What are the most commonly used methods to validate antibody specificity?

Updated: Apr 11

In 2016, the International Working Group for Antibody Validation (IWGAV) convened to outline the best approaches to validate antibodies to ensure reproducibility in common research applications. They proposed five approaches, termed the five pillars for antibody validation. These five pillars include the orthogonal approach, independent antibody approach, recombinant expression approach, captured mass spectrometry approach, and genetic approach (Uhlen et al., 2016). The following table summarizes the features, strengths, and weaknesses of each approach (Edfors et al., 2018; Pillai-Kastoori et al., 2020; Uhlen et al., 2016).


Table 1. Comparison of the five pillars for antibody validation in terms of their features, strengths and weaknesses

Table 2. Comparison of the five pillars for antibody validation in terms of their antibody specificity score, technical challenges, and directness (Edfors et al., 2018; Pillai-Kastoori et al., 2020; Uhlen et al., 2016)

References

Edfors, F., Hober, A., Linderback, K., Maddalo, G., Azimi, A., Sivertsson, A., Tegel, H., Hober, S., Szigyarto, C.A., Fagerberg, L., et al. (2018). Enhanced validation of antibodies for research applications. Nat Commun 9, 4130.

Pillai-Kastoori, L., Heaton, S., Shiflett, S.D., Roberts, A.C., Solache, A., and Schutz-Geschwender, A.R. (2020). Antibody validation for Western blot: By the user, for the user. J Biol Chem 295, 926-939.

Uhlen, M., Bandrowski, A., Carr, S., Edwards, A., Ellenberg, J., Lundberg, E., Rimm, D.L., Rodriguez, H., Hiltke, T., Snyder, M., et al. (2016). A proposal for validation of antibodies. Nat Methods 13, 823-827.

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