Reverse immunoaffinity chromatography: application to the purification of the 60- to 90-kDa gastric parietal cell autoantigen associated with autoimmune gastritis.
Previous attempts to purify autoantigens by affinity chromatography using the total IgG from autoantibody-positive sera have not been very successful. We describe here a novel method using parietal cell autoantibodies, isolated on a crude autoantigen immunoabsorbent column, for the purification of the 60- to 90-kDa gastric parietal cell antigen targeted in autoimmune gastritis. The 60- to 90-kDa parietal cell antigen was first enriched 2.5-fold from a 0.5% Triton X-100 extract of total pig gastric mucosal membranes by DEAE-Sepharose 4B chromatography. The fraction containing the 60- to 90-kDa antigen was directly coupled to CNBr-activated Sepharose 4B to produce an immunoadsorbent column for the "reverse" purification of the specific parietal cell autoantibodies from pooled gastric parietal cell autoantibody-positive human sera. A 0.5% Triton X-100 extract of pig gastric mucosal membranes was initially precleared by sequential passage through Protein A-Sepharose 4B and normal human Ig-Sepharose 4B columns. The 60- to 90-kDa gastric parietal cell antigen (approximately 10 micrograms) was then purified from this precleared Triton X-100 membrane extract by affinity chromatography on a 1-ml column of purified autoantibody-Sepharose 4B, followed by preparative sodium dodecyl sulfate-polyacrylamide gel electrophonesis. This method, incorporating a "reverse" immunoaffinity chromatography step, has general applicability for the purification of tissue autoantigens using highly specific autoantibodies present in the sera of humans and experimental animals with a variety of other autoimmune diseases.
Goldkorn
I
I
Department of Pathology and Immunology, Monash University Medical School, Prahran, Victoria, Australia.
Gleeson
P A
PA
Toh
B H
BH
D000818:Animals; D001324:Autoantigens / Q000302:isolation & purification*; D001327:Autoimmune Diseases / Q000276:immunology*; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D005756:Gastritis / Q000276:immunology*; D006801:Humans; D015151:Immunoblotting; D008970:Molecular Weight; D010295:Parietal Cells, Gastric / Q000276:immunology*; D013552:Swine
D001324:Autoantigens