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Journal of Histochemistry and Cytochemistry, Vol. 49, 1537-1546, December 2001, Copyright © 2001, The Histochemical Society, Inc.

ARTICLE

Characterization of a Polyclonal Antibody to Human Pituitary Tumor Transforming Gene 1 (PTTG1) Protein

Sham S. Kakara, Leilei Chena, Rashmi Puria, Shawn E. Flynna, and Lothar Jennesb
a James Graham Brown Cancer Center, University of Louisville, Louisville, Kentucky
b Department of Anatomy/Neurobiology, University of Kentucky, Lexington, Kentucky

Correspondence to: Sham S. Kakar, Dept. of Medicine, 570 South Preston Street, Baxter Biomedical Research Building, Room 204 C, U. of Louisville, Louisville, KY 40202. E-mail: sskaka01@louisville.edu

Pituitary tumor transforming gene 1 (PTTG1), recently cloned from human testis, is a potent oncogene that is expressed in most tumors. However, assessment of its potential value as a prognostic marker is dependent on the development of a suitable antibody. We have developed a rabbit polyclonal antibody, SK601, that is highly specific for the PTTG1 gene product using recombinant PTTG1 protein (24 kD) containing an N-terminal His6 tag as the immunogen. The antiserum is capable of detecting recombinant PTTG1 protein in ELISA assays at a titer of 1:100,000. Use of the antibody as the probe in Western blotting analyses revealed a single band with the anticipated relative molecular weights of 52 kD from E. coli expressing the GST-PTTG1 recombinant protein, and 56 kD from COS-7 cells transfected with the PTTG1–GFP chimeric construct. A single band with a relative molecular weight of 28 kD was observed in extract of COS-7 cells transfected with PTTG1 cDNA. The antiserum immunoprecipitated a protein of relative molecular weight of 56 kD from the extracts of COS-7 cells transfected with the PTTG1–GFP chimeric construct. Immunohistochemical analysis of COS-7 cells transfected with this construct confirmed that the antibody detected and was specific for expressing the PTTG1–GFP recombinant protein. Screening of various normal human tissues (testis, ovary, and breast) by immunohistochemistry indicated that these tissues did not exhibit staining with the exception of testis, a tissue that had previously been shown to express PTTG1 mRNA. In contrast all of the tumor tissues (testicular tumor, ovarian tumor, and breast tumor) that were assessed exhibited intense staining. The results suggest that antiserum SK601 is highly specific for the PTTG1 protein and therefore should prove useful in further analysis of the expression and interactions of this protein, including its potential application as an immunohistochemical marker of human tumors. (J Histochem Cytochem 49:1537–1545, 2001)

Key Words: PTTG1, cancer, transforming gene, antibody


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