Lack of Specificity of Commercially Available Antisera: Better Specifications Needed

Abstract

The ideal antiserum for immunohistochemical (IHC) applications contains mono-specific high-affinity antibodies with little nonspecific adherence to sections. Many commercially available antibodies are “affinity” purified, but it is unknown if they meet “hard” specificity criteria, such as absence of staining in tissues genetically deficient for the antigen or a staining pattern that is identical to that of an antibody raised against a different epitope on the same protein. Reviewers, therefore, often require additional characterization. Although the affinity-purified antibodies used in our study on the distribution of muscarinic receptors produced selective staining patterns on sections, few passed the preabsorption test, and none produced bands of the anticipated size on Western blots. More importantly, none showed a difference in staining pattern on sections or Western blots between wild-type and knockout mice. Because these antibodies were used in most studies published thus far, our findings cast doubts on the validity of the extant body of morphological knowledge of the whole family of muscarinic receptors. We formulate requirements that antibody-specification data sheets should meet and propose that journals for which IHC is a core technique facilitate consumer rating of antibodies. “Certified” antibodies could avoid fruitless and costly validation assays and should become the standard of commercial suppliers.

Strategies for immunohistochemical protein localization using antibodies: What did we learn from neurotransmitter transporters in glial cells and neurons

“…We therefore fully agree with those who argue that commercial antibody producers should test their antibodies more rigorously before selling them to scientists or pathologists who often lack the resources or expertise to evaluate acquired antibodies (Rhodes and Trimmer, 2006;Pradidarcheep et al, 2008;Couchman, 2009;Boenisch, 2006;Kalyuzhny, 2009;Bohmer et al, 2014;Baker, 2015).…”

Expression and distribution of immunoglobulin G in the normal liver, hepatocarcinoma and postpartial hepatectomy liver

“…22 Rabbit anti-human IgG antibody (1:4000 diluted as working solution, Dako) and different concentration of human IgG (as an antigen from human serum, Sigma, St Louis, MO, USA) were mixed. Then, the mixed solution was used as the primary antibody.…”

How reliable are G-protein-coupled receptor antibodies?

“…A second soft criterion used in the past is that antibodies against different subtypes of a given receptor exhibit distinct staining patters in tissues. However, studies with both knock-out mice (Everaerts et al 2009;Jensen et al 2009;Jositsch et al 2009;Lu and Bartfai 2009;Pradidarcheep et al 2008) or immunoblots comparing staining to cloned receptor subtypes (Bodei et al 2009;Hamdani and van der Velden 2009;Pradidarcheep et al 2009) demonstrate that this also is insufficient to prove selectivity. Rather we propose that at least one of the following four conditions must be met to consider an antiserum to indeed be selective.…”

“…Several studies demonstrate that many available antibodies fail to meet this test (Everaerts et al 2009;Jensen et al 2009;Jositsch et al 2009;Lu and Bartfai 2009;Pradidarcheep et al 2008). Secondly, animals or cell lines can be treated with genetic tools to knock-down expression of a given receptor, e.g.…”

“…Doubts about the usefulness of antibodies against GPCR (O'Connell et al 2006;Pradidarcheep et al 2008;Rhodes and Trimmer 2006), receptors from the ligand-gated ion channel family (Herber et al 2004) or transmitter transporters (Chen et al 2004) have been raised in the past and have even led to the retraction of papers (Hawes and Picciotto 2005). However, these reports have largely been thought to represent isolated cases and did not raise more fundamental questions about the use of receptor antibodies.…”