Antibodies are one of the most frequently used research tools in science and clinical assays. However, there are no guidelines or standardized methods that are universally accepted for establishing the validity of the reagents.
There is a lack of target specificity for antibodies, resulting in a call for more stringent antibody validation criteria. Determining the specificity of an antibody is largely dependent on the kind of immunogen involved.
Antibody validation means that it must be specific, selective, and reproducible for the application it is to be used in. This article will outline the six key pillars of antibody validation, their benefits, and their applications.
Independent Antibodies
In this technique of antibody validation, two or more antibodies must be targeted at a range of epitopes of a protein and produce similar outcomes, such as similar staining patterns on the same tissue or similar results on a western blot. Most Bethyl antibodies are validated using in-house, independent antibodies.
Complementary Assays
This technique of antibody validations is based on the premise that many antibody-dependent assays form complementary results. One example of this is cell line-specific staining from an antibody that shows comparable specificity on a western blot.
Orthogonal Characteristics
In this area of antibody validation, antibody-independent and antibody-dependent assays are required to form correlative results. Mass spectrometry or mRNA transcript analysis of samples is required for antibody-independent methods and a relationship between the data and immunocytochemistry or western blot produced is studied.
Biological Characteristics
This pillar of antibody validation capitalizes on some protein targets and the unique biology associated with them. The antibody of interest identifies a protein target only accessible through specific conditions. This antibody validation method is often used for phosphor-specific antibodies.
Protein OE/Epitope Tags
This antibody validation technique requires over-expressed proteins to validate antibodies against targets in which a natively expressing cell line cannot be identified. This technique is also used to dismiss cross-reactivity with certain proteins. This method of antibody validation must always be used in conjunction with one or more other methods.
Genetic Strategies
The genetic strategies antibody validation pillar employs gene knockdown/knockout to minimize the amount of target protein accessible for detection. Knockout target protein should generally lead to a decrease in band intensity. This method of antibody detection is not commonly used for Bethyl antibodies.
Find Out More about Antibody Validation
At Fortis Life Sciences, we know that improperly validated antibodies on the market can be extremely challenging and expensive for scientists. Our antibodies are held in high esteem due to the strict antibody validation standards they must achieve before customers access them.
Find out more about the benefits of our Antibody Validations and how our techniques can put your mind at ease.
