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

In This Article

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 receptor (e.g., cDNA or a plasmid).
Obtain DNA material that expresses the transmembrane and the intracellular domains of the mouse CD3 ....

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Results

PCR diagram showing receptor and CD3ζ fusion with primers and domain annotations.
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' pri.......