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

Published: June 29, 2023

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…

開示

The authors have nothing to disclose.

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

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記事を引用
ELISA-Based Reporter Assay to Study the Receptor-Ligand Interaction in BW Cells. J. Vis. Exp. (Pending Publication), e21452, doi: (2023).

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