Evaluating Commercially Available Antibodies for Rat α7 Nicotinic Acetylcholine Receptors.

Alpha7 nicotinic acetylcholine receptors (7 nAChRs) are important drug targets in neurological disorders and inflammation, making their detection and localization by validated antibodies highly desirable. However, tests in knockout animals raised questions about specificity of antibodies to mouse 7 nAChRs. To date, methods for validating antibodies for rat or human 7 nAChR have not been reported. We developed a gel-shift assay for western blots using GH4C1 cells expressing either native rat receptors or 7 nAChR-green fluorescent protein (GFP) chimeras to evaluate seven commercially available 7 nAChR antibodies. Blots with anti-GFP antibody detected GFP or 7 nAChR-GFP expressed in GH4C1 cells, and I-125--bungarotoxin binding and RNA analysis demonstrated 7 nAChR expression. Validated samples were used to evaluate 7 nAChR antibodies by western blot and immunofluorescence studies. These methods confirmed that two of seven 7 nAChR antibodies identify gel-shifts for 7 nAChR/nAChR-GFP but only one antibody demonstrated low background and significant immunofluorescence differences between wild-type and 7 nAChR expressing GH4C1 cells. However, that polyclonal antibody displayed lot-to-lot variability. Our findings suggest that careful validation methods are required for all 7 nAChR receptor species and antibody lots and that the gel-shift assay may allow for relatively rapid antibody screening.