3.A Reactivity with the target antigen
- 1. Reactivity with the immunogen: Antibodies are commonly screened for reactivity against the immunogen using a high-throughput technique such as, enzyme linked immunoabsorbant assay (ELISA), flow cytometry or Western blotting. Screening may extend no further than an ELISA assay using the immunising peptide. This is insufficient to validate an antibody and such information does not guarantee reactivity with the endogenous protein.
- 2. Recognition of a recombinant target antigen: Antibody reactivity with transfected cells overexpressing the target protein or purified recombinant proteins are used to demonstrate binding to the target antigen. Tagged expression constructs or even lysates for most proteins are now commercially available to assist in validation. Be warned that this information still does not guarantee reactivity with the endogenous protein.
- 3. Recognition of the endogenous protein: This is generally achieved by testing antibody labelling in positive and negative controls (cells and tissues that express versus those that lack the target antigen). Ideally start by testing the antibody reactivity (usually in multiple techniques and ideally with more than one antibody) with at least two positive and two negative cell lines and/or tissues (usually transcript positive/negative). Recognition of the endogenous protein can also be achieved by modulating expression of the endogenous protein, e.g. comparing tissues from a gene knock out or gene targeted silencing experiment. The multiple positive and negative controls should be tested in the same experiment and are essential to enable confirmation of detection of the endogenous protein and to show that the antibody does not bind in the absence of the target protein.
- 4. Evidence of specificity for the target antigen: Detection of the target antigen does not exclude the possibility that the antibody is cross-reactive with another protein(s). While sequence similarity can be used to predict potential cross-reactivity with related proteins containing linear epitopes, the antibody may also recognise conformational epitopes whose cross-reactivity cannot be predicted from amino acid sequence comparison. Antibody cross-reactivity with related proteins that share significant sequence identity should be tested experimentally. This is commonly performed using transiently transfected cells (e.g. COS-7, HEK293 cells) expressing different members of the related proteins of the same gene family.
- 5. Expression pattern: Test that the antibody has the predicted/expected cell or tissue labelling pattern and ideally detects the appropriate subcellular localisation of the antigen. Choosing positive controls with different levels of protein expression for your target antigen, e.g. strong versus moderate/weak expression, can inspire added confidence in the validation, the specificity of the antibody, and help to identify those antibodies with the highest affinity for the target antigen.
- 6. Corroboration from using multiple reagents: Testing several different antibodies in parallel and observing any common patterns of antibody reactivity can significantly strengthen confidence in the validation data generated. This is particularly important when unexpected or novel findings are observed.