Analysis of TRAIL receptor expression using anti-TRAIL death receptor-5 monoclonal antibodies

To establish hybridomas that produce anti-death receptor-5 (DR5) monoclonal antibodies (mAbs) and check the surface expression of DR5 (sDR5) on cell lines.

The cDNA of human DR5 was cloned into pGAPZalpha. Recombinant Pichia pastoris clones generated via homologous recombination secreted high levels of sDR5. The sDR5 was purified using a nickel ion column. BALB/c mice were immunized with sDR5 and spleen cells were fused with the SP2/0-Ag 14. Monoclonal antibodies were tested by ELISA for their abilities binding to sDR5 and by flow cytometry for the reactivities to surface DR5 of Jurkat cells. Surface expression of the TRAIL receptor was determined by flow cytometric analysis measuring the binding of anti-DR5 mAb.

Isotypes of mAbs were determined to be IgG(1) and IgM, all of which were reactive to sDR5 as observed through ELISA. It was discovered using flow cytometry that only IgG was able to bind to DR5 on the plasma membrane of Jurkat cells. sDR5 was found to completely inhibit anti-DR5 mAb binding to Jurkat cells. Approximately 95% of Jurkat cells, 98% SW480, 99% U937, 100% U87, 86% HCT116, 64% HL-60, 47% HeLa and 13% K562 cells express membrane DR5.

These results demonstrate that anti-DR5 mAb is able to specifically bind to DR5 and that DR5 is expressed at high levels on Jurkat, SW480, U87, U937 and HCT116 cell lines, and at medium levels on HL-60 and HeLa cell lines. The expression of DR5 on K562 cell line is low.