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Simultaneous immunoenzyme double labelling using two different enzymes linked directly to monoclonal antibodies or with biotin‐avidin

A novel and rapid method for immunoenzyme double staining with monoclonal antibodies (McAb) is described. The principles of this method are the simultaneous application of primary antibodies and the unrelatedness of the two detection systems. One McAb is directly labelled with horseradish peroxidase and the other McAb is labelled with biotin. This second McAb is thereafter detected using an avidin‐alkaline phosphatase conjugate. This conjugate was prepared by a new method using a heterobifunctional reagent. Double staining of cell surface membranes of human tonsil was studied in cryosections using various combinations of McAbs to lymphoid cell markers. As expected, suppressor T cells were found to be contained within the pan T cell population on the basis of the distinguishable intermediate colour produced. Similarly, it was shown that most of the suppressor T cells were not HLA‐DR activated in this tonsil. In cryostat sections of human skin T6 positive Langerhans cells in the epidermis were shown to carry the HLA‐DR antigen.