Detection of colonic antigen(s) in tissues from ulcerative colitis using purified colitis colon tissue-bound IgG...

In our report of a disease-specific colonic tissue-bound antibody (CCA) from patients with ulcerative colitis (Proc Natl Acad Sci USA 1978;75:4528), crude CCA was largely fragmented, and the yield was small. We have modified the extraction procedure to increase the yield of intact IgG present in CCA by sequential elution, storage of tissues in presence of a protease inhibitor, 2 mM Phenylmethylsulfonyl Fluoride, and use of Phenylmethylsulfonyl Fluoride in extraction buffers. Intact CCA-IgG was purified using protein A-Sepharose 4B affinity chromatography. 125I-CCA-IgG formed immune complexes in vitro with the aqueous extracts of colonic mucosa from 5 patients with ulcerative colitis but not from 6 patients with Crohn's disease and 6 normal colons from patients with carcinoma (p less than 0.01). We also performed reverse experiments by iodination of the colonic mucosal extracts with Bolton-Hunter reagent and incubating them with several preparations of CCA-IgG. Mucosal extracts of colon from 3 patients with ulcerative colitis bound significantly with CCA-IgG when compared with the identical extracts from 3 patients with Crohn's disease, 3 with a normal colon (p less than 0.005), and with the control human IgG (p less than 0.025). These studies demonstrate a better method of extraction and purification of intact CCA-IgG. Intact CCA-IgG binds to a specific protein(s) present in the homogenates of colonic mucosa from patients with ulcerative colitis but not from patients with Crohn's disease and normal colon.