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Validation of antibodies for the specific detection of human TRPA1

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journal contribution
posted on 23.04.2020, 10:12 by HS Virk, MZ Rekas, MS Biddle, AKA Wright, J Sousa, CA Weston, L Chachi, KM Roach, P Bradding
The transient receptor potential cation channel family member ankyrin 1 (TRPA1) is a potential target for several diseases, but detection of human TRPA1 (hTRPA1) protein in cells and tissues is problematic as rigorous antibody validation is lacking. We expressed hTRPA1 in a TRPA1-negative cell line to evaluate 5 commercially available antibodies by western blotting, immunofluorescence, immunocytochemistry and flow cytometry. The three most cited anti-TRPA1 antibodies lacked sensitivity and/or specificity, but two mouse monoclonal anti-TRPA1 antibodies detected hTRPA1 specifically in the above assays. This enabled the development of a flow cytometry assay, which demonstrated strong expression of TRPA1 in human lung myofibroblasts, human airway smooth muscle cells but not lung mast cells. The most cited anti-TRPA1 antibodies lack sensitivity and/or specificity for hTRPA1. We have identified two anti-TRPA1 antibodies which detect hTRPA1 specifically. Previously published data regarding human TRPA1 protein expression may need revisiting.

Funding

We thank the MRC for funding a clinical research training fellowship for HSV (award reference MR/N02074X/1) and the Midland Asthma and Allergy Research Association for award of a consumables grant.

History

Citation

Scientific Reports, 2019, 9, 18500

Author affiliation

Department of Respiratory Sciences

Version

VoR (Version of Record)

Published in

Scientific Reports

Volume

9

Issue

1

Pagination

18500

Publisher

Nature Research

issn

2045-2322

eissn

2045-2322

Acceptance date

22/11/2019

Copyright date

2019

Publisher version

https://www.nature.com/articles/s41598-019-55133-7#Abs1

Spatial coverage

England

Language

eng