Lack of specificity of commercially available antisera: better specifications needed.
The ideal antiserum for immunohistochemical (IHC) applications contains monospecific 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.
Pradidarcheep
Wisuit
W
AMC Liver Center, Academic Medical Center, University of Amsterdam, Meibergdreef 69-71, 1105 BK Amsterdam, The Netherlands.
Labruyère
Wil T
WT
Dabhoiwala
Noshir F
NF
Lamers
Wouter H
WH
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D000939:Epitopes; D041981:Gastrointestinal Tract / Q000378:metabolism; D007106:Immune Sera; D007150:Immunohistochemistry; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D051381:Rats; D017208:Rats, Wistar; D011976:Receptors, Muscarinic / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D012680:Sensitivity and Specificity; D014551:Urinary Tract / Q000378:metabolism
D000939:Epitopes; D007106:Immune Sera; D011976:Receptors, Muscarinic
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