Heather O'Neill (Presenter)
Caris Life Sciences
Authorship: Heather O’Neill, Annemarie Benton, Melissa Richards, Phil Ellis, Peggy Teresi, Zoran Gatalica, David Spetzler, Inga Rose
Caris Life Sciences
| Short Abstract Antibody specificity is a potential source of variability in immunohistochemical (IHC) clinical assays. To verify specificity for downstream IHC testing of TLE3 protein, anti-TLE3 antibodies from three separate vendors were analyzed by western blot and subsequent mass spectrometry of gel-excised bands to confirm target specificity. All three anti-TLE antibodies identified a common ~83 kD band by western blot, in addition to several bands of differential molecular weights. High-resolution mass spectrometry was used to verify specific target bands and identify which antibodies resulted in detection of non-specific targets. The results demonstrate the utility of western blot combined with mass spectrometry as a supplemental quality control for IHC testing. |
Long Abstract
It is well known that variability in immunohistochemistry (IHC) clinical assay performance can occur due to multiple factors. Referral laboratories performing predictive IHC tests face specific challenges not adequately covered by standard processes, nor regulated by the major clinical laboratory oversight agencies. Performing quality control of reagents by IHC and the use of tissue controls has been thoroughly discussed, but further examination of the antibody’s specificity to the protein of interest could serve as an additional quality control measure. Inconsistent protein specificity was observed by western blot across three different vendors of anti-TLE3 antibody with observed bands at not only ~83kDa, where you would expect to detect TLE3, but also at additional and varying molecular weights across the three antibodies. IHC analysis confirmed variable staining of the three antibodies. To further investigate the variable reactivity and more confidently confirm each antibody’s reactivity with targeted TLE3 protein, high-resolution mass spectrometry was used to examine the bands observed in the western blot. The gel bands from a Coomassie stained gel of HeLa nuclear extract, specified by a western blot run in parallel, were used to verify which antibody was detecting the correct target protein. A gel slice that corresponded to the region of the western blot where the specific TLE3 band was detected (~60-100kDa) with antibodies from all three vendors resulted in detection of ~1000 protein groups and ~9700 peptide groups with a 60 minute RP gradient on a 2µm EASY-Spray, 50cm x 75µm, PepMap RSLC C18, column combined with a TOP20 data dependent discovery with a 10 sec dynamic exclusion run on an UltiMate 3000 RSLCnano System coupled to a Thermo Scientific™ Q-Exactive™ HF hybrid quadrupole-Orbitrap mass spectrometer. Three tryptic peptides of the low abundance TLE target protein (~10 ng TLE3/µg of HeLa nuclear extract) were identified with high confidence in the ~60-100kDa band but not in the ~40-60kDa, ~100-220kDa, or >220kDa gel slices where other potential non-specific bands were detected by western blot in 2/3 of the anti-TLE3 antibody lots. These findings confirmed that the bands observed, in addition to the ~83kDa band, are not specific to TLE3 protein and could interfere with downstream IHC processing and interpretation. Quantitative proteomic workflows can be further developed from the identified peptides to determine the absolute abundance of the target proteins in the extract and assist with determination of antibody sensitivity in addition to specificity. The results demonstrate how western blot and mass spectrometry can be utilized as a supplement to traditional IHC quality controls aiding in the selection of optimal antibodies for use in IHC assay development, assay validation and for ongoing antibody quality control and highlight the necessity for rigorous quality control of IHC predictive markers.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | Caris Life Sciences |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | no |