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Assay Tutorials : Oct 15, 2010 (Vol. 30, No. 18)

Novel Antibody-Binding Detection Assay

Tag-lite® Technology Was Designed to Increase Flexibility of Cell-Surface Receptor Research
  • Jean-Luc Tardieu, Ph.D.

Tag-lite is a cellular screening platform from Cisbio Bioassays that enables the investigation of natural ligand, small molecule, or antibody binding to cell-surface proteins such as GPCRs and RTKs. This technology, which combines HTRF and SNAP-tag technologies, can be applied to the discovery and development of therapeutic antibodies against cell-surface proteins. 

Tag-lite’s high-throughput format is ideal for primary and secondary screening of antibodies and can be applied to a variety of assay formats for pharmacological characterization.

Tag-lite was investigated to screen therapeutic antibodies for their binding to cell-surface protein targets and characterize  their affinities. Different assay formats were applied to determine the binding of antibodies to two different receptors: CXCR4, a GPCR belonging to the chemokine family, and EGFR, a tyrosine kinase receptor.

Binding of the antibodies to their targets was detected using Tag-lite. Specific cell lines were designed for each receptor, allowing either binding experiments, functional assays (cAMP, Phospho-Erk) or internalization assays.

Assay Principle

Tag-lite starts with the cloning of the gene encoding the receptor of interest in a plasmid containing the SNAP-tag sequence. This SNAP-receptor plasmid is then transfected into HEK cells to produce Tag-lite cells. These cells can be used to investigate the activity of the receptor via functional tests or can be specifically labeled on the SNAP-tag using SNAP-Lumi4 Tb substrate. The labeled cells are then used for different applications, like ligand-binding assays, dimerization, and internalization (Figure 1).

To detect the binding of antibodies on cell-surface receptor targets, different assay formats were designed. Antibodies were screened by displacing different types of ligands—a labeled natural ligand or a labeled antibody—away from the receptor of interest. In this case, antibodies were detected by their binding to the ligand site. Antimouse Fc-d2 labeled antibody was also used to detect the binding in an indirect assay format (Figure 2).

 

Material & Methods

Reagents: SNAP-Lumi4-Tb, antimouse Fc-d2, SNAP-CXCR4, and SNAP-EGFR plasmids and the labeling medium were from Cisbio Bioassays. Lipofectamin 2000 was purchased from Invitrogen, and the cell-dissociation buffer was from Sigma Aldrich. Anti-CXCR4 antibodies (12G5 and 44717) and anti-CXCR7 antibody (11G8) were from R&D Systems.

Covalent labeling of SNAP-receptor cells: A solution of Tag-lite SNAP-Lumi4-Tb substrate at 100 nM was prepared in Tag-lite labeling medium. After aspiration of the cell culture medium, 3 mL of this solution was added to the cells expressing the SNAP-receptor in the T175 flask, followed by a one hour incubation at 37ºC. The cells were then washed four times to remove the excess of SNAP-Lumi4-Tb and detached using the cell dissociation buffer. The cells were frozen under 1 million cells/vial in culture medium and 10% DMSO.

Ligand binding assay: The SNAP-Lumi4-Tb labeled cells were thawed and washed with PBS, then resuspended in labeling buffer. Binding assays were performed in a total volume of 20 µL in 384-small volume white plates. 10,000 cells/well were incubated for one hour at room temperature with different concentrations (0–200 nM) of labeled ligand (SDF1-red, mAb anti-CXCR4-red) or unlabeled ligand (mAbs anti-CXCR4). 100 nM of antimouse Fc-d2 was added for the detection of unlabeled mAbs.

Competition binding assay: Competition binding assays were carried out in a final volume of 20 µL in 384-small volume white plates. 10,000 labeled cells were incubated with red-labeled SDF1 or red-labeled EGF (final concentration of 12.5 nM or 10 nM respectively) in the presence of different concentrations of antibodies (0–100 nM). Following incubation, the plates were measured on a TR-FRET reader.

cAMP functional assay with cAMP dynamic 2 kit: The functional tests were performed in 20 µL final volume in a 384-small volume plate. 10,000 cells were incubated with Forskolin (2 µM final concentration) and different concentrations of SDF1 or 12G5 antibody (0–1 µM final concentration). The plates were incubated 30 minutes at room temperature before being read on a TR-FRET reader.

Internalization Assay: Internalization assays were carried out in a final volume of 20 µL in 384-small volume white plates. 10,000 labeled cells were incubated at room temperature with a high concentration of internalization reagent in the presence or not of different concentrations of SDF1. The plate was measured after a two-hour incubation.

Results

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.

Internalization Tests

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.