Detection and quantitation of HLA class II molecules on keratinocytes by quantitative immunofluorescence

Abstract

The paper describes measurements of HLA-DR antigen expression on normal human keratinocytes in culture using anti-HLA-DR antibodies and fluorescent Protein A or fluorescent second antibody methods and a low-light level video camera and image analysis program to quantitate the fluorescence output. The measurements are ultimately quantitated in terms of molecules of Ia antigen reacting material per cell. The method has a sensitivity at a S/N ratio of 10:1 of 180 molecules/μm 2. The results show that normal keratinocytes do indeed express class II antigens on their surfaces at levels well above background. We confirm that treatment of the cells with γ-interferon produces enhanced expression of DR antigens within 4 days. The method of quantitation is applicable to fluorescence and other low light level images.