Vance B A, Karlson K H, Morganelli P M, Guyre P M
Department of Physiology, Dartmouth Medical School, Hanover, NH 03756.
J Immunol Methods. 1989 Mar 31;118(2):287-96. doi: 10.1016/0022-1759(89)90018-5.
Interferon-gamma (IFN-gamma) activation of human monocytes in vitro results in enhanced phagocytosis and cellular cytotoxicity. These enhanced effector functions are attributable, at least in part, to increased expression of recognition molecules on the plasma membrane. In this article we report a rapid screening procedure for the primary selection of monoclonal antibodies (mAbs) which bind to cell surface molecules, the expression of which is increased or decreased by IFN-gamma. The procedure is based on flow cytometric analysis of mixed cell populations. Mouse mAbs were prepared using human monocytes cultured for 40 h with 400 U/ml of IFN-gamma as the immunogen. The hybridoma supernatants were screened using mixtures of six cell populations, some of which were pretreated with IFN-gamma for 40 h. Cells included in the mixture were chosen for their distinctive light scatter profiles. mAbs of interest were identified by preferential binding to monocytes and increased or decreased binding to monocytes treated with IFN-gamma. This procedure allowed us to screen several hundred clones per day, and to immediately eliminate mAbs that bound to B cells, T cells, neutrophils, and several cell lines. We selected ten mAbs which bound to surface molecules on monocytes that were modulated by IFN-gamma. Further characterization of five of the initial ten mAbs revealed that mAb gamma M phi 22.2 and mAb gamma M phi 197.1 bind to the high affinity Fc receptor for IgG (Fc gamma RI). mAb gamma M phi 28.3 appears to bind to a class II histocompatibility antigen and mAb gamma M phi 150.3 and mAb gamma M phi 195.18 appear to have binding patterns to human leukocytes and cell lines which are distinct from previously described mAbs. This rapid and specific procedure for screening mAbs has broad application for selecting mAbs that are specific for any given cell type and/or for surface molecules that are modulated by any cytokine and other hormone.
体外γ干扰素（IFN-γ）激活人单核细胞可增强吞噬作用和细胞毒性。这些增强的效应功能至少部分归因于质膜上识别分子表达的增加。在本文中，我们报告了一种快速筛选程序，用于初步筛选与细胞表面分子结合的单克隆抗体（mAb），这些分子的表达会因IFN-γ而增加或减少。该程序基于混合细胞群体的流式细胞术分析。使用以400 U/ml IFN-γ培养40小时的人单核细胞作为免疫原制备小鼠单克隆抗体。使用六个细胞群体的混合物筛选杂交瘤上清液，其中一些细胞群体用IFN-γ预处理40小时。混合物中包含的细胞因其独特的光散射特性而被选择。通过优先结合单核细胞以及与用IFN-γ处理的单核细胞的结合增加或减少来鉴定感兴趣的单克隆抗体。该程序使我们每天能够筛选数百个克隆，并立即排除与B细胞、T细胞、中性粒细胞和几种细胞系结合的单克隆抗体。我们选择了十种与单核细胞表面分子结合的单克隆抗体，这些分子受IFN-γ调节。对最初十种单克隆抗体中的五种进行的进一步表征表明，单克隆抗体γMφ22.2和单克隆抗体γMφ197.1与IgG的高亲和力Fc受体（FcγRI）结合。单克隆抗体γMφ28.3似乎与II类组织相容性抗原结合，单克隆抗体γMφ150.3和单克隆抗体γMφ195.18似乎对人白细胞和细胞系具有与先前描述的单克隆抗体不同的结合模式。这种快速且特异的单克隆抗体筛选程序在选择针对任何给定细胞类型和/或受任何细胞因子和其他激素调节的表面分子的单克隆抗体方面具有广泛应用。
