Simultaneous detection of two different cell surface antigens by electron microscopy by means of multivalent hybrid antibody with double specificity.

Multivalent hybrid antibody (MHA) complexes with dual specificity were prepared by combining two antibodies of different specificities, one against ferritin (Fer), the other against horseradish peroxidase (HRP), with protein A of Staphylococcus aureus (SpA). Electron microscopy of mouse spleen lymphocytes and thymocytes (previously coated with mouse IgG anti-Thy-1 antibody) treated with IgG anti-Fer/SpA/IgG anti-mouse Ig complex and Fer gave better resolution and higher accuracy than previously obtained with IgG anti-HRP/SpA/IgG anti-mouse Ig complex and HRP (Mandache et al., 1980). Surface Thy-1 alloantigen and Fc receptor (charged with human IgG) treated with a mixture of IgG anti-Fer/SpA/IgG anti-Thy-1 and IgG anti-HRP/SpA/IgG anti-human Fab could be simultaneously detected on the thymocyte surface by either light or electron microscopy using Fer and HRP. The concomitant visualization of Thy-1 alloantigen (with Fer) and FcR (with HRP) on mouse thymocyte clearly shows that their distribution is largely independent and that the amount of Thy-1 antigen is greater. These results show that electron microscopy with a mixture of MHA is a useful technique for simultaneous location of two antigenic markers on the cell surface.