Characterization and epitope mapping of several new anti-P-glycoprotein monoclonal antibodies.
Monoclonal antibodies (MAbs) were raised against partially purified Class I P-glycoprotein from multidrug-resistant Chinese hamster ovary CHRB30 cells. Fifteen stable monoclonal hybridoma cell lines were established, and the secreted antibodies were classified into 8 groups on the basis of banding pattern on immunoblots of P-glycoprotein digested with cyanogen bromide or partially digested with proteases. One representative of each group was tested further for several activities. Six of the 8 recognized human P-glycoprotein in the multidrug-resistant SKVLBI cell line. None of the antibodies recognized P-glycoprotein in unfixed cells, suggesting that all recognize cytoplasmic epitopes or extracellular epitopes not accessible in native P-glycoprotein. All 8 antibodies were able to immunoprecipitate P-glycoprotein from non-denaturing detergent solution. The linear epitopes of the antibodies were mapped to 11-27 amino acids. Two of the antibodies had epitopes in the linker region, 3 in the N-terminal nucleotide binding domain, 2 in the C-terminal nucleotide binding domain and 1 in the predicted cytoplasmic loop between predicted transmembrane helices 8 and 9. These antibodies, with known epitopes, could have uses for P-glycoprotein detection, structure/function studies, purification and quantitation.
Shapiro
A B
AB
Division of Molecular and Structural Biology, Ontario Cancer Institute/Princess Margaret Hospital, Toronto, Canada.
Duthie
M
M
Childs
S
S
Okubo
T
T
Ling
V
V
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000145:classification / Q000276:immunology*; D000918:Antibody Specificity; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D003488:Cyanogen Bromide; D018432:Drug Resistance, Multiple; D000939:Epitopes / Q000737:chemistry* / Q000276:immunology*; D005260:Female; D006801:Humans; D015151:Immunoblotting; D007163:Immunosorbent Techniques; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology; D014407:Tumor Cells, Cultured
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments; D003488:Cyanogen Bromide