Detection of P-glycoprotein in cell lines and leukemic blasts: failure of select monoclonal antibodies to detect clinically significant Pgp levels in primary cells.

Multidrug resistance (MDR) is a salient feature of chemotherapy failure in pediatric patients. One of the most common and well-studied mechanisms implicated in causing MDR is P-glycoprotein (Pgp), an ATP-dependent, transmembrane drug efflux pump. Accurate and reproducible detection of this MDR protein is necessary as it may have important clinical implications. In this study comparing the directly conjugated anti-Pgp monoclonal antibodies UIC2-PE and 15D3-PE to the unconjugated anti-Pgp mAb MRK16, we analyzed cell lines, normal peripheral blood cells, and bone marrow cells from pediatric patients diagnosed with acute myeloid leukemia and acute lymphoblastic leukemia; all samples were also analyzed for Pgp function using rhodamine 123 in order to correlate results from antibody staining with functional activity. For all patient samples evaluated, only MRK16 correlated well with the rhodamine 123 assay. Both the directly conjugated antibodies UIC2-PE and 15D3-PE failed to detect Pgp in almost all cases. Pre-treatment of cells with neuraminidase did not provide a consistent enhancement of antigen detection. Based on these results, we suggest that while UIC2-PE and 15D3-PE may be able to detect the very high levels of Pgp expressing laboratory-cultured cell lines, they are not suitable for clinical application in their currently available conjugated form. When assaying patient samples for Pgp expression and function using flow cytometry, the rhodamine 123 functional assay should be performed in concert with staining with MRK16.