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Characterization of a New Antibody Raised against the NH₂ Terminus of P-Glycoprotein

Authors' Affiliations: Departments of ¹ Biochemistry and Molecular Biology and ² Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska

Requests for reprints: U. Subrahmanyeswara Rao, Department of Pharmaceutical Sciences, 1300 Coulter, Texas Tech University Health Sciences Center, Amarillo, TX 79106. Phone: 806-356-4015 ext. 245; E-mail: usrao{at}unmc.edu.

Purpose: Cancers exposed to chemotherapy develop multidrug resistance, a major cause for chemotherapy failure. One mechanism of multidrug resistance development is due to overexpression of P-glycoprotein (Pgp) in these cancer cells. Thus, a prechemotherapy evaluation of Pgp in cancer cells aids in the design of alternative regimens that can circumvent such failure. As few Pgp-specific antibodies are available in detecting low levels of Pgp, there is a need for preparing an antibody that allows the detection of Pgp by various immunologic methods.

Experimental Design: We selected the amino acid stretch 11 to 34 in the cytoplasmically located NH₂ terminus of Pgp as antigen, which was chemically synthesized and used to raise an antibody in a rabbit, termed NH₂11 antibody. We compared the properties of NH₂11 antibody with that of the well-characterized Pgp-specific antibody, C219, by Western blotting, immunoprecipitation, immunocytochemistry, and immunohistochemistry.

Results: Immunoblotting analysis suggested that NH₂11 antibody efficiently interacts with both recombinant and constitutively expressed Pgp in cancerous and noncancerous human cells. Immunoprecipitation reactions indicated that the NH₂11 antibody selectively immunoprecipitates Pgp. Immunocytochemical analyses indicated that the NH₂11 antibody detects Pgp in drug-resistant breast cancer cells as well as in human prostate and breast adenocarcinoma tissue sections.

Conclusion: As the NH₂11 antibody detects Pgp present in cells and tissues, we conclude that the amino acid sequence to which this antibody was raised is highly antigenic and the antibody is useful in the detection of Pgp by a variety of immunologic methods.