Single step screening of monoclonal antibodies against interferon-γ-induced surface molecules on human monocytes

Abstract

Interferon-γ (IFN-γ) 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-γ. 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-γ as the immunogen. The hybridoma supernatants were screened using mixtures of six cell populations, some of which were pretreated with IFN-γ 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-γ. 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-γ. Further characterization of five of the initial ten mAbs revealed that mAb γMθ 22.2 and mAb γMθ 197.1 bind to the high affinity Fc receptor for IgG (FcγRI). mAb γMθ 28.3 appears to bind to a class II histocompatibility antigen and mAb γMθ 150.3 and mAb γMθ 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.