An immunoenzymatic staining assay (ISA) for the rapid screening of monoclonal antibodies detecting membrane and cytoplasmic antigens

Abstract

We report a rapid and very sensitive immunoenzymatic staining assay (ISA) for the determination of the fine specificity of monoclonal antibodies directed against cellular antigens. Reactivity is analysed at the single cell level by light microscopy using alkaline phosphatase-conjugated second and third antibodies on cells bound to Terasaki plates. Reactive cells can be defined by their morphology even in preliminary screening procedures. Only small numbers of cells are necessary for this assay which is comparable to radioimmunoassays in its sensitivity. Plates with fixed cells can be stored at 4°C so that a permanent ‘library’ of various cell types is always immediately available for use. The test is also suitable for human blood cell phenotyping. Moreover, a simple modification of the procedure by short pretreatment of the cells with the detergent Brij allows the detection of antigens within the cytoplasm. The importance of evaluating the cytoplasmic compartment in order to define antibody specificity and study the distribution of different cytoplasmic and membrane antigens is emphasized.