Antibody Characterization and Validation

Antibody validation is the determination of the following characteristics:

  • Specificity:  (Off-targets and on-targets)
  • Sensitivity and  Affinity: the strength with which an antibody binds an epitope and a quantitative measurement of antibody affinity (KD)
  • Reproducibility:   Problems with antibodies are thought to have led to misidentificaton of candidates for drug development and generated a numerous misleading or irreproducible scientific results

To validate an antibody, it must be shown to be specific, selective, and reproducible in the context for which it is to be used.

Funding agency requirements

Requirements for NIH grant applications are described in: “Authentication of Key Biological and/or Chemical Resources”

This document describes methods to ensure the identity and validity of key biological and/or chemical resources (may include cell lines, specialty chemicals, antibodies, and other biologics), now must include validation data.

How can antibodies be validated?

Optimization of experimental parameters for every new antibody is still required to ensure confidence in results. AbET offers assays for antibody validation and characterization to meet the needs of investigators from very different areas

  1. ELISA
  2. Western blots
  3. Immunoprecipitation (pull down)
  4.  Antibody-antigen affinity determined by Bio-Layer Interferometry on a fortéBIO Octet.

The fortéBIO system is similar to surface plasmon resonance systems in measuring changes with time in optical properties when one molecule binds to another immobilized on a solid surface to determine rates of association and dissociation and hence a dissociation constant.

Useful links

For general antibody information, consult the following databases:

  • Antibodypedia — The Human Proteome Organization (HUPO)’s catalog of validated antibodies against human proteins (
  • Human Protein Atlas — HUPO’s high-resolution images of protein expression profiles in normal and cancerous tissue (
  •  1degreebio — Read and submit independent reviews of antibodies and antibody-related services. Vendors subscribe to have their the products and services listed on the website (
  • MIAPAR — Minimum Information about A Protein Affinity Reagent released through a working group at HUPO (doi:10.1038/nbt0710-650)

Resources and Articles