Hapten-sandwich labeling. III. Bifunctional reagents for immunospecific labeling of cell surface antigens.

Bifunctional reagents have been prepared which permit extensive coupling of haptens to antibodies with retention of antibody-binding capacity. This modification procedure increases the sensitivity with which cell-surface antigens can be labeled for fluorescent or electron microscopy by the hapten-sandwich method to a level comparable to or greater than may usually be achieved by conventional indirect techniques. With two different haptens, and amplifying with anti-hapten antibodies bearing fluorescent or other markers, different antigens can be distinguished readily on a single cell or on separate cells. Mouse alloantigens detected only with difficulty by conventional fluorescence can be discerned clearly and with high specificity. The reagent, either methyl hydroxybenzimidate (HB) or methyl 3,5-dihydroxybenzimidate (DHB), is first reacted with a diazonium phenyl hapten. Since the resulting azo-derivative retains the imido ester function, the reaction solution can be used directly to amidinate antibodies. Both HB and DHB are easily prepared and may be stored for use as needed. With these reagents, the hapten-sandwich procedure may be applied to proteins other than immunoglobulins, e.g., hormones and mitogens. In addition to labeling antigens for fluorescent or electron microscopy, haptenamidinated antibodies or other ligands may be used in conjunction with appropriately modified antihapten amplifiers for a variety of purposes in cell biology, such as radioisotope studies, selective cell killing or suppression, and isolation of membrane antigens.