An Enzyme-Linked Immunosorbent Assay for the Measurement of Circulating Antibodies to Polymorphic Epithelial Mucin (MUC1)

Abstract

Introduction: About one-third of breast and ovarian carcinoma patients have circulating antibodies reactive with polymorphic epithelial mucin (MUC1), either free or bound to immune complexes. While the presence of these immune complexes has prognostic significance in breast cancer patients, the significance of free MUC1 antibodies is less clear. The objective of this study was to develop a reliable assay for the accurate determination of circulating free antibodies to MUC1. Material and Methods: We developed an enzyme-linked immunosorbent assay (ELISA) (PEM.CIg) employing a 60 mer peptide (a triple tandem repeat sequence of the MUC1 peptide core) conjugated to bovine serum albumin and peroxidase-labeled antihuman immunoglobulin G or M antibodies. The assay was standardized and its analytical performance evaluated. A total of 492 serum samples were obtained from 40 healthy men, from 201 healthy women (including 55 women without a history of pregnancy and 45 pregnant women), and (before primary treatment) from 62 benign breast tumor patients and 190 breast cancer patients. MUC1 serum levels were determined with commercial CA 15-3 tests. Results: Circulating antibodies to MUC1 are present both in healthy subjects and in breast cancer patients. The within- and between-assay coefficients of variation were, respectively, 2 and 12% for the IgG determinations and 1.2 and 3% for the IgM determinations. Correlation coefficients for serially diluted serum samples ranged from 0.9998 to 0.9920 for IgG and from 0.9996 to 0.9818 for IgM determinations. The reactivity of serum samples was partially blocked by the addition of various MUC1 peptides and by MUC1 mucin. The inhibiting effect of modified 60 mer peptides suggests the presence of antibodies directed to more than one epitope. Conclusions: The PEM. CIg assay is a reliable ELISA for measuring free MUC1 antibodies in serum. We are in the process of relating the results obtained in the breast cancer group to disease outcome to evaluate its prognostic significance. In addition, the assay may become a useful tool for vaccine therapy monitoring.

Identification of Tumor-Specific Antibodies in Patients With Breast Cancer Vaccinated With Gene-Modified Allogeneic Tumor Cells

“…In some cases preabsorption of sera with FBS failed to eliminate binding to the lysate-coated plate in prevaccination sera. We attribute this to the possibility of antimucin antibodies that have been found in the sera of healthy women and breast cancer patients (20), or possibly antibodies to other breast cancer antigens. In conclusion, we found that a whole cell vaccine employing MB-231 transfected with CD80 was unable to induce high-titered antibody responses to breast cancer-associated antigens in a majority of patients.…”

“…The samples were tested for reactivity against a bovine serum albumin (BSA)-conjugated 60-mer MUC1 as described previously (20).…”

A Human Immunoglobulin G1 Antibody Originating from an in Vitro-Selected Fab Phage Antibody Binds Avidly to Tumor-Associated MUC1 and Is Efficiently Internalized

“…A Sensor Chip SA was coated with a biotinylated MUC1 15-mer, containing the epitope PAP [Ac-PDTRPAPGSTAPPAK-NH 2 (with a K as last amino acid for amino group-based biotinylation), 50 RU and 320 RU] and 60-mer [NH 2 -(VTSAPDTRPAPGSTAPPAHG) 3 -COOH, 17 50 RU] in Hepes Buffered Saline-EDTA P20 HBS-EP buffer (BIAcore). A surface, blocked with biotin (15 RU), was used as a negative control.…”

Antibody-dependent cell-mediated cytotoxicity can be induced by MUC1 peptide vaccination of breast cancer patients

“…23,45 MUC1 Ab detection in the plasma of the patients MUC1 Abs in the plasma of the patients were measured by the PEM.CIg assay. 46 In short, a BSA-conjugated MUC1 60-mer tandem-repeat peptide (250 ng/well in PBS) and 1% BSA were used to coat 96-well ELISA plates (Costar) in alternate duplicate columns overnight at room temperature. After washing with PBS/ 0.1% Tween-20, the plates were incubated with 1% BSA (Grade V; Sigma) for 3 hr at 37°C, washed and incubated overnight with the plasma samples in PBS ϩ 1% BSA ϩ 0.02% azide.…”