Detection of circulating anti-mucin 1 (MUC1) antibodies in breast tumor patients by indirect enzyme-linked immunosorbent assay using a recombinant MUC1 protein containing six tandem repeats and expressed in Escherichia coli

Abstract

Mucin 1 (MUC1), a tumor-associated antigen, is a transmembrane glycoprotein expressed by normal epithelial cells and overexpressed by carcinomas of epithelial origin. Autoantibodies against MUC1 are often found in circulation, either free or bound to immune complexes, which might contribute to limit tumor outgrowth and dissemination by antibody-dependent cell-mediated cytotoxicity, and were found favorably predictive of survival in early breast cancer patients. There is no commercial enzyme-linked immunosorbent assay (ELISA) kit for detecting the anti-MUC1 antibodies in human serum thus far. To detect circulating anti-MUC1 antibodies, we established an indirect ELISA (I-ELISA) using a recombinant MUC1 protein containing six tandem repeat sequences of MUC1 after the antigenicity and specificity of the protein were confirmed. The I-ELISA had a sensitivity of 91.3% and a specificity of 94.1% when a competitive I-ELISA was used as a reference test. The results showed that more patients with benign breast tumors (P = 0.001) and breast cancer patients before primary treatment (P = 0.010) were found to have anti-MUC1 IgG than healthy women; anti-MUC1 IgG before primary treatment was found more than after primary treatment (P = 0.016) in breast cancer patients. Interestingly, the anti-MUC1 IgG serum level was reversely correlated to that of CA15-3 antigen in advanced-stage patients (r = -0.4294, P = 0.046). Our study has demonstrated the suitability of the established I-ELISA for detecting circulating anti-MUC1 antibodies in human serum. Furthermore, we found that circulating anti-MUC1 antibodies may still bind MUC1 shed into blood in stage IV breast cancer, which can support the use of MUC1-target immune therapy strategies.

FIG. 1.

Construction, expression, and identification of recombinant 8R-MUCPT. (A) Structure of 8R-MUCPT protein. (B) Analysis of recombinant 8R-MUCPT protein expressed in E. coli on an SDS-10% PAGE gel. Lane M, marker; lane 1, lysate of E. coli induced by IPTG; lane 2, lysate of E. coli without induction. (C) Analysis of purified 8R-MUCPT protein on an SDS-10% PAGE gel. Lane M, marker; lane 1, purified 8R-MUCPT protein. (D) Identification of purified 8R-MUCPT protein by Western blotting with anti-MUC1 VNTR MAb. The arrows indicate 8R-MUCPT protein.

FIG. 2.

Analysis of the antigenicity and specificity of 8R-MUCPT as detecting antigen. (A) Immunohistochemical staining of breast cancer sections by anti-MUC1 VNTR MAb (left) or anti-8R-MUCPT serum (right). (B) Dot blot and inhibition test. 8R-MUCPT was spotted onto a nitrocellulose filter in four dosages. The serum reactivity to 8R-MUCPT could be abolished by preincubation with anti-MUC1VNTR MAb but not by preincubation with anti-HBSAg MAb. (C) Blocking experiment. The sera of four patients with breast cancer were mixed with 8R-MUCPT, poly-R, or poly-H in equal molar rations, and then their anti-MUC1 antibody levels were detected by the I-ELISA. The serum reactivity to 8R-MUCPT in an I-ELISA could be blocked by being mixed with 8R-MUCPT, but poly-R or poly-H could not block this reactivity. (D) CI-ELISA. 8R-MUCPT was captured by anti-MUC1 VNTR MAb as a coating antigen. The serum samples were directly added into the wells or mixed with three different concentrations of anti-MUC1 VNTR MAb (as a competitive antibody), respectively, before addition to the well. Each point represents the average OD of triplet wells. There was growing inhibition with increasing concentrations of competitive antibody, and the inhibition rate induced by the competitive antibody at 3 μg/ml was at least 30% for the positive serum. There was no inhibition for the negative serum.

FIG. 3.

Relationship between circulating CA15-3 antigen and anti-MUC1 IgG in patients with the advanced stage. The levels of anti-MUC1 IgG in CA15-3 antigen-positive and -negative sera from the advanced-stage patients were measured by I-ELISA using 8R-MUCPT as a coating antigen. The horizontal line represents the cutoff value of CA15-3 antigen. The vertical line represents the cutoff value of anti-MUC1 IgG (r = 0.4294, P = 0.046 [serum CA15-3 antigen versus anti-MUC1 IgG]).