一是，从庞大的抗体库中筛选催化抗体需要耗费大量人力的程序。在此，我们通过利用抗体对仅包含过渡态结构元件的短过渡态类似物的亲和力来筛选潜在的催化抗体，并详细评估了其富集催化抗体的效率。针对膦酸酯衍生物TSA1产生的杂交瘤细胞培养上清液，通过三步筛选过程进行筛选：步骤1，针对TSA1 - BSA的酶联免疫吸附测定（ELISA）；步骤2，针对短TSA4的ELISA；步骤3，短TSA2的竞争性抑制。结果发现，步骤1中的阳性单克隆抗体（mAb）仅有22.8%具有催化活性。使用步骤1加步骤2的筛选方法，催化抗体的比例增至45.7%，而使用所有三个筛选步骤时则达到83.3%。这清楚地表明，我们的筛选方案是一种筛选潜在催化抗体的有效方法。此外，我们还详细表征了催化抗体和非催化抗体的特性。与非催化抗体相比，催化抗体对TSA1和短TSA2的解离常数（Kd）往往更低。还观察到，催化抗体对含有d - 苯丙氨酸的底物6表现出明显的对映体特异性，而非催化抗体则没有。对这些抗体的动力学和结合参数进行详细分析，能让我们对催化抗体有更深入的了解。
