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Measurement of Local M_r 97,000 and 250,000 Protein Antigen Concentration in Sections of Human Melanoma Tumor Using in Vitro Quantitative Autoradiography¹

Nuclear Medicine Department, Clinical Center, NIH, Bethesda, Maryland 20892 [S. D., J. C. R., R. G. B., R. D. N., J. A. C., S. M. L.], and Oncogen, Inc., Seattie, Washington [I. H.]

An assay method that uses ¹²⁵I-labeled monoclonal antibody (MoAb) and in vitro quantitative autoradiography was developed to determine the local concentration of tumor-associated antigens in tissue sections. Human melanoma biopsy specimens were evaluated for the expression of the M_r 97,000 and 250,000 protein antigens using MoAb-96.5 and MoAb-9.2.27, respectively. Tissue sections were incubated in solutions of increasing concentration of ¹²⁵I-labeled MoAb with or without an excess of unlabeled antibody. Quantitative autoradiography was performed on the sections and compared with ¹²⁵I standards to determine tumor-bound radioactivity and calculate bound pmol of MoAb per g of tumor. The total binding, nonsaturable binding, and specific binding of ¹²⁵I-labeled MoAb to tumor were then computed. Specific binding of MoAbs to tumor tissue was saturable in all antigen-positive tumors. The maximal concentration of specific binding of antibody to tissue (B_max) represented the tissue antigen concentration. Estimates of the K_a of antigen/antibody binding were also made. The reliability of the measurements was confirmed by testing sections from mixtures of antigen-positive and antigen-negative cells.

¹ Preliminary report at the European Nuclear Medicine Congress, August 25, 1987.

² Permanent address: Istituto di Medicina Nucleare, II Facolta' di Medicina e Chirurgia, Napoli, Italy.

³ To whom requests for reprints should be addressed, at NIH, Building 10, Room 1C-401, 9000 Rockville Pike, Bethesda, MD 20892.

Received 1/21/88. Revised 6/25/88. Accepted 7/ 5/88.

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