Quantitative immunoautoradiography at the cellular level. II. Absolute measurements using labeled standard cells as a source of reference.

A quantitative autoradiographic method is presented for determining absolute amounts of 125I-labeled compounds on the surface of individual cells. Autoradiographic evaluation of single cell radioactivity is accomplished by comparing the silver grain densities over the specimen and a radioactive standard being exposed simultaneously. In order to obtain a reference source of comparable physical properties, surface-radioiodinated human erythrocytes are used, the radioactivity of which is determined in a crystal counter. A simple enzymatic method for preparing such standard erythrocytes of very uniform label density is described. Numerous experimental advantages derived from the use of the standard are discussed and demonstrated by examples employing various exposure times and different radioactive standards. Hereby, very similar results were obtained when the number of A-antigenic sites was quantified on single erythrocytes in different experiments. The quantification of membrane-bound IgM on single human lymphocytes is shown as another application of this scheme.