Development and Characterization of a New Polyclonal Antibody Specifically against Tissue Inhibitor of Metalloproteinases 4 in Human Breast Cancer

• Published in: Biochemical and biophysical research communications Vol. 281; no. 1; pp. 166 - 171
• Main Authors: Hurst, Douglas R., Li, Hui, Xu, Xinyun, Badisa, V.Lakshmi D., Shi, Y.Eric, Amy Sang, Qing-Xiang
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 16.02.2001
• Subjects: Antibodies; Apoptosis; Breast Neoplasms - immunology; Cell Division; Dose-Response Relationship, Drug; Dose-Response Relationship, Immunologic; enhanced chemiluminescence (ECL); enzyme regulation; Enzyme-Linked Immunosorbent Assay; enzyme-linked immunosorbent assay (ELISA); extracellular matrix (ECM); Extracellular Matrix - metabolism; Female; human breast cancer; Humans; Immunoblotting; Immunoglobulin G - metabolism; Immunohistochemistry; Luminescent Measurements; Matrix Metalloproteinase 1 - metabolism; matrix metalloproteinases (MMPs); new TIMP-4-specific antibody; proliferation and apoptosis; Tissue Inhibitor of Metalloproteinase-1 - immunology; Tissue Inhibitor of Metalloproteinase-2 - immunology; Tissue Inhibitor of Metalloproteinase-4; Tissue Inhibitor of Metalloproteinases - immunology; tissue inhibitors of metalloproteinase 4; tissue inhibitors of metalloproteinases (TIMPs); Transfection; Tumor Cells, Cultured; matrix metalloproteinases (MMPs); extracellular matrix (ECM); proliferation and apoptosis; enzyme-linked immunosorbent assay (ELISA); tissue inhibitors of metalloproteinases (TIMPs); new TIMP-4-specific antibody; enzyme regulation; human breast cancer; enhanced chemiluminescence (ECL); immunohistochemistry
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1006/bbrc.2000.4319

Tissue inhibitors of metalloproteinases (TIMPs) may regulate extracellular matrix turnover and cellular functions by modulating matrix metalloproteinase (MMP) activity and cell proliferation and apoptosis. To investigate the locations and functions of TIMP-4 in human breast cancer, a highly specific polyclonal anti-TIMP-4 peptide antibody (pAb-T4-S61) was developed. The potency and specificity of the purified IgG were characterized by an enzyme-linked immunosorbent assay, immunoblot, and immunohistochemistry. The optimal IgG concentration range was 0.1–10 μg/ml. pAb-T4-S61 did not cross-react with TIMP-1 and TIMP-2 and should not react with TIMP-3 according to the sequence analysis. Parental MDA-MB-435 breast cancer cells were TIMP-4 negative and a TIMP-4 transfected clone, TIMP-4-435-12, produced TIMP-4. Membrane type-1 MMP was detected although TIMP-2 was not found in these cells. Interestingly, the TIMP-4 protein was detected by immunohistochemical staining in infiltrating breast carcinoma cells in tumor tissues. Thus, pAb-T4-S61 is a useful tool to investigate expression patterns and functions of TIMP-4 in cancers.