There are many commercial providers of many tens of thousands of antibodies. At face value one would think that there are antibodies to most proteins and that they are not a limiting resource for research and development. However, the real truth is the “dirty little secret” of the antibody world. It is surprisingly common to purchase a catalog antibody product and not have it work in the desired application.
The source of this problem derives from multiple issues, some of which rest on the user and some on the antibody provider. Most people blame the antibody. Achieving success in an immunoassay depends on three things (assuming the immunoassays are technically executed appropriately);
1) Nature of the epitope that the antibody is directed against (unique or common to the protein, linear or conformational, buried or surface exposed, subject to post-translational modifications. This is a complex topic and responsibility lies on both sides of customer and provider. The customer should carefully review the details on how the antibody was designed to see if the epitopes recognized would be suitable for the application (eg. if the antibody recognizes denatured epitopes but the immunoassay displays folded epitopes). The antibody provider should design the antigen carefully in order to generate antibodies to recognize the desired form and position of the epitope.
2) The quality and nature of the sample. The responsibility lies with the customer. Are the samples degraded, denatured, or otherwise modified? Is it in the appropriate buffer? Is the protein present at a suitable concentration? Are there inhibitory factors present? Are there cross-reacting proteins present?
3) The quality and nature of the antibody. The responsibility here lies with the antibody provider, although antibodies must be handled and stored appropriately to ensure consistent performance.
References
1) Saper CB. An open letter to our readers on the use of antibodies.
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493: 477–478, 2005.2) Rhodes KJ, Trimmer JS. Antibody-based validation of CNS ionchannel drug targets.131: 407–413, 2008.3) Saper CB, Sawchenko PE. Magic peptides, magic antibodies:guidelines for appropriate controls for immunohistochemistry.465: 161–163, 2003.4) Jennifer Bordeaux, Allison W. Welsh, Seema Agarwal, Elizabeth Killiam, Maria T. Baquero, Jason A. Hanna, Valsamo K. Anagnostou, and David L. Rimm. Antibody validation48:197-209 (March 2010)5) John R. Couchman Commercial Antibodies: The Good, Bad, and Really Ugly. Volume 57(1): 7–8, 2009. Journal of Histochemistry & Cytochemistry6) Jean-Marc Fritschy. TECHNICAL SPOTLIGHT: Is my antibody-staining specific? How to deal with pitfalls of immunohistochemistry. European Journal of Neuroscience, Vol. 28, pp. 2365–2370, 2008.7) Martin C. Michel & Thomas Wieland & Gozoh Tsujimoto. How reliable are G-protein-coupled receptor antibodies? Naunyn-Schmied Arch Pharmacol (2009) 379:385–388