title: Biochemical and histochemical evidence of nonspecific binding of alpha7nAChR antibodies to mouse brain tissue.
abstract: Alpha 7 nicotinic acetylcholine receptors are involved in learning and memory, and are implicated in the pathology of Alzheimer's disease and schizophrenia. Detection of alpha7 subunits can be accomplished via immunodetection or alpha-bungarotoxin-binding techniques. Standard protocols for immunohistochemistry and Western blotting were followed using several commercially available antibodies. Various mice were evaluated, including non-transgenics, APP, PS1, APP+PS1, and alpha7 knockouts. Initial results with amyloid-depositing mice revealed alpha7 immunolabeled astrocytes, in addition to expected neuronal staining. Subsequent studies with intrahippocampal injections of lipopolysaccharide (LPS) into alpha7 knockout mice showed that both neuronal and astrocytic labeling by alpha7 antibodies was nonspecific. On Western blots of mouse brain proteins, none of the bands detected with antibodies directed against alpha7 subunits diminished in the alpha7 knockout mice. Although LPS-related changes in the expression of some bands were found, these also were unaffected by the alpha7 genotype of the mice. In general, the Western staining patterns for these antibodies revealed few overlapping bands. These immunodetection data are in contrast to genotyping results and mRNA analyses that confirmed the disruption of the alpha7 allele and lack of alpha7 message in the knockouts. These findings suggest caution in interpreting results when using several commercially available alpha7 nicotinic receptor antibodies.
authors.affiliation: Department of pharmacology and Therapeutics, University of South Florida, 12901 Bruce B. Downs Blvd., MDC Box 9, Tampa, FL 33612-4799, USA.
authors.forename: Donna L
authors.initials: DL
authors.lastname: Herber
authors.forename: Emily G
authors.initials: EG
authors.lastname: Severance
authors.forename: Javier
authors.initials: J
authors.lastname: Cuevas
authors.forename: Dave
authors.initials: D
authors.lastname: Morgan
authors.forename: Marcia N
authors.initials: MN
authors.lastname: Gordon
mesh_terms: D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D000918:Antibody Specificity; D015153:Blotting, Western / Q000379:methods; D001921:Brain / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics; D005838:Genotype; D007150:Immunohistochemistry / Q000379:methods; D008070:Lipopolysaccharides / Q000494:pharmacology; D008565:Membrane Proteins / Q000235:genetics; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D053764:Presenilin-1; D012313:RNA / Q000032:analysis; D011978:Receptors, Nicotinic / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D064569:alpha7 Nicotinic Acetylcholine Receptor
chemical_list: D000906:Antibodies; D002352:Carrier Proteins; C579166:Chrna7 protein, mouse; D008070:Lipopolysaccharides; D008565:Membrane Proteins; D053764:Presenilin-1; D011978:Receptors, Nicotinic; D064569:alpha7 Nicotinic Acetylcholine Receptor; D012313:RNA