Abstract
A double fluorescent antibody method for quantitating human complement-fixing antibody to lymphocyte subclasses has been developed. The indicators in this system are a C6-deficient serum as a non-lytic source of complement, rhodamine-labeled anti-C3 and fluorescein-labeled murine monoclonal antibodies to human lymphocyte subsets. The basic procedure is to incubate lymphocytes with the unknown serum and then to add C6-deficient serum. The binding of C3 is indicated by staining with rhodamine-labeled anti-C3 and the subset class of the lymphocyte so stained is determined by binding of fluorescein-tagged anti-OKT4 or OKT8 antibodies. The occurrence of both redand green cell surface fluorescence denotes the presence of a complement-binding antibody to the lymphocyte subset defined by the monoclonal antibody. In addition to defining the specificity of complement-fixing anti-lymphocyte antibodies this technique is more sensitive than the microcytotoxicity assay.
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