Dataset Information

Fit-for-purpose based testing and validation of antibodies to amino- and carboxy-terminal domains of cannabinoid receptor 1.

ABSTRACT: Specific and selective anti-CB₁ antibodies are among the most powerful research tools to unravel the complex biological processes mediated by the CB₁ receptor in both physiological and pathological conditions. However, low performance of antibodies remains a major source of inconsistency between results from different laboratories. Using a variety of techniques, including some of the most commonly accepted ones for antibody specificity testing, we identified three of five commercial antibodies against different regions of CB₁ receptor as the best choice for specific end-use purposes. Specifically, an antibody against a long fragment of the extracellular amino tail of CB₁ receptor (but not one against a short sequence of the extreme amino-terminus) detected strong surface staining when applied to live cells, whereas two different antibodies against an identical fragment of the extreme carboxy-terminus of CB₁ receptor (but not one against an upstream peptide) showed acceptable performance on all platforms, although they behaved differently in immunohistochemical assays depending on the tissue fixation procedure used and showed different specificity in Western blot assays, which made each of them particularly suitable for one of those techniques. Our results provide a framework to interpret past and future results derived from the use of different anti-CB₁ antibodies in the context of current knowledge about the CB₁ receptor at the molecular level, and highlight the need for an adequate validation for specific purposes, not only before antibodies are placed on the market, but also before the decision to discontinue them is made.

SUBMITTER: Echeazarra L

PROVIDER: S-EPMC8604870 | biostudies-literature | 2021 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Fit-for-purpose based testing and validation of antibodies to amino- and carboxy-terminal domains of cannabinoid receptor 1.

Echeazarra Leyre L García Del Caño Gontzal G Barrondo Sergio S González-Burguera Imanol I Saumell-Esnaola Miquel M Aretxabala Xabier X López de Jesús Maider M Borrega-Román Leire L Mato Susana S Ledent Catherine C Matute Carlos C Goicolea María Aranzazu MA Sallés Joan J

Histochemistry and cell biology 20210827 5

Specific and selective anti-CB<sub>1</sub> antibodies are among the most powerful research tools to unravel the complex biological processes mediated by the CB<sub>1</sub> receptor in both physiological and pathological conditions. However, low performance of antibodies remains a major source of inconsistency between results from different laboratories. Using a variety of techniques, including some of the most commonly accepted ones for antibody specificity testing, we identified three of five c ...[more]

PMID: 34453219

Similar Datasets

Project description:Background: The freezing process of tissue samples is crucial for the preservation of morphological and molecular features. Several biobanking guidelines describe freezing techniques for optimal outcomes. As the Vetbiobank standard freezing protocol does not comply with those recommendations in detail, a process validation was performed to demonstrate that samples are suitable for downstream applications. Here we give a formal example of a process validation in the biobanking setting, as required by the biobanking guideline ISO 20387 (2018). Methods: Three different freezing protocols, freezing in liquid nitrogen, freezing via isopentane precooled on dry ice and freezing via liquid nitrogen vapor, were assessed based on morphological integrity of mouse liver and muscle tissue samples. Samples were either frozen in cryotubes (without Optimal Cutting Temperature compound, OCT) or in cryomolds (with OCT). The protocol providing the best results was validated for reproducibility and robustness in terms of defined acceptance criteria for morphological evaluability, A260/A280 ratio, and RNA integrity number values (RIN). In addition, performance tests were run by gene expression analyzes of selected, tissue specific biomarkers to confirm that processed samples are fit for purpose. Results: From the three applied freezing protocols, freezing in liquid nitrogen generated best results. Reproducibility acceptance criteria were met for both, morphological integrity and RNA quality. The freezing method was robust for the tested tissue types and the application of OCT, with exception of liver tissue, where it led to a significant decrease of the RIN value. Gene expression analyzes showed good comparability of results regardless of the applied freezing method. Conclusion: Freezing of tissue samples in liquid nitrogen provides samples of adequate quality for subsequent RNA investigations. A negative impact of OCT on the RIN value of liver samples was observed, which was independent from the applied freezing protocol and showed no impact on subsequent gene expression analysis.

Project description:BACKGROUND: Mass spectrometry (MS) is a very sensitive and specific method for protein identification, biomarker discovery, and biomarker validation. Protein identification is commonly carried out by comparing MS data with public databases. However, with the development of high throughput and accurate genomic sequencing technology, public databases are being overwhelmed with new entries from different species every day. The application of these databases can also be problematic due to factors such as size, specificity, and unharmonized annotation of the molecules of interest. Current databases representing liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based searches focus on enzyme digestion patterns and sequence information and consequently, important functional information can be missed within the search output. Protein variants displaying similar sequence homology can interfere with database identification when only certain homologues are examined. In addition, recombinant DNA technology can result in products that may not be accurately annotated in public databases. Curated databases, which focus on the molecule of interest with clearer functional annotation and sequence information, are necessary for accurate protein identification and validation. Here, four cases of curated database application have been explored and summarized. FINDINGS: The four presented curated databases were constructed with clear goals regarding application and have proven very useful for targeted protein identification and biomarker application in different fields. They include a sheeppox virus database created for accurate identification of proteins with strong antigenicity, a custom database containing clearly annotated protein variants such as tau transcript variant 2 for accurate biomarker identification, a sheep-hamster chimeric prion protein (PrP) database constructed for assay development of prion diseases, and a custom Escherichia coli (E. coli) flagella (H antigen) database produced for MS-H, a new H-typing technique. Clearly annotating the proteins of interest was essential for highly accurate, specific, and sensitive sequence identification, and searching against public databases resulted in inaccurate identification of the sequence of interest, while combining the curated database with a public database reduced both the confidence and sequence coverage of the protein search. CONCLUSION: Curated protein sequence databases incorporating clear annotations are very useful for accurate protein identification and fit-for-purpose application through MS-based biomarker validation.

Dataset Information

Fit-for-purpose based testing and validation of antibodies to amino- and carboxy-terminal domains of cannabinoid receptor 1.

Publications

Fit-for-purpose based testing and validation of antibodies to amino- and carboxy-terminal domains of cannabinoid receptor 1.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets