Nature of antigens and antibodies in immune complexes isolated by staphylococcal protein A from plasma of melanoma patients

Abstract

Immune complexes (IC) were isolated from plasma of melanoma patients by absorption to staphylococcal protein A and subsequent elution with MgCl2. The isolated ICs were purified by precipitation with polyethylene glycol and sucrose density gradient ultracentrifugation after radioiodination with 125I. The purified ICs were dissociated and radiolabeled antigen/antibody components were separated by ultracentrifugation at low pH (2.6). Under these conditions, about 72% radioactivity of the purified IC remained in the light-density region as a wide band. After neutralization, 26%-60% radioactivity in the region of 5S sedimentation bound to immobilized autologous immunoglobulins, as opposed to a maximum of 23% to immobilized immunoglobulins from human normal serum. Significant levels (73%-77%) of radioactivity in 7S region bound to rabbit anti-human IgG immunobeads. Immunoprecipitation of the antigen fraction by allogeneic anti-melanoma and rabbit anti-melanoma antibodies followed by SDS-polyacrylamide gel electrophoresis revealed the presence of a fetal antigen (FA) and a melanoma tumor-associated antigen (TAA). In addition, the presence of auto-antigen(s) was indicated by using autologous antibody in immunoprecipitation. Immunoglobulins (IgG) isolated from purified IC bound to cultured melanoma, sarcoma, and normal fibroblasts, although the binding to sarcoma and normal fibroblasts could be inhibited by preincubation of isolated IgG with soluble FA but not with soluble melanoma TAA. Thus, results of this investigation provide evidence that circulating IC in melanoma patients are composed of at least IgG and different antigens, and some of these antigens are produced by their tumor.

Selective expression of inhibitory Fcγ receptor by metastatic melanoma impairs tumor susceptibility to IgG‐dependent cellular response

“…In addition, expression of FcγRIIB was found in a very high percentage of metastases from the liver, an organ with one of the highest levels of NK cell activity 45, 46. Surprisingly, the presence of elevated levels of circulating immune complexes has been reported in some studies to be positively correlated with disease status, tumor burden and worse prognosis47–51 suggesting that anti‐tumor IgG might be not protective per se . Several hypotheses could explain these unexpected observations.…”

Assay, Isolation and Characterization of Circulating Immune Complexes from Serum of Gastrointestinal Cancer, Stage III and IV Melanoma and Chronic Inflammatory Bowel Disease Patients

“…7-9 (2) pancreas (2) ' Values in parentheses are numbers of patients. albumin (64-72 kD) [17], each serum revealed a faslmigrating band consistent with a putative of approximate ly 71-76 kD. In 4 additional sera, a fast-migrating band wasdemonslrated in the region of 30-49 kD.…”

“…In recent years, a number of specific cellsurface human melanoma antigens (M A) [4][5][6][7][8][9], as w'ell as human melanoma tumor-associated antigens (MTAA) [10][11][12][13] have been isolated, characterized and purified us ing a variety of different techniques [13][14][15][16][17], In addition, high levels of circulating immune complexes (CIC) fre quently occur in malignant disease. In some cases, this is associated with advanced neoplastic disease or cancer re lapse [18], even though the relationship between tumor burden and CIC concentrations remains unclear [12][13][14][15], Moreover, the detection of antigen-specific CIC in sera from cancer patients provides an approach for defining tumor antigens able to induce a response from the host, and they could prove to be useful in so-called immunoprognosis [12,19,20].…”