An open letter to our readers on the use of antibodies

Abstract

The use of immunohistochemistry has become ubiquitous in neuroscience. A large majority of papers now published in The Journal of Comparative Neurology use immunohistochemistry, and some papers may employ a battery of ten or more antibodies to examine issues of colocalization or cell typing. This pattern has resulted in a flood of new information. …but also a flood of misinformation. The Journal has repeatedly, over the last few years, received distressed communications from authors, who have had to withdraw papers because an antibody against a novel marker was found to stain tissue in knockout animals, who lack that target protein. In many cases these papers contained careful characterization of the antibodies and immunocytochemical controls. This issue has sensitized the Editors to the problem of antibody specificity, and we soon realized that many of the papers we were publishing had very limited characterization or controls for antibodies that were used. Subsequently the Editors noticed that a number of commercially available antisera, particularly against G-protein coupled receptors, gave staining patterns that did not match mRNA distributions, and that these antibodies still stained tissue from animals in which the receptor had been knocked out. We felt that the integrity of scientific communication was being threatened by the proliferation of poorly characterized antibodies that produce artifactual staining patterns, and therefore came up with a minimal set of rules for identification, characterization, and controls for immunohistochemistry that we are attempting to apply to all manuscripts that we publish (Saper and Sawchenko, 2003). The result has been a substantial degree of confusion about what information is necessary for a paper in JCN (or any other journal, for that matter), to provide a reasonable level of assurance that an antibody is actually recognizing what it is supposed to be staining. In the interest of demystifying this procedure further, we present the following brief description of the three basic elements that are necessary in describing an antibody for use in neuroscience: 1.) Complete information on the antibody. It is important to recognize that antibodies are not simple reagents that always identify the same thing. Thus, to describe an antibody as completely as possible, so that other scientists can replicate your work, you need to provide information of two kinds: —Identification: What was the source of the antibody? If another lab has donated the antibody, give the antiserum code number, and if possible, the bleed. If it was obtained from commercial sources, give the catalog, and if possible the lot number. —Preparation of the antibody: What was the antibody actually raised against? Give the precise structure of the immunizing antigen, not just vague information about the part of the molecule that was used. What species was the antiserum raised in? Was it a polyclonal or monoclonal preparation? Please note that some antibody manufacturers deliberately try to obscure the information about the structure of the antigen. We have received complaints from authors that several manufacturers have claimed that sequence information on the antigen was “proprietary,” and they would not provide it. Because work using these antibodies is inherently not repeatable, such papers are not acceptable for publication. Our advice is: NEVER BUY ANTIBODIES FOR WHICH THE MANUFACTURER WILL NOT DISCLOSE THE STRUCTURE OF THE IMMUNIZING ANTIGEN. THESE REAGENTS ARE NOT FIT FOR SCIENTIFIC WORK, AND THE WORK YOU DO WITH THEM WILL NOT BE PUBLISHABLE. Examples of antibody descriptions that would be acceptable: “This rabbit antiserum (Company XYZ #30248) was prepared against a synthetic peptide representing amino acids 121-142 from tyrosine hydroxylase.” “This mouse monoclonal antibody, kindly donated by Dr. John Smith, University of Alabama, was raised against human placental choline acetyltransferase.” 2.) How has the specificity of the antibody been characterized? If the antiserum is against a large protein, it is important to know what it stains on a gel from the tissue and species you are using. Ideally, an antibody should stain a single band (or several bands if the antigen has several known molecular configurations) of appropriate molecular weight. This information is often included by the manufacturer in the technical information, and can be cited, as can previous studies that provided this information. However, it is not sufficient just to state “this antiserum was previously characterized (Jones et al., 2002).” You need to provide a reasonably critical reviewer with information on what characterization was done, and what it showed. Examples of antibody characterization that would be acceptable: “The antiserum stains a single band of 55 kD molecular weight on Western blot (manufacturer's technical information).” “This antiserum stains the 150kD but not the 130kD or 110kD forms of the molecule on Weste