The sequential method of adsorption analysis was applied to antiglioma radioantibodies adsorbed by intact monolayer cultures of the C6 rat astrocytoma cell line. A noncentrifugal technique was devised to permit transfer of radioantibody solutions from one culture to the next in a sequence and to effect removal of unbound radioglobulins. Primary binding of 125I-labeled antibody immunoglobulin G (IgG) was readily measured in a particularly sensitive and reproducible manner over and above control IgG. With proportions varying from one serum to the next, several individual antiglioma radioantibody preparations, after preadsorption three times with rat liver, still retained cross-reactivity for fibroblast cultures and, after preadsorption three times with fibroblasts, still retained selective reactivity for glioma cells. When adsorbed with glioma cells first, the bulk of all binding activity remained with the glial cells. When unlabeled antibody IgG in varying amounts was mixed with fixed quantities of labeled antibody IgG and glioma cells, an equivalence point was established by competitive binding curves and, independently, by microscopic examination of the monolayer-antibody complexes after light trypsinization: cell clumps in excess antibody, free cells otherwise. parameters of IgG binding in one antibody-cell combination were Ka = 2.1 × 106 m-1; a = 0.75; and s = 6.9 × 107 antigenic sites/cells. When two cloned sublines of the S-77 line of the C6 astrocytoma were used sequentially along with fibroblasts to adsorb a radioantibody preparation from one particular anti-S-77 antiserum in nine steps, four different antigenic moieties were clearly distinguished: a cross-reaction between fibroblasts and glioma cells; a cross-reaction between the two sublines that was not shared by fibroblasts; and a distinctive reaction in each of the two sublines that was not shared by the other subline or by fibroblasts.
This work was supported by Grants CA-14651 and CA-11898 from the National Cancer Institute and by Grant NS-10237 from the National Institute of Neurologic Diseases and Stroke, NIH, Bethesda, Md.