The quantitation of HLA-DR expression on human cells using immunocytochemistry

An immunocytological method that can be used to quantify the comparative expression of Class II HLA-DR antigens on human cells has been developed. Tissue sections or cytospins are incubated with the monoclonal antibody RFDR1 (anti-HLA-DR), conjugated to the enzyme glucose oxidase (GO). The bound McAb is identified by incubation with the substrate beta-D-glucose and capture of the reducing equivalents generated by a tetrazolium salt forming an insoluble formazan. Under standard conditions the amount of formazan precipitated is proportional to the amount of McAb bound to the cells or tissue and thus the amount of HLA-DR expressed. The reaction has been quantified both by spectrophotometry (on tissue sections) and by microdensitometry (on cell spreads). Computer analysis has been used to relate density of HLA-DR expression to cell area. Repeated 'blind' measurements by separate investigators have confirmed the reproducibility of the results. It is suggested that this method adds a new dimension to immunohistological/cytological analysis of tissues and single cells.