This is a summary of the five enhanced validation principles in Antibodypedia. The validation principles are based on the five “pillars” described by the International Working Group for Antibody Validation (IWGAV), but with requirements for particular applications, including Western blot (WB), immunohistochemistry (IHC), immunocytochemistry (ICC), cell flow sorting (FS) and protein assays (ELISA. Enhanced validation of an antibody has to include primary data (most often images) comparing side-by side detection of the target in biological samples that confirm specificity towards the target and absence of cross-reactivity.
The five enhanced validation principles
1. Genetic validation. This method is based on the knock-down or knock-out of the target protein using genetic methods, such as CRISPR or siRNA, in a suitable cell line. The staining of the antibody is evaluated by the antibody-based method through analyses of samples from cell lysates before and after knock-down of the corresponding target gene.
2. Recombinant expression validation. This method is based on over-expression of the target protein in a cell line preferably not expressing the target protein. The staining of the antibody is evaluated by the antibody-based method through analyses of samples from cell lysates with and without overexpression of the target protein. Alternatively, the target protein is expressed using a tag and the staining using an antibody directed towards the target protein is compared to detecting the tag itself (In some cases using a tag-specific antibody).
3. Independent antibody validation. This method is based on comparing the staining pattern using two independent antibodies with no overlapping epitopes. The staining of the two antibodies is compared through analyses of samples from at least two cell lysates preferably expressing the target protein at different levels.
4. Orthogonal validation. This method is based on comparing the protein analysis achieved by the antibody-based method with levels of the target protein detected by an antibody-independent method. At least two cell or tissue samples must be used and the target protein should normally express the target at different levels. The levels of the target protein in the different samples determined by the two independent methods should correlate. This method can also be used to compare the protein levels determined by the antibody with the corresponding RNA.
5. Capture MS validation. This method is based on mass spectrometry analysis either by analysing the proteins captured by a particular antibody or in the case of Wester blot applications, comparing the staining pattern of the antibody and protein size obtained from the Western blot with the size obtained by a capture MS method in which multiple gel slices are cut out from the electrophoretic separation and analysed separately for the presence of peptides corresponding to the target protein. IN the latter case, the band detected by the antibody should be equivalent to the same size as the gel slice with the detected peptide(s) corresponding to the target protein.