Abstract
So-called “non-specific bindings” are troublesome problems in flow cytometric surface marker assay with monoclonal antibodies. Especially in the detection of weakly positive populations and in the fine evaluation of fluorescence intensity, it should be resolved for practical purpose.
We examined the reactivities of mouse serum IgG fractions and mouse monoclonal isotypic control antibodies to some cell lines bearing Fc receptor, and also referred these subclass specificities to other monoclonal antibodies against defined surface markers. Well controlled staining procedures and use of selected monoclonal antibodies and fluorochromic anti-mouse antibodies disclosed that most cause of the non-specific binding was thought to be deduced to the antibody binding activities of Fc receptors.
We attempted to eliminate Fc receptor derived non-specific binding by adding a step to the conventional staining procedures. The pretreatment of the cells with commercial human gamma globulins was quite effective in the neglection of false positivities of control antibodies. However this simple but useful technique might have some adverse possibilities that it decreases the fluorescence intensity of some special markers, for example Leu-11a (antibody directed against Fc receptor).
Negative control staining with control antibodies is essential for practical surface marker assay and non-specific binding can be prohibited by blocking of Fc receptor with human globulins in most cases.
