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 Mr 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 (Bmax) represented the tissue antigen concentration. Estimates of the Ka 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.
↵3 To whom requests for reprints should be addressed, at NIH, Building 10, Room 1C-401, 9000 Rockville Pike, Bethesda, MD 20892.
- Received January 21, 1988.
- Revision received June 25, 1988.
- Accepted July 5, 1988.
- ©1988 American Association for Cancer Research.