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

https://doi.org/10.1006/bbrc.2000.4319Get rights and content

Abstract

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.

References (20)

  • K. Brew et al.

    Biochim. Biophys. Acta

    (2000)
  • A.Y. Strongin et al.

    J. Biol. Chem.

    (1995)
  • G.S. Butler et al.

    J. Biol. Chem.

    (1998)
  • T. Kinoshita et al.

    J. Biol. Chem.

    (1998)
  • G.I. Goldberg et al.

    J. Biol. Chem.

    (1992)
  • H.F. Bigg et al.

    J. Biol. Chem.

    (1997)
  • W.G. Stetler-Stevenson et al.

    J. Biol. Chem.

    (1990)
  • M. Wick et al.

    J. Biol. Chem.

    (1994)
  • J. Greene et al.

    J. Biol. Chem.

    (1996)
There are more references available in the full text version of this article.

Cited by (15)

  • Protein engineering and properties of human metalloproteinase and thrombospondin 1

    2002, Biochemical and Biophysical Research Communications
    Citation Excerpt :

    Antibody production and purification. According to our protocol previously reported for producing and characterizing anti-tissue inhibitor of metalloproteinase-4 Ab [24], the pre-immunization sera were collected from the New Zealand white rabbits 4 weeks before the initial injection. The peptide antigens (one milligram of each) were coupled to Keyhole Limpets Hemocyanin (Sigma Chemical) with 2% glutaraldehyde in 0.2 M phosphate buffer at a pH of 7.3.

  • Inhibitory antibodies against endopeptidase activity of human adamalysin 19

    2001, Biochemical and Biophysical Research Communications
View all citing articles on Scopus

Supported in part by grants from the National Institutes of Health (CA78646), the Elsa U. Pardee Foundation, the Gustavus and Louise Pfeiffer Research Foundation (to Q.-X.A.S.), and the Department of Defense Predoctoral Fellowship (DAMD17-00-1-0243, to D.R.H.).

☆☆

Abbreviations used: Ab, antibody; ECL, enhanced chemiluminescence; ECM, extracellular matrix; ELISA, enzyme-linked immunosorbent assay; MMP, matrix metalloproteinase; MMP-2, matrix metalloproteinase 2, gelatinase A; MT1-MMP, membrane type 1 matrix metalloproteinase; pAb-T4-S61, a polyclonal antibody against a TIMP-4 peptide starting from residue Ser61; TIMP, tissue inhibitor of metalloproteinases.

1

To whom correspondence should be addressed at Department of Chemistry, 203 Dittmer Laboratory of Chemistry Building, Florida State University, Tallahassee, FL 32306-4390. Fax: (850) 644-8281. E-mail: [email protected].

View full text