ELISA-Based Reporter Assay to Study the Receptor-Ligand Interaction in BW Cells

Abstract

Source: Elias, S. et al., A BW Reporter System for Studying Receptor-Ligand Interactions. J. Vis. Exp. (2019)

This video demonstrates the reporter assay-based detection of the receptor-ligand interaction. The successful receptor-ligand interaction activates the downstream signaling pathway, causing the generation and release of cytokines as secretory reporter molecules in the solution, which is confirmed using ELISA.

Protocol

1. Generation of a Plasmid that Expresses the Chimeric Construct NOTE: The aim is to generate a plasmid that expresses the extracellular domain of the receptor of interest fused to the transmembrane and intracellular domains of the mouse CD3ζ chain (Figure 1A). Retrieve the sequence of the extracellular domain of the receptor of interest including the signal peptide. Obtain DNA material that is expected to express this recepto…

Representative Results

Figure 1: The structure of the chimeric receptor-CD3ζ and the design of PCR primers. (A) General structure of an extracellular receptor fused to the transmembrane and intracellular domains of mouse CD3ζ. (B) The extracellular domain of the receptor was amplified with a 3' primer which also included nucleotides of CD3ζ. (C) The intracellular and transmembrane domains of CD3&#950…

Materials

AccuPrep, Plasmid Mini Extraction Kit	Bioneer	K-3030
Anti-mouse IL-2	BioLegend	503702
Biotin anti-mouse IL-2	BioLegend	503804
ELISA plates	De-groot	60-655061
Fetal Bovine Serum	Sigma	F7524-500ml
G418	Mercury	MBS3458105GM
Gene Pulser II	Bio-Rad	165-2105, 165-2106, 165-2107, 165-2108, 165-2109, 165-2110
L-Glutamine	Biological industry	03-020-1B
MEM Non-Essential Amino Acids Solution	Biological industry	01-340-1B
Penicillin-Streptomycin Solution	Biological industry	03-031-1B
Peroxidase streptavidin	Jackson ImmunoResearch	016-030-084
PureLink HiPure Plasmid Filter Maxiprep Kit	ThermoFisher Scientific	K210016
RPMI-1640 Medium	Biological industry	30-2001
Sodium Pyruvate	Biological industry	03-042-1B
TMB	SouthernBiothech	0410-01