Enzyme Linked Immunosorbent Assay (ELISA) and Immunoprecipitation

224 Gut 1994; 35:224-230 Enzyme linked immunosorbent assay (ELISA) and immunoprecipitation studies on anti-goblet cell Gut: first published as 10.1136/gut.35.2.224 on 1 February 1994. Downloaded from antibody using a mucin producing cell line in patients with inflammatory bowel disease T Hibi, M Ohara, K Kobayashi, W R Brown, K Toda, H Takaishi, Y Hosoda, A Hayashi, Y Iwao, M Watanabe, S Aiso, Y Kawai, M Tsuchiya Abstract epithelial cells in the colon and mediates specific Circulating anti-goblet cell antibody and its cytotoxicity to colonic epithelial cells.>" Except corresponding antigen in patients with inflam- for a 40 kD colitis colon associated antigen matory bowel disease were investigated. Anti- described by Das et al,'21'4 no other colon specific goblet cell antibody in the serum was examined antigens have been clarified yet. We have by immunocytochemistry and enzyme linked reported the high incidence of anti-colon anti- immunosorbent assay (ELISA), using a body, which reacts with the surface of isolated colonic cancer cell line, HT29-18-N2, which rat colonic epithelial cells and the presence of differentiates into intestinal goblet cells. The antibody dependent cell mediated cytotoxicity frequencies of anti-goblet cell antibody activity in the serum samples from patients with detected by immunocytochemistry were 14 in ulcerative colitis.'0 48 patients with ulcerative colitis (29%) and The other autoantibodies, anti-neutrophil five in 15 patients with Crohn's disease (33%). cytoplasmic antibodies (ANCA), have been By ELISA, the frequencies of anti-goblet cell recently found in inflammatory bowel disease.'5 antibody were 38% in ulcerative colitis and ANCA were present in most patients with 33% in Crohn's disease. This antibody did not ulcerative colitis and a small subset of patients relate directly to anti-neutrophil cytoplasmic with Crohn's disease. antibodies (ANCA), although the serum The significance of these antibodies in samples positive for anti-goblet cell antibody the pathogenesis of ulcerative colitis is still were commonly positive for ANCA in ulcera- uncertain, and some investigators insist that the tive colitis. Immunoprecipitation and SDS production of circulating autoantibodies in polyacrylamide gel electrophoresis (PAGE) inflammatory bowel disease is an epipheno- http://gut.bmj.com/ study showed that the antibody in the ELISA menon. Characterisation of the antigens recog- positive serum samples recognised a >200 kD nised by the autoantibodies is essential for goblet cell antigen, which remained unchanged determination of the pathogenetic importance of after reduction, indicating that it consists of the antibodies. In addition, the identification of single chain polypeptides. These results sug- the antigens may allow us to establish more gest that there is a subgroup of inflammatory precise diagnostic methods for this disease. bowel disease that has circulating anti-goblet Extraction of the colon specific antigens, how- on October 1, 2021 by guest. Protected copyright. Departments of Internal cell antibody reactive with a >200 kD antigen. ever, from human colonic tissue is often difficult Medicine The antibody detected by newly established because colonic tissue contains many kinds of T Hibi ELISA will be a disease marker for this group other substances. Thus, cell lines containing the M Ohara K Kobayashi and the identification of the corresponding antigens may be more useful. A human colonic K Toda antigens may be important for the understand- cancer cell line, HT28-18-N2,'6 which differenti- H Takaishi ing ofthe underlying immune abnormalities. ates into goblet cells producing mucin in con- Y Hosoda A Hayashi (Gut 1994; 35: 224-230) fluent cell culture, is a good source of the Y Iwao antigens recognised by an anti-goblet cell anti- M Watanabe body. Y Kawai M Accumulative evidence suggests that ulcerative There have been no studies on the characteri- Tsuchiya colitis is one of the autoimmune diseases in sation of the antigens recognised by anti-goblet and Anatomy, School of which various autoantibodies are often found.' cell antibody and the relation between ANCA Medicine, Keio In we University, Japan In ulcerative colitis, two kinds of autoantibodies and anti-goblet cell antibody. this study, S Aiso to colonic epithelial cells have been proposed. focused on anti-goblet cell antibody and com- One is the classic antibody that reacts with pared this antibody with the other autoanti- Department of Medicine mucin colonic of the Veterans phenol water extracts of fetal colonic tissue and bodies, ANCA. Using producing Administration Medical mucopolysaccharide in colonic goblet cells.' epithelial cells (HT29-18-N2), we developed an Center and University of Since this anti-goblet cell (anti-mucin) autoanti- improved method for detecting serum anti- Colorado School of was and Perlmann goblet cell antibody and characterised the Medicine, Denver, USA body proposed by Broberger W R Brown in 1959,' numerous studies on this kind of antigen detected by this antibody in inflamma- Correspondence to: antibody have been performed by various tory bowel disease. Dr T Hibi, Department of investigators."8 Nevertheless, any simple assay Internal Medicine, School of Medicine, Keio University, for this antibody has not been developed and the 35 Shinanomachi, characteristics of the corresponding antigens Patients and methods Shinjuku-ku, Tokyo 160, Japan. remain unclear. The other antibody is anti- Accepted for publication colonic epithelial cell antibody, which mainly SERUM SAMPLES 16 June 1993 reacts with the membrane of the surface Ulcerative colitis or Crohi's disease was diag- ELISA andanti-goblet cellantibody inIBD 225 nosed on the basis of typical clinical, radio- serum) overnight in a moist chamber and washed graphic, and pathological features. Serum in phosphate buffered saline. They were then samples were obtained from 48 patients with incubated with peroxidase labelled rabbit anti- ulcerative colitis, 15 patients with Crohn's human IgG F(ab')2 (Dako, Santa Barbara, CA, Gut: first published as 10.1136/gut.35.2.224 on 1 February 1994. Downloaded from disease, five patients with colon cancer, seven USA) for 30 minutes. After further washing, patients with colon polyp, six patients with colon peroxidase activity was developed with diamino- diverticulitis, 12 patients with infectious colitis benzidine and hydrogen peroxide, and the slides (four Campylobacter colitis, two colon tuber- were examined by light microscopy in a blinded culosis, and six amoebic colitis), five patients manner. with rheumatoid arthritis, and two patients with systemic lupus erythematosus. The mean age of the patients with ulcerative colitis was 32 years PREPARATION OF HORSERADISH PEROXIDASE (range 17-52). Thirty three of the patients had LABELLED F(ab')2 OF POSITIVE AND NEGATIVE relapsing type and 15 patients had chronic SERUM SAMPLES continuous type of disease. Nineteen patients One strongly positive serum from a patient with were in active stage and 29 patients in inactive ulcerative colitis and a negative serum were stage. The disease activity was calculated accord- separately precipitated in 50% ammonium sul- ing to a previous report17 in which the scores were fate and purified by adsorption to a protein A- made by frequencies of bowel movement, blood sepharose CL-4B column and elution with 0-1 M in stool, body temperature, abdominal pain, and acetic acid. Immunoglobulins were digested by pulse rate. The scores ranged from 0 to 8. The reaction with pepsin and (1:50 wt/wt pepsin: patients with scores more than three were protein) in 0-05 M acetate buffer, pH 4-5, and regarded as in active stage. The mean age of the chromatography on sephacryl S-200. F(ab')2 was patients with Crohn's disease was 29 years conjugated to horseradish peroxidase type 6 as (range 15-36). All of them had active disease described. 8 Direct immunocytochemistry (Crohn's disease activity indices of >150) when against HT29-18-N2 and immunohistochemistry the serum samples were obtained. Seven against colonic biopsy specimens using HRP- patients had colitis disease type and eight ileo- F(ab')2 was performed. colitis type. None of the patients received corticosteroids. Control serum samples were obtained from 35 healthy hospital workers whose ELISA FOR THE DETECTION OF ANTI-GOBLET CELL sex and age distribution was comparable with ANTIBODY that of the patient group. Serum samples were A mucin rich fraction was obtained from a stored at -80°C without preservatives and confluent culture ofHT29-18-N2 cells. Cells that centrifuged at 105 000 g for 90 minutes before differentiated into mucin producing cells in two use. weeks after they were confluent in culture, were http://gut.bmj.com/ sonicated and centrifuged at 105 000 g for 60 minutes to remove nuclei. The supernatant was IMMUNOCYTOCHEMISTRY FOR THE DETECTION OF dialysed against deionised water and then used as CIRCULATING ANTI-GOBLET CELL ANTIBODY the solid phase antigen for ELISA after the Human colonic cancer cell line, HT29, and its protein concentration was measured and pH was subclone, HT29-18-N2, were cultured in 8 well fixed to 7-2. EIA plates (Flow Laboratories, Lab-Tec chambers (Nunc Inc, Naperville, IL, McLean, Virginia, USA) were coated with this on October 1, 2021 by guest. Protected copyright. USA) on slide glass. At confluence, cells were mucin rich fraction at 0v 1 [ig protein/ml over- fixed in ethanol solution for one second. After night at 4°C. After being washed, the plates were being washed, the slides were incubated with incubated with 200 RI of blocking buffer (1% phosphate buffered saline containing 10% rabbit bovine serum albumin in 0- 1 M sodium bicarbo- serum and then with 1:50 diluted serum in a nate, pH 8 2) at 22°C for four to six hours. The moist chamber at room temperature for 30 plates were washed three times with phosphate minutes. The slides were washed three times by buffered saline, then incubated with 200 p1 of immersion in excess phosphate buffered saline.

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