title: 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.
authors.affiliation: Lindsley F. Kimball Research Institute, New York Blood Center, New York, New York, 10021, USA.
authors.forename: Qian
authors.initials: Q
authors.lastname: Zhao
authors.forename: Hong
authors.initials: H
authors.lastname: Lu
authors.forename: Dominique
authors.initials: D
authors.lastname: Schols
authors.forename: Erik
authors.initials: E
authors.lastname: De Clercq
authors.forename: Shibo
authors.initials: S
authors.lastname: Jiang
mesh_terms: D019380:Anti-HIV Agents / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology*; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology*; D015704:CD4 Antigens / Q000378:metabolism; D002459:Cell Fusion; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005434:Flow Cytometry; D015658:HIV Infections / Q000821:virology; D015497:HIV-1 / Q000378:metabolism / Q000472:pathogenicity; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D019713:Receptors, CCR5 / Q000378:metabolism; D019718:Receptors, CXCR4 / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism; D012680:Sensitivity and Specificity
chemical_list: D019380:Anti-HIV Agents; D000911:Antibodies, Monoclonal; D015704:CD4 Antigens; D019713:Receptors, CCR5; D019718:Receptors, CXCR4