Development of a cell-based enzyme-linked immunosorbent assay for high-throughput screening of HIV type 1 entry inhibitors targeting the coreceptor CXCR4
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Published on: June 23, 2011
Qian Zhao1, Hong Lu, Dominique Schols
1Lindsley F. Kimball Research Institute, New York Blood Center, New York, New York, 10021, USA.
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.
Insights
A new cell-based ELISA assay specifically detects CXCR4 binding, enabling efficient screening of HIV-1 entry inhibitors targeting this crucial coreceptor for virus entry.
Area of Science:
- Virology
- Immunology
- Biotechnology
Background:
- CXCR4 is a key coreceptor for HIV-1 X4 virus entry into host cells.
- Inhibiting CXCR4 binding is a strategy for developing antiviral agents against HIV-1.
- Existing methods for studying CXCR4-ligand interactions can be improved for efficiency and specificity.
Purpose of the Study:
- To develop and validate a specific, sensitive, and convenient cell-based ELISA for detecting CXCR4 antagonists.
- To assess the utility of this assay for high-throughput screening of novel HIV-1 entry inhibitors targeting CXCR4.
Main Methods:
- Development of a cell-based ELISA using U373-MAGI-CXCR4(CEM) cells and an anti-CXCR4 monoclonal antibody (12G5).
- Validation of assay specificity by testing binding to CCR5-expressing cells and cells lacking both receptors.
- Evaluation of known CXCR4 antagonists (T22, T14012, AMD3100) and other HIV-1 inhibitors for their effect on 12G5 binding.
Main Results:
- The developed ELISA specifically detected 12G5 binding to CXCR4-expressing cells.
- Known CXCR4 antagonists effectively inhibited 12G5 binding, confirming assay validity.
- The assay demonstrated stability with long-term frozen storage of prepared plates, enhancing convenience.
Conclusions:
- The cell-based ELISA is a specific, sensitive, convenient, rapid, and economic method for studying CXCR4-ligand interactions.
- This assay is suitable for high-throughput screening of HIV-1 entry inhibitors targeting the CXCR4 coreceptor.
- The assay facilitates the discovery of new antiviral strategies against HIV-1.
