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Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells
Published on: June 5, 2015
Development of monoclonal antibodies to the human breast carcinoma cell line PMC42
Abstract:
In an attempt to identify antigens expressed during breast differentiation, three murine monoclonal antibodies, CIBr2, CIBr7, and CIBr18, were produced against the human pleomorphic breast carcinoma cell line PMC42. All three monoclonal antibodies reacted with previously undescribed antigenic determinants on the PMC42 cell line. Antibody CIBr18 reacted only with the immunizing cell line PMC42, whereas antibodies CIBr2 and CIBr7 showed minimal reactivity toward a panel of 34 human leukemia- and solid tumor-derived cell lines. The antigenic determinants detected by the three antibodies were distinct, and each showed variable expression in PMC42 monolayer and organoid cultures. The heterogeneity of staining seen on PMC42 cultures may reflect the fact that this cell line contains up to eight morphologically distinct cell types. Antigen expression correlated with cell type in some instances, whereas in other instances phenotypic subdivision within a cell type was apparent. Antigens recognized by antibodies CIBr7 and CIBr18 were characterized biochemically. In Western blotting, antibody CIBr7 identified a single band of an apparent molecular weight of 38,000 within PMC42 cell lysates. Sodium dodecyl sulfate-polyacrylamide gel analysis of polypeptides immunoprecipitated by antibody CIBr18 from [35S]methionine-labeled PMC42 cell lysates identified two glycoproteins of apparent molecular weights of 115,000 and 120,000, respectively. No biochemical data for the CIBr2 antigen are yet available. All three antigens were detected in human mammary epithelium and some non-breast tissues. The expression of these antigens in normal and neoplastic mammary epithelia is discussed in terms of antigen heterogeneity and changes in antigen expression upon conversion to the malignant state.
Insights
Researchers identified novel breast cancer antigens using monoclonal antibodies against the PMC42 cell line. These antigens show variable expression, potentially reflecting cell type heterogeneity in breast differentiation and malignancy.
Area of Science:
- Immunology
- Oncology
- Cell Biology
Background:
- Identifying specific antigens during breast differentiation is crucial for understanding breast cancer.
- The human pleomorphic breast carcinoma cell line PMC42 presents a unique model for studying breast-specific antigens.
Purpose of the Study:
- To produce and characterize novel monoclonal antibodies targeting antigens expressed during breast differentiation.
- To investigate the expression patterns and biochemical properties of these newly identified breast cancer antigens.
Main Methods:
- Production of three murine monoclonal antibodies (CIBr2, CIBr7, CIBr18) against the PMC42 cell line.
- Reactivity testing against a panel of human tumor cell lines and analysis of expression in PMC42 cultures.
- Biochemical characterization using Western blotting and immunoprecipitation.
Main Results:
- Three novel antigenic determinants were identified on the PMC42 cell line.
- Antibodies CIBr2 and CIBr7 showed limited reactivity with other tumor cell lines, while CIBr18 was specific to PMC42.
- Antigen expression varied across different cell types within the PMC42 line, indicating heterogeneity.
- Biochemical analysis revealed a 38 kDa antigen for CIBr7 and two glycoproteins (115 kDa, 120 kDa) for CIBr18.
- All identified antigens were found in human mammary epithelium and some non-breast tissues.
Conclusions:
- The study identified novel antigens associated with breast differentiation and cancer.
- Antigen heterogeneity in breast carcinoma cell lines reflects cellular diversity.
- These antigens may serve as biomarkers for breast cancer research and diagnostics.
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