Annual BVC/ABC (Belgische vereniging voor cytometrie - Association belge de cytométrie) symposium: Imaging technology in microbiology: cytometric and molecular approaches location:Brussels, Belgium date:18 November 2005
Abstract p. 46
In order to infect a target cell, the HIV envelope glycoprotein gp120 has to interact with both the cellular CD4 receptor and a chemokine receptor, CCR5 or CXCR4, the so-called HIV co-receptors. Several flow cytometric/fluorometric assays are developed in our lab and are very helpful in deciphering the mode of action and interaction site of several classes of HIV entry inhibitors, such as the chemokine receptor antagonists.
Chemokine receptor antagonists can be identified by their ability to inhibit ligand binding to their receptor. Chemokine binding assays are mostly performed with radiolabeled chemokine ligands. Recently, we have developed a flow cytometric technique using an Alexa Fluor 647 conjugate of CXCL12 (CXCL12AF647), the specific ligand of CXCR4. CXCR4 inhibitors blocked the binding of this chemokine at concentrations that are that are comparable with their anti-HIV activity. Also analysis of the chemokine binding in heterogeneous cell populations, such as PBMCs, via multi parameter flow cytometry is feasible. This assay circumvented the poor stability, high backgrounds and the expensive labeling of the radio-active chemokine and the cost/problems of the radioactive waste. In addition, chemokine receptor expression levels and dose-dependent inhibition of compounds can be measured by monitoring the binding of specific fluorescent-labeled anti-CXCR4 and CCR5 mAbs. Mabs recognizing different epitopes on the receptor give already information on the interaction site(s) of the compound in the receptor. Also, chemokine-induced intracellular calcium signaling, with fluorescent probes, will provide further information on the specificity of the compounds and their potency in inhibiting the interaction of the chemokine with the receptor. Thus, flow cytometric/fluorometric assays are very valuable with regard to the intense search for CXCR4 and CCR5 inhibitors to combat HIV transmission and infection.