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AIM: To detect multiple H pylori antibodies in serum samples of individuals who carryH pylori by protein array. METHODS: Recombinant H pylori antigens, urease B subunit (UreB), vacuolating toxin A (VacA) and cytotoxin associated gene A protein (CagA), were prepared and immobilized in matrixes on nitrocellulose membrane by robotics to bind the specific immunoglobulin G (IgG) antibodies in serum. Staphylococcus protein A (SPA) labeled by colloid gold was used to integrate the immuno-complex and gave red color signal. The scanner based on charge-coupled device (CCD) could collect the image signal and convert it into digital signal. RESULTS: When human IgG was printed on the membrane in increasing concentrations and incubated with immunogold, a linear dose response curve was obtained and the detection limit for IgG was about 0.025 ng. The cutoff values, which were defined as the mean grey level plus 3 times of standard deviation, were 27.183, 28.546 and 27.402, for anti-UreB IgG, anti-CagA IgG and anti-VacA IgG, respectively, as 400 human serum samples with negative H pylori antibodies were detected by the protein array. When 180 serum samples from patients in hospital were employed for detection of IgG against UreB, CagA and VacA, the sensitivity of the protein array was 93.4%, 95.4%, 96.0%, and the specificity was 94.8%, 94.4% and 97.5%, respectively, as compared with the results obtained by ELISA. The assay also showed high reproducibility, uniformity and stability, and the results were available within 30 min. CONCLUSION: The protein array is a very practical method for rapid detection of multiple antibodies in serum samples. It is especially useful for large scale epidemiological investigation of the infection of H pylori.

AIM: The genomes of Helicobacter pylori (H. pylori) from different individuals are different. This project was to identify the strain specific DNA sequences between two clinical H. pylori isolates by suppression subtractive hybridization (SSH). METHODS: Two clinical H. pylori isolates, one from gastric ulcer (GU, tester) and the other from non-ulcer dyspepsia (NUD, driver), were cultured and the genomic DNA was prepared and submitted to Alu I digestion. Then two different adaptors were ligated respectively to the 5’-end of two aliquots of the tester DNA fragments and SSH was made between the tester and driver DNA. The un-hybridized tester DNA sequences were amplified by two sequential PCR and cloned into pGEM-T-Easy Vector. The tester strain specific inserts were screened and disease related DNA sequences were identified by dot blotting. RESULTS: Among the 240 colonies randomly chosen, 50 contained the tester strain specific DNA sequences. Twenty three inserts were sequenced and the sizes ranged from 261 bp to 1036 bp. Fifteen inserts belonged to the H.pylori plasmid pHPO100 that is about 3.5 kb and codes a replication protein A. Other inserts had patches of homologous to the genes of H.pylori in GenBank. Various patterns of dot blots were given and no GU strain unique DNA sequences were found when 4 inserts were used as probes to screen the genomic DNA from 27 clinical isolates, 8 from GU, 12 from duodenum ulcer (DU), 4 from GU-DU, 2 from NUD and 1 from gastric cancer (GC). But a 670 bp DNA fragment (GU198) that was a bit homologous to the 3’-end of the gene of thymidylate kinase was positive in 7 GU strains (7/8), 3 GU-DU strains (3/4) and 3 DU strains (3/12). A 384 bp fragment (GU79) of the replication gene A (repA) was positive only in 4 H.pylori isolates, 2 from GU and 2 from GU-DU. CONCLUSION: Differences exist in the genes of different H.pylori isolates. SSH is very effective to screen H.pylori strain specific DNA sequences between two clinical isolates, and some of these sequences may have clinical significance.

Hepatitis C is a world-wide epidemic disease stemming from the hepatitis C virus (HCV). HCV is not only the pathogenic factor of hepatitis C but also plays an important role in the process of triggering and development of cirrhosis and liver cancer. There are two indicators for detecting HCV infection: one is HCV RNA which is the gene of the virus itself and the other is anti-HCV which is the antibody produced when the HCV antigen stimulates the body. The HCV antibody detection includes the detection of HCV IgG and HCV-IgM antibody. At present, EIA and the recombinant immunoblot assay (RIBA) are mainly used for detecting anti-HCV-IgG. By using the colloidal gold labeling technique to fix the structural and the non-structural domains of HCV recombinant antigen on the millipore membrane, a dot immunogold filtration assay for detecting anti-HCV-IgG was successfully established.

Helicobacter pylori (Hp) plays an important role in the upper digestive tract diseases. It can be divided into two main groups (toxic and non-toxic Hp) according to the production of vacuolating cytotoxin (VacA). The toxic bacteria also produce cytotoxin associated protein A (CagA) which might have something to do with the transcription, folding, transportation or the function of VacA. Studies showed that CagA positive Hp (CagA+Hp) accounted for more than 50% of all kinds of Hp, and peptic ulcer and gastric cancer were closely related to their infection[1-7]. Therefore, detection of the infection of CagA+Hp is of great significance. This study was carried out to express CagA gene in E.coli and develop an immuno-assay for the rapid detection of CagA+Hp.

The hepatitis A virus specific immunoglobulin M (IgM) antibody is a specific serological marker for early diagnosis of hepatitis A. At present, the methods used at home or abroad for detecting anti-HAV IgM are RIA, ELISA and SPHAI. The dot immunogold combination assay that has been developed since 1989 is a new technique with the property of simple and rapid immunological detection, by using the red colloidal gold particles to label the antibodies as indicator, and the millipore filtering membrane coated with antigen as the carrier. Affected by filtration and condensation, the antigen antibody reaction is enabled to go on rapidly. When the reaction is positive, red dots appear on the membrane. It takes about 2 min to 4 min for the whole reaction to be carried out. With the above technique, we have established the dot immunogold filtration assay (DIGFA) for rapid detection of anti-HAV IgM with comparatively satisfactory results.