Long Ton That Ai, Kalantarov Gavreel, Chudner Alexandra, Trakht Ilya
Division of Experimental Therapeutics, Department of Medicine, College of Physicians and Surgeons, Columbia University in New York City, New York 10032, USA.
Monoclon Antib Immunodiagn Immunother. 2013 Aug;32(4):246-54. doi: 10.1089/mab.2013.0003.
There are many methods for evaluating the cytotoxic effect of monoclonal antibodies (MAbs) against cancer cells. Most of these methods require either purified MAbs or biological solutions (e.g., cell culture supernatants, ascitic fluids) containing high concentrations of MAbs. This makes the primary screening of antibody-producing hybridomas for specific cytotoxic antibodies a challenging task. Addressing this issue, this work introduces a high throughput screening method, which enables the identification of cytotoxic antibodies using primary hybridoma populations without prior antibody concentration and/or purification. The method is comprised of a dual-chamber system, where antibody-producing hybridomas and target cancer cells are co-cultured but separated by a porous membrane in which the pore size is sufficient for the diffusion of antibody molecules. The MAbs produced in the system continuously diffuse through the membrane between the two chambers and interact with the target cells placed on the other side of a membrane, resulting in death or proliferation arrest of these cells, if MAbs are cytotoxic or cytostatic. The cytotoxic/cytostatic effect can be registered by measuring the viability of target cells. The advantage of this method is that purification or concentration of antibodies secreted by hybridomas is not required. In addition, this method does not require MAb-secreting hybridomas, which are subcloned or have a high level of MAb production. The method may serve as an effective primary high throughput screening for cytotoxic antibodies.
评估单克隆抗体（MAb）对癌细胞的细胞毒性作用有多种方法。这些方法大多需要纯化的单克隆抗体或含有高浓度单克隆抗体的生物溶液（如细胞培养上清液、腹水）。这使得针对特异性细胞毒性抗体的产生抗体的杂交瘤的初次筛选成为一项具有挑战性的任务。为了解决这个问题，这项工作引入了一种高通量筛选方法，该方法能够使用原始杂交瘤群体来鉴定细胞毒性抗体，而无需事先进行抗体浓缩和/或纯化。该方法由双室系统组成，其中产生抗体的杂交瘤和靶癌细胞共同培养，但由多孔膜隔开，膜的孔径足以使抗体分子扩散。系统中产生的单克隆抗体持续通过膜在两个腔室之间扩散，并与置于膜另一侧的靶细胞相互作用，如果单克隆抗体具有细胞毒性或细胞抑制作用，会导致这些细胞死亡或增殖停滞。细胞毒性/细胞抑制作用可以通过测量靶细胞的活力来记录。该方法的优点是不需要对杂交瘤分泌的抗体进行纯化或浓缩。此外，该方法不需要分泌单克隆抗体的杂交瘤是亚克隆的或具有高水平的单克隆抗体产生。该方法可作为一种有效的细胞毒性抗体高通量初次筛选方法。
