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

Background. in studies on surface membrane antigen expression using immunof luorescence techniques, it is commonly observed that direct staining gives weaker signals than the signals following indirect staining with fluorochro me-conjugated secondary antibodies. This is most marked when cells have als o been permeabilized in order to stain intracellular protein. The commonly accepted explanation for this observation is that fluorochrome-conjugated s econdary antibodies bind to a higher number of binding sites on the primary antibody, as compared to the binding of conjugated primary antibodies to t he membrane antigens. Another hypothesis might be that the antibody/antibod y complexes formed on the membranes when using the indirect technique may h ave an augmented ability to bind the membrane epitopes. The present study w as 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 seco nd-step application of nonconjugated goat anti-mouse IgG antibodies, follow ed by different fixation and permeabilization methods. The cells were analy zed by flow cytometry. Results. A second-step application of nonconjugated goat anti-mouse IgG ant ibodies following direct staining with fluorochrome-conjugated anti-CD anti bodies gave a significant increase in membrane antigen expression on permea bilized 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-m ouse antibodies showed effects, while Fab fragments did not. Conclusions. Nonlabeled secondary antibodies are able to influence the bind ing of primary, specific antibodies to cell membrane antigens on cells trea ted with permeabilizing agents necessary for staining intracellular protein s. The improved membrane antigen expression seems to be due to the formatio n of a network of primary and secondary antibodies on the cell surface, wit h increased ability for maintaining binding to CD antigens. Cytometry 45: 1 87-193, 2001. (C) 2001 Wiley-Liss, Inc.