At Atlas Antibodies, we have always worked extensively with antibody validation and with producing highly characterized antibodies our customers can trust.
All our antibodies are rigorously evaluated for specificity and performance and are characterized in several applications. The second layer of validation, Enhanced Validation, is performed following the recommendations of the International Working Group for Antibody Validation (IWGAV ) published in Nature Methods. Continue reading to learn about the concepts of Enhanced Validation and how the methods are employed to ensure the specificity of our antibodies.
Characterization and Validation of Antibodies
For our antibodies, specificity starts with the design. The antibodies are developed against recombinant human Protein Epitope Signature Tags (PrESTs) of approximately 50 to 150 amino acids. The PrEST sequences are designed to have the lowest possible identity to other human proteins.
The specificity and purity are validated by protein array against 364 human recombinant protein fragments.
Functional validation and characterization
All Triple A Polyclonals and PrecisA Monoclonals offered by Atlas Antibodies are validated and characterized in each application the specific antibody is approved for (IHC, ICC-IF, WB), in the majority of normal and cancerous human tissues as well as on a subcellular level. Each antibody is accompanied with a large number of images on the Human Protein Atlas showing the result of these validation assays, each manually annotated by researchers. In IHC, each antibody is evaluated in 44 normal tissues and the 20 most common cancers.
Antibody specificity is further evaluated and final approval takes into consideration the literature, bioinformatics and RNA sequencing data.
We believe that transparency and open access are crucial for research. All characterization data including images, are presented on the Atlas Antibodies and the Human Protein Atlas websites. Unlike most antibodies suppliers, we provide you the exact immunogen sequence used to raise the antibody.
In addition to the extensive validation and characterization always performed for our antibodies, we perform application-specific Enhanced Validation. The enhanced validation follows the guidelines proposed by the International Working Group for Antibody Validation (IWGAV) in Nature Methods.
The working group was formed by distinguished researchers from around the world to “formulate the best approaches for validating antibodies used in common research applications and to provide guidelines that ensure antibody reproducibility.”1 IWGAV proposed five conceptual pillars for antibody validation, to be used in an application-specific manner. At least one of the pillars must be used for an antibody to be validated in a specific application. At Atlas Antibodies, we base the Enhanced Validation on Human Protein Atlas' interpretation of these pillars, explained here.
Learn about Enhanced Validation
Enhanced validation offers increased security of antibody specificity in a defined context. This is ensured by using the most relevant validation method for each combination of protein, sample, and application.
In addition to performing a thorough validation, it is of vital importance to ensure antibody reproducibility. Each new lot of an antibody has to perform equally compared to a previous lot so that the experiments using the antibody can be repeated with the same results. At Atlas Antibodies we test all new antibody lots in parallel to existing lots in a large set of samples.
1 The group of distinguished researchers from Stanford University, Yale University, MIT, UCSD, and NIH in the USA, University of Toronto in Canada, the EMBL in Germany, Niigata University in Japan and the Science for Life Laboratory in Sweden, was formed to develop formal guidelines for antibody validation and reproducibility to be used by antibody manufacturers to validate antibodies in a standardized manner (Nature Methods, September 2016).