Generation of a monoclonal antibody to P-glycoprotein peptides using tuberculin-PPD as a carrier

Abstract

A novel immunization protocol together with stringent selection criteria have been employed to generate a new murine monoclonal antibody ("D8", isotype IgG1, kappa) which specifically recognizes the human p170 drug resistance glycoprotein. This antibody is directed towards a defined peptide sequence located in the -COOH terminal region of the first external loop of the molecule. It is reactive with its epitope within the intact native glycoprotein in formalin-fixed and conventionally processed histological tissues, in flow-cytometric preparations and by Western blotting. The antibody precipitates its target peptide sequence from solution, and thus may be a useful reagent with which to establish an ELISA, RIMA or other similar assay. The peptide epitope recognized by this monoclonal antibody is restricted to the human MDR1 gene product and is not contained within the rodent homologue of the P-170 molecule. Immunohistochemistry has consistently failed to detect this epitope in rodent tissues, thus confirming that it does not exhibit the cross-reactivity of other currently available anti-P-glycoprotein monoclonal antibodies. The experience of this study emphasizes the value of the tuberculin-PPD (purified protein derivative) immunization protocol as a powerful strategy when generating monoclonal antibodies to small synthetic peptides. The resulting monoclonal antibody (D8) will be an invaluable reagent with which to analyse P-170 glycoprotein expression when assessing the role of multidrug resistance in human cancers.