The development of a membrane-based screening method to detect antibodies to intermediate filament proteins.
Autoantibodies directed against the intermediate filament proteins (IF) arise in a variety of disease states. The authors have investigated the binding of the IF to solid membrane supports in a dot blot format in an attempt to develop a simple procedure to detect antibodies (ab) to IF. Commercially obtained, purified IF were utilized. These were: vimentin (VIM), cytokeratin 8 (CYK), glial fibrillary acidic protein (GFA), desmin (DES), and the neurofilament triplet proteins (68, 160, and 200 KDa, respectively designated LMW, MMW, and HMW). Murine monoclonal antibody (mAb) probes were used to detect the presence and immunoreactivity of IF. The mAb were visualized with HRP-anti-mouse conjugates using alpha-chloronaphthol/H 2O 2 as substrate. The membranes studied were nitrocellulose (NC), and two of modified nylon. Nitrocellulose provided the most reproducible binding; no advantage was found to ensue from the use of the other membranes with regard either to quantitative binding or improved capping. Among the IF studied, VIM, GFA, LMW, MMW, and HMW bound well to NC; optimal mass/dot was 1 mug. Filtered, non-fat dry milk is a better capping agent than either albumin or fetal calf serum, but interferes with ab binding to GFA. Binding of CYK and DES is weak at neutral pH. Standard densitometric techniques provide the possibility of quantitation. We conclude that dot and slot blot assays may be practical methods to detect ab to IF antigens.
Rheumatology Laboratory, St. Joseph's Hospital and Medical Center, Paterson, NJ 07503.
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DE
Moretti
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Saini
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Abner
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LB
Keil
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DeBari
D001323:Autoantibodies / Q000032:analysis*; D003101:Collodion; D006801:Humans; D015151:Immunoblotting / Q000295:instrumentation / Q000379:methods*; D007381:Intermediate Filament Proteins / Q000276:immunology*; D008567:Membranes, Artificial; D009757:Nylons