Q&A Part 5: Antibody Validation Practices

Firstly, validation strategies for antibodies are immunoassay specific and strong performance in a given assay does not ensure strong performance in all assays. Secondly, the proper standards and controls must be included in all validation experiments. Thirdly, the validation strategy should be in accordance with the five “conceptual pillars” for antibody validation that were described in a report published by Nature Methods entitled “A Proposal for Validation of Antibodies” on September 5, 2016. For example, acceptable validation strategies for Western blotting include the use of a knockdown or knockout cell line or a lysate prepared from cells treated with a drug to enhance or diminish expression of the target. Other assays such as IHC or IP can then be performed using the established controls to further demonstrate correctness. Validation performed in parallel with a second or third antibody recognizing epitopes of the same target is exceptionally good.

Our product development process includes testing on a combination of primary cells, cell lines and/or transfectant cell models with relevant controls using multiple immunoassays to ensure biological accuracy. We also perform multiplexing with additional antibodies to interrogate antibody staining in multiple cell populations.

Internally, we have stringent quality systems that monitor each production lot. During validation, we make use of any technology that allows us to develop the most relevant model systems and controls. These include knock-downs, knock-outs, blocking and competition assays, bench-marking to existing clones, and staining on endogenous targets. The data generated is made publicly available through our website. We have also begun citing contra-indicated applications when we have demonstrated an antibody is not suitable for a particular application.

CST’s application-specific antibody validation begins at the time of target selection. Once a target is identified, each application team evaluates the biology of the target and potential testing models to determine the most appropriate and desired applications to test. These decisions will then determine the antigen and project design. Together with the antigen design team and the application team, the development scientist leading the project is responsible for ensuring the initiation of the most appropriate experiments for each chosen application. All CST antibodies are validated in biological models relevant to the target of interest.

For all applications, CST uses five general categories of validation testing to verify antibody specificity, sensitivity, reactivity, and performance:

• Specificity
  • Binary Models & Genetic Inactivation: Using multiple cell lines with known target expression levels and recombinant controls, and also employing siRNA knockdown, knockout models, and other tools
  • PTM-specificity: Using appropriate kinase-specific activators, inhibitors, and phosphatases, etc.
  • Localization: Assessing subcellular localization and treatment-induced localization
  • Biologically Relevant Treatments: Testing cell lines treated with growth factors, cytokines, or chemical activators/inhibitors to knowingly modify target expression
  • Tissue Screening: Analyzing multiple normal and diseased tissues to assess performance across a broad spectrum of tissues
• Sensitivity
  • Detection of endogenous protein levels
  • Optimized application validation and specialized formulations for WB, IHC, IF, flow, and ChIP
  • Titration testing in cell and tissue-based arrays
• Consistency
  • Highly reproducible recombinant monoclonal antibody production method to ensure lot-to-lot consistency and stability
  • Annual quality control re-validation for all products
  • Multiple rounds of independent validation analysis by CST scientists for all product lots
• Methodology
  • Product-specific optimized protocols
  • Recommendations for optimal dilutions and buffers
• Support
  • Top-ranked global technical support—assurance by the scientists who validated the antibody that it will work in your lab

Even using this method, we’re never finished validating our antibodies. As new protocols, methods, models, etc. become available, CST continues to validate its antibodies, both internally and by working with our international community of researcher customers. Our development, application, and production scientists are constantly reading the literature, attending scientific conferences, and speaking with customers. This allows us to understand how our products are used, adapt them to new applications, and enhance our validation data.

At the end of the day, the antibody product has to do what it's supposed to do. This is tested during the development process as well as during the production of each batch. Of course, the tests depend on the application. Generally, we develop specific protocols for validation experiments and see if the results meet our pre-defined specifications. We routinely compare our products with those of our competitors, if there are any.

Our antibody validation process is centered on the customer’s needs, which include demonstrating specificity to a target and in some cases sub-cellular location of the target in the context of different physiological states. Based on these criteria, the first validation may be Western blotting or ELISA where we use control cell lines. We may also use, in parallel, lysates from wild-type and knockout cells or cell lines that are typically null for the target protein. In some cases, cells may be treated with known activators or inhibitors of a particular target molecule, and lysate pre-tested for a specific post-translational modified state, such as phosphorylation, acetylation, or glycosylation. Further validation is conducted using immunohistochemistry, immunocytochemistry, or flow cytometry techniques to confirm specificity and cellular location of the target molecule.