Catalase, a specific antigen in the feces of human subjects infected with Helicobacter pylori

Abstract

Recently, we reported the production of three new monoclonal antibodies with high specificity for a Helicobacter pylori antigen suitable for diagnosis of H. pylori infection. The aim of the present study was to identify the antigen recognized by these monoclonal antibodies concerning both H. pylori and the feces of human subjects infected with H. pylori. The cellular antigen was purified from an H. pylori cell extract by immunoaffinity column chromatography with the monoclonal antibody as a ligand. The amino-terminal amino acid sequences (eight residues) of the purified antigen and H. pylori catalase were the same. The molecular weights of native and subunit, specific catalase activity, and UV and visible spectra of the purified antigen were in good agreement with those of H. pylori catalase. The human fecal antigens were purified from two fecal samples of two H. pylori-positive subjects by ammonium sulfate precipitation, CM-Sephadex C(50) chromatography, and the same immunoaffinity chromatography used for the H. pylori cellular antigen. The fecal antigens had catalase activity. The amino-terminal amino acid sequences (five residues) of the human fecal antigen and H. pylori catalase were the same. The monoclonal antibodies reacted with the native cellular antigen, but did not react with the denatured antigen, human catalase, and bovine catalase. The results show that the target antigen of the monoclonal antibodies is native H. pylori catalase and that the monoclonal antibodies are able to specifically detect the antigen, which exists in an intact form, retaining the catalase activity in human feces.

FIG. 1.

Purification of H. pylori cellular antigen by immunoaffinity chromatography with MAb 21G2 as a ligand. (A) Five milliliters of the soluble fraction (20 mg of protein) from H. pylori ATCC 43504 cells was loaded onto an immunoaffinity column. The column was washed with PBS, and then the antigen was eluted with 0.2 M glycine-HCl buffer (pH 3.0). Fractions of 10 ml were collected. The arrow indicates the start of elution. ○, antigenicity (dilution, 1:2,000); •, protein; ▵, catalase activity. (B) Ten microliters of the eluate fractions was mixed with an equal volume of sample buffer. The samples were boiled for 5 min and electrophoresed on an SDS-polyacrylamide gel (4 to 20% gradient), and then the gel was visualized with silver stain. M, marker proteins for molecular weight determination (phosphorylase b, 94,000; bovine serum albumin, 67,000; ovalbumin, 43,000; carbonic anhydrase, 30,000; soybean trypsin inhibitor, 20,100; and α-lactalbumin, 14,400).

FIG. 2.

Purification of H. pylori fecal antigen from subject A (A) and subject B (B) by immunoaffinity chromatography with MAb 21G2. Six milliliters of partially purified fecal antigen (2.4 mg of protein and 1 mg of protein for panels A and B, respectively) was loaded onto an immunoaffinity column. The column was washed with PBS, and the antigen was eluted with 0.2 M glycine-HCl buffer (pH 3.0). Fractions of 10 ml were collected. The arrows indicate the start of elution. ○, antigenicity (1:10 dilution for panel A and no dilution for panel B); ▵, catalase activity.