The feces from these patients were collected using the stool collection devices, and were stored at −20°C. All the subjects provided their written informed consent, and the use of fecal samples and isolated H. pylori strains for this study was approved by the ethics committee of Hirosaki University. The following laboratory bacterial strains were used: check details H. pylori ATCC 43504, H. hepaticus ATCC 51448, H. felis ATCC 49179, H. mustelae ATCC 43772, H. cinaedi ATCC 35683, Campylobacter jejuni ATCC 29428, Escherichia coli ATCC 25922, Bacteroides vulgatus IFO 14921, Bifidobacterium breve JCM 1192, and Bifidobacterium infantis JCM 1222. H. pylori strains isolated
from 1344 Japanese patients who underwent gastro-duodenoscopy at Hyogo College of Medicine Hospital were also tested. Culturing and whole-cell disruption of all of the bacterial strains was carried out as described previously.8 The standard H. pylori cellular antigen was prepared from H. pylori ATCC 43504 and cultured on Difco Brain Heart Infusion Agar (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) plates containing 5% horse blood in a microaerophilic environment (Anaero Pack Helico System; Mitsubishi Gas Chemical Co. Inc., Tokyo, Japan).
The culture plate was incubated for 7 days in an anaerobic environment (Anaero Pack Anaero System; Mitsubishi Gas Chemical Co. Inc.). Bacterial cells R788 clinical trial were harvested, washed with PBS, suspended in PBS containing 0.5% formalin, and then incubated at 4°C for overnight. The bacterial cells were washed three times with PBS and disrupted by sonication (Biom’c Model 7250, Seiko Instruments & Electronics, Ltd, Tokyo, Japan). H. pylori ATCC 43504 antigen was obtained from the supernatant upon centrifugation of the sonicated suspension and was stored at −30°C until use. The protein concentration of antigens from the clinical strains was adjusted to 5 µg/mL with the diluent buffer. The protein concentration of the laboratory strains was adjusted to 40 000, 8000, 1600, 320, and 64 ng/mL for TPAg EIA test,
and adjusted to 10 µg/mL for Rapid TPAg test. Catalase activity of the bacterial 3-oxoacyl-(acyl-carrier-protein) reductase antigen prepared from 127 H. pylori clinical strains was determined at 25°C by spectrophotometry15 using a molar absorption coefficient of 43.48 L/mol/cm at 240 nm.16 The protein concentration was determined with a bicinchoninic acid protein assay reagent (PIERCE., Rockford, IL, USA) with bovine serum albumin as the standard. TPAg EIA and Rapid TPAg were stored at 30°C for 12 months. The diagnostic performances of the TPAg EIA and Rapid TPAg were examined using the H. pylori ATCC 43504 antigen and five clinical fecal samples. Three of the fecal samples were from H. pylori-positive patients, and two samples were from H. pylori-negative patients. Correlation between catalase activity and the absorbance value of TPAg EIA was calculated by Pearson’s correlation coefficient.