Simultaneous detection of two independent antigens by double staining with two mouse monoclonal antibodies

Simultaneous detection of two antigens by immunostaining usually requires primary antibodies from two different species or a hapten modification of one of the antibodies if they are from the same species. A novel double staining method is described for immunodetection of two independent antigens using two mouse monoclonal antibodies. The principle of the method is the following: The first antigen is detected by a monoclonal antibody that is diluted so extensively that it cannot be recognized with conventional detection systems. A highly sensitive biotin-tyramide amplification system is used to identify this antibody. The second antigen is stained with a monoclonal antibody by dilution and detected by conventional immunostaining. The method was tested for both alkaline-phosphatase staining on paraffin sections and immunofluorescence staining on cultured cells in cytospin preparation. The absence of cross-reaction in the former system was demonstrated by the mutually exclusive detection of T- and B-cells in human lymph nodes or T-cells and carcinoma cells in nasopharyngeal carcinoma biopsies. Similarly, the EBV encoded EBNA2 and ZEBRA proteins showed a mutually exclusive staining by immunofluorescence on B95-8 cells. The method could be used to demonstrate co-expression of two independent antigens in the same cells, such as PCNA and keratin in carcinoma cells in paraffin sections and for EBNA2 and LMP1 EBV proteins in immunofluorescence preparations of B95-8 cells.