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

AIDS Res Hum Retroviruses. 2003 Nov;19(11):947-55. doi: 10.1089/088922203322588297.

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.

Publication types

  • Evaluation Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Anti-HIV Agents / immunology
  • Anti-HIV Agents / metabolism
  • Anti-HIV Agents / pharmacology*
  • Antibodies, Monoclonal / immunology
  • Antibodies, Monoclonal / metabolism
  • Antibodies, Monoclonal / pharmacology*
  • CD4 Antigens / metabolism
  • Cell Fusion
  • Cell Line
  • Enzyme-Linked Immunosorbent Assay / methods
  • Flow Cytometry
  • HIV Infections / virology
  • HIV-1 / metabolism
  • HIV-1 / pathogenicity
  • Humans
  • Microbial Sensitivity Tests / methods
  • Receptors, CCR5 / metabolism
  • Receptors, CXCR4 / antagonists & inhibitors
  • Receptors, CXCR4 / immunology
  • Receptors, CXCR4 / metabolism
  • Sensitivity and Specificity

Substances

  • Anti-HIV Agents
  • Antibodies, Monoclonal
  • CD4 Antigens
  • Receptors, CCR5
  • Receptors, CXCR4