THE UNLABELED ANTIBODY ENZYME METHOD OF IMMUNOHISTOCHEMISTRY MOLECULAR IMMUNOCYTOCHEMISTRY OF ANTIBODIES ON THE ERYTHROCYTE SURFACE

DENNIS M. HINTON ¹, JOHN P. PETRALI ¹, HOWARD G. MEYER ¹, and LUDWIG A. STERNBERGER ²

¹ Basic Sciences Department, Medical Research Laboratory, Edgewood Arsenal, Maryland 21010
² Basic Sciences Department, Medical Research Laboratory, Edgewood Arsenal, Maryland 21010 and Department of Microbiology, The Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205

The number of discrete unit stains on the surface of sheep erythrocytes enumerated by electron microscopy after preembedding staining using ¹²⁵I antierythrocyte antibody (anti-E) and the unlabeled antibody enzyme method was correlated with enzyme activity and radioactivity of lysed cell suspensions. Purified anti-E was applied in a concentration range resulting in the binding of 10-2,600,000 molecules/cell. Sheep serum antirabbit immunoglobulin and peroxidase-antiperoxidase complex were used in excess. With a new light-scattering assay for peroxidase the sensitivity of measurement of enzyme activity on suspensions of cell stromata exceeded that of radioactivity assay allowing measurement of as little as 5 x 10^–13 mmole antibody/ml. Discrete unit stains on cell sections were visualized to 1.5 x 10⁴ anti-E/cell. Above this number, staining occurred in patches of increasing size but was still discontinuous to 3 x 10⁴ anti-E bound/cell. Above 2-3 x 10⁴ anti-E bound/cell continuous staining of the membrane was observed. Assuming random distribution of bound anti-E, the number of discrete unit stains visualized per cell section was formulated as a probability function. The observed numbers of unit stains per section agreed well with the values so formulated to 8,000 anti-E/cell. Thus, enzyme activity of peroxidase-antiperoxidase, in suspensions or by enumeration of unit stains by electron microscopy, served as a sensitive quantitative measure of specific antibody bound per cell. The data permit the interpolation that staining of sparsely distributed cell surface antigens by the unlabeled antibody enzyme method with excess antibody permits the enumeration of antigen sites. In addition, mobility of surface antigens may be quantitated by comparison of observed and predicted numbers of discrete unit stains per section.

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