Nonlabeled secondary antibodies augment/maintain the binding of primary, specific antibodies to cell membrane antigens

Background In studies on surface membrane antigen expression using immunofluorescence techniques, it is commonly observed that direct staining gives weaker signals than the signals following indirect staining with fluorochrome‐conjugated secondary antibodies. This is most marked when cells have also been permeabilized in order to stain intracellular protein. The commonly accepted explanation for this observation is that fluorochrome‐conjugated secondary antibodies bind to a higher number of binding sites on the primary antibody, as compared to the binding of conjugated primary antibodies to the membrane antigens. Another hypothesis might be that the antibody/antibody complexes formed on the membranes when using the indirect technique may have an augmented ability to bind the membrane epitopes. The present study was performed in order to check this hypothesis. Materials and Methods Peripheral blood mononuclear cells were stained with fluorochrome‐conjugated anti‐CD antibodies directly without or with a second‐step application of nonconjugated goat anti‐mouse IgG antibodies, followed by different fixation and permeabilization methods. The cells were analyzed by flow cytometry. Results A second‐step application of nonconjugated goat anti‐mouse IgG antibodies following direct staining with fluorochrome‐conjugated anti‐CD antibodies gave a significant increase in membrane antigen expression on permeabilized cells as compared to direct staining alone. The secondary antibody must be bivalent, since whole IgG or F(ab′) 2 fragments of the goat anti‐mouse antibodies showed effects, while Fab fragments did not. Conclusions Nonlabeled secondary antibodies are able to influence the binding of primary, specific antibodies to cell membrane antigens on cells treated with permeabilizing agents necessary for staining intracellular proteins. The improved membrane antigen expression seems to be due to the formation of a network of primary and secondary antibodies on the cell surface, with increased ability for maintaining binding to CD antigens. Cytometry 45:187–193, 2001. © 2001 Wiley‐Liss, Inc.