Development of a cell-based enzyme-linked immunosorbent assay for high-throughput screening of HIV type 1 entry inhibitors targeting the coreceptor CXCR4.

Abstract:

CXCR4, a coreceptor for human immunodeficiency virus type 1 (HIV-1) X4 virus, plays an important role in virus entry into the target cells. Antiviral agents that bind to CXCR4 are expected to inhibit HIV-1 entry. A cell-based enzyme-linked immunosorbent assay (ELISA) was developed utilizing an anti-CXCR4 monoclonal antibody, 12G5, and cells expressing CD4 and CXCR4, U373-MAGI-CXCR4(CEM) cells. Using this assay, we demonstrated that 12G5 specifically binds to the CXCR4-expressing cells, but not to CCR5-expressing cells and cells without CXCR4 and CCR5, consistent with the results obtained by using flow cytometry. The well-characterized CXCR4 antagonists, T22, T14012 (a downsized analog of T-22), and AMD3100, effectively inhibited 12G5 binding to CXCR4-expressing cells, while HIV-1 entry inhibitors targeting CD4 and gp41 as well as HIV-1 reverse transcriptase and protease inhibitors did not block the binding of 12G5 to U373-MAGI-CXCR4(CEM) cells. The prepared plates containing the fixed cells could be stored at -80 degrees C for at least 5 months without affecting the cell reactivity with 12G5, which enhances the convenience of this method. This suggests that the cell-based ELISA is specific, sensitive, convenient, rapid, and economic. With a robotic sample processing system, this assay can be used for high-throughput screening of HIV-1 entry inhibitors targeted to the HIV-1 coreceptor CXCR4.

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