Ligand binding assay to screen mouse anti-CXCR4 antibodies: Ligand binding assays were performed on a CXCR4 receptor; direct and indirect assay formats were tested. The direct format is a competition assay where either SDF1-red or mAb anti-CXCR4-red were used as labeled ligands. In this format a competition occurs between the labeled ligand and the specific anti-CXCR4 antibody to be screened. In the indirect assay format the anti-CXCR4 antibody were revealed in a sandwich assay by using an antimouse Fc-d2.
cAMP functional assay: The functional activation of CXCR4 receptor by increasing concentrations of SDF1 or anti-CXCR4 antibody (12G5) was assessed using the cAMP kit after stimulation of the cells with Forskolin. A positive dose-response curve was obtained with SDF1, while no activation was observed with the antibody.
The labeled cells expressing CXCR4 were incubated with a large excess of internalization reagent and different concentrations of SDF1. A dose-response curve showing the internalization process was observed in the presence of SDF1. The same assay was performed by using 12G5 antibody, but no internalization of the receptor was observed in these conditions.
Tag-lite technology offers a lot of flexibility in terms of assay format for detecting antibody binding on cell-surface receptors. This enables the detection of different binder types, the determination of their binding affinity, as well as their ranking.
Tag-lite is HTS friendly and highly miniaturizable. It also has been shown to screen therapeutic antibodies in a cost-effective way. Moreover, frozen prelabeled Tag-lite cells can be stored and used for daily experiments and HTS campaigns. The implementation of Tag-lite assay is straightforward, and the technology has a broad application potential in the field of biotherapeutic screening and characterization, from the early R&D stage, through to production and quality control phases.