Validation of antibodies for the specific detection of human TRPA1

Abstract

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.

Figure 1

Validation of positive and negative control cells by qPCR and patch clamp electrophysiology. (a) Real time quantitative PCR of HEK293T cell RNA for TRPA1 mRNA. The negative control cells were transduced with an empty GFP vector (Vector), the positive control cells were transduced with dual promoter TRPA1 and GFP lentiviral vector (TRPA1), data presented are mean and standard error of mean of two independently generated cell lines. (b,c) Whole cell recordings by patch clamp electrophysiology of negative control cells (n = 3) or positive control cells (n = 5) stimulated with the specific TRPA1 agonist JT-010. GFP fluorescence was confirmed in all cells prior to recording. The left two panels show representative baseline and JT-010 stimulated currents. The right panel shows the mean current voltage relationship of all recordings, including following addition of the specific TRPA1 antagonist A967079, which was added when a JT-010 stimulated current was observed (only in positive controls). These experiments demonstrate low or no expression of TRPA1 mRNA in negative control cells, which corresponds to no functional response. The positive controls display high mRNA and strong functional responses.

Figure 2

Evaluation of anti-TRPA1 antibody specificity by western blotting and immunofluorescence. (a) Lysates derived from HEK293T cells transiently transfected with the TRPA1 and GFP expression construct (TRPA1 OE) or TRPM2 and GFP negative control (TRPM2 OE) were probed with the antibodies indicated. Additional untransfected and GFP only controls are presented in the first blot (6G8). Uncropped full length blots are displayed, gaps between the blots are present to indicate the use of a different antibody. Mouse mAbs C-5 and 6G8, and NB110-40763 can detect TRPA1 only in the membrane fraction of lysates at the expected molecular weight (127.5 kDa). Several TRPA1-specific bands are observed above this weight. NB110-40763 also detects several other antigens. Ab58844 and ACC- 037 only appear to detect antigens other than TRPA1 in the conditions used. (b) The performance of the same antibodies was evaluated by immunofluorescence (red) in comparison to the expression of the GFP reporter (green), or control TRPM2 and GFP overexpressing cells as for western blotting. Mouse mAb C-5 again shows high specificity with a strong correlation between the expression of the GFP reporter and antibody staining, and no staining in TRPM2 and GFP overexpressing controls. NB110-40763 also demonstrates some sensitivity but has high background that likely does not correspond to Fc binding of the antibody, as the other two rabbit polyclonal antibodies or isotype control did show different staining patterns. Ab58844 and ACC- 037 only appear to detect antigens other than TRPA1 in the conditions used.

Figure 3

Validation of flow cytometry and immunocytochemistry hTRPA1 assays using mouse mAb C-5. (a) Dot plot of transiently transfected live HEK293(T) cells stained with Alexa Flour 647 (AF647) primary conjugated mouse mAb C-5. Cells were transfected with a TRPA1 overexpression construct with a roGFP2 reporter or a control roGFP2 vector. Only cells transfected with TRPA1 stained and the intensity of expression of the reporter correlated with TRPA1 staining intensity. (b) Paraffin embedded cell pellets of untransfected HEK293T cells, transiently transfected HEK293T cells (vector control and TRPA1) stained with mouse mAb C-5. Further uncropped images with more cells are presented in the supplemental information (Fig. s3).

Figure 4

Expression profiling of TRPA1 in human lung and airway derived cells and cell lines. Flow cytometry comparing hTRPA1 staining between negative control cell line HEK293T and (a) human lung mast cells (HLMCs, no staining observed, representative of 4 donors), human lung myofibroblasts (HLMFs, staining observed in 4 out of 4 donors) and human airway smooth muscle cells (HASM, staining observed in 7 out of 7 donors). (b) HMC-1 cell line, HeLa cell line and A549 cell line. (c) expression of mRNA for TRPA1 pattern matches expression pattern observed by flow cytometry in HLMF, HASM and cell lines. (d) Mean current voltage relationships of whole cell currents by patch clamp electrophysiology show TRPA1 currents in HASM cells (3 cells from 3 donors) but not HLMCs (representative of 8 cells in 2 donors). Example raw currents are display in the supplemental information (Fig. s5).