Antibody validation

Abstract

Antibodies are among the most frequently used tools in basic science research and in clinical assays, but there are no universally accepted guidelines or standardized methods for determining the validity of these reagents. Furthermore, for commercially available antibodies, it is clear that what is on the label does not necessarily correspond to what is in the tube. To validate an antibody, it must be shown to be specific, selective, and reproducible in the context for which it is to be used. In this review, we highlight the common pitfalls when working with antibodies, common practices for validating antibodies, and levels of commercial antibody validation for seven vendors. Finally, we share our algorithm for antibody validation for immunohistochemistry and quantitative immunofluorescence.

Reproducibility crisis: Blame it on the antibodies

“…This is something that Rimm is passionate about: he has developed a multistep flowchart for effective validation 6 , which he shares with anyone who will listen. But the process is time consuming -Rimm recommends control experiments that involve engineering cell lines to both express and stop expressing the protein of interest, for example.…”

Reproducibility: Standardize antibodies used in research

“…Alarmingly, there are serious flaws in the reliability of antibodies, the most widely used class of protein-binding reagent 2,3 .…”

Preanalytical variables and phosphoepitope expression in FFPE tissue: quantitative epitope assessment after variable cold ischemic time

“…29 In brief, each antibody was titrated on test TMAs, consisting of 40 breast cancer samples. The breast cancer samples represented on the test TMA are formalin fixed and paraffin embedded, and were collected from the Yale Archives (New Haven, CT, USA).…”