Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India.
J Immunol Methods. 2010 May 31;357(1-2):26-32. doi: 10.1016/j.jim.2010.03.008. Epub 2010 Mar 17.
Antigen specific monoclonal antibodies present in crude hybridoma supernatants are normally screened by ELISA on plates coated with the relevant antigen. Screening for inhibitory monoclonals to enzymes would require the evaluation of purified antibodies or antibody containing supernatants for their inhibition of enzyme activity in a separate assay. However, screening for inhibitory antibodies against DNA transacting enzymes such as topoisomerase I (topo I) cannot be done using hybridoma supernatants due to the presence of nucleases in tissue culture media containing foetal calf serum which degrade the DNA substrates upon addition. We have developed a simple and rapid screening procedure for the identification of clones that secrete inhibitory antibodies against mycobacterial topo I using 96 well ELISA microtiter plates. The principle of the method is the selective capture of monoclonal antibodies from crude hybridoma supernatants by topo I that is tethered to the plate through the use of plate-bound polyclonal anti-topo I antibodies. This step allows the nucleases present in the medium to be washed off leaving the inhibitor bound to the tethered enzyme. The inhibitory activity of the captured antibody is assessed by performing an in situ DNA relaxation assay by the addition of supercoiled DNA substrate directly to the microtiter well followed by the analysis of the reaction products by agarose gel electrophoresis. The validity of this method was confirmed by purification of the identified inhibitory antibody and its evaluation in a DNA relaxation assay. Elimination of all enzyme-inhibitory constituents of the culture medium from the well in which the inhibitory antibody is bound to the tethered enzyme may make this method broadly applicable to enzymes such as DNA gyrases, restriction enzymes and other DNA transaction enzymes. Further, the method is simple and avoids the need of prior antibody purification for testing its inhibitory activity.
在含有相关抗原的平板上，通过 ELISA 从粗杂交瘤上清液中筛选抗原特异性单克隆抗体。筛选针对酶的抑制性单克隆抗体需要评估纯化的抗体或含有抗体的上清液，以评估它们在单独的测定中对酶活性的抑制作用。然而，由于胎牛血清中存在核酸酶，组织培养培养基中的核酸酶会在添加后降解 DNA 底物，因此无法使用杂交瘤上清液筛选针对拓扑异构酶 I（topo I）等 DNA 转导酶的抑制性抗体。我们开发了一种简单快速的筛选程序，用于鉴定分泌针对分枝杆菌 topo I 的抑制性抗体的克隆，使用 96 孔 ELISA 微量滴定板。该方法的原理是通过使用板结合的多克隆抗拓扑异构酶 I 抗体将拓扑异构酶 I 固定在平板上，从粗杂交瘤上清液中选择性捕获单克隆抗体。这一步骤允许将存在于培养基中的核酸酶洗掉，使抑制剂与固定化酶结合。通过将超螺旋 DNA 底物直接添加到微量滴定孔中，进行原位 DNA 松弛测定，评估捕获抗体的抑制活性，然后通过琼脂糖凝胶电泳分析反应产物。通过纯化鉴定的抑制性抗体并在 DNA 松弛测定中进行评估，验证了该方法的有效性。从与固定化酶结合的抗体所在的孔中消除培养基中所有的酶抑制成分，可能使该方法广泛适用于 DNA 回旋酶、限制酶和其他 DNA 转导酶等酶。此外，该方法简单，避免了在测试其抑制活性之前对抗体进行纯化的需要。
