Characterization of a new antibody raised against the NH2 terminus of P-glycoprotein.
OBJECTIVE
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.


METHODS
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 NH2 11 antibody. We compared the properties of NH2 11 antibody with that of the well-characterized Pgp-specific antibody, C219, by Western blotting, immunoprecipitation, immunocytochemistry, and immunohistochemistry.


RESULTS
Immunoblotting analysis suggested that NH2 11 antibody efficiently interacts with both recombinant and constitutively expressed Pgp in cancerous and noncancerous human cells. Immunoprecipitation reactions indicated that the NH2 11 antibody selectively immunoprecipitates Pgp. Immunocytochemical analyses indicated that the NH2 11 antibody detects Pgp in drug-resistant breast cancer cells as well as in human prostate and breast adenocarcinoma tissue sections.


CONCLUSIONS
As the NH2 11 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.
Rao
Prema S
PS
Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska, USA. usrao@unmc.edu
Govindarajan
Rajgopal
R
Mallya
Kavita B
KB
West
William
W
Rao
U Subrahmanyeswara
US
D018435:ATP Binding Cassette Transporter, Subfamily B / Q000737:chemistry / Q000276:immunology*; D000595:Amino Acid Sequence; D000906:Antibodies / Q000032:analysis / Q000276:immunology*; D000918:Antibody Specificity; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000276:immunology; D002460:Cell Line; D045744:Cell Line, Tumor; D000939:Epitopes / Q000276:immunology; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology; D011471:Prostatic Neoplasms / Q000276:immunology
D018435:ATP Binding Cassette Transporter, Subfamily B; D000906:Antibodies; D000939:Epitopes; D010446:Peptide Fragments