Background and Aim: Helicobacter pylori infection has been identified as a major cause of peptic ulcer diseases, including gastric and duodenal ulcers, gastritis, chronic and gastric carcinoma, and even gastric lymphoma. In vitro studies using Western blotting analysis, hemagglutination test, adherence inhibition assays, and immunocytochemical staining revealed that the 49.6-kDa subunit pili protein of H. pylori was considered an immunogenic protein. This study aimed to develop a serological diagnostic test using 49.6 kDa for detecting antibodies against H. pylori proteins in an early phase of the infection.

Materials and Methods: An in-house immunochromatographic test (ICT) kit was developed and used to test a panel of sera sample obtained from a randomly selected symptomatic patient, in which 40 sera were H. pylori positive and 40 sera were H. pylori negative.

Results: The results showed that ICT with 49.6 kDa as an antigen was highly sensitive and specific for detecting anti-H. pylori immunoglobulin G antibodies in human serum, with a high negative predictive value.

Conclusion: The developed test could be used to exclude H. pylori infection in symptomatic patients.

Keywords: 49.6-kDa pili protein, Helicobacter pylori, immunochromatographic test.

1. Parsonnet, J., Friedman, G.D., Vandersteen, D.P., Chang, Y., Vogelman, J.H., Orentreich, N. and Sibley, R.K. (1991) Helicobacter pylori infection and the risk of gastric carcinoma. N. Engl. J. Med., 325(16): 1127-1131. [Crossref] [PubMed]

2. Kusters, J.G., van Vliet, A.H.M. and Kuipers, E.J. (2006) Pathogenesis of Helicobacter pylori infection. Clin. Microbiol. Rev., 19(3): 449-490. [Crossref] [PubMed] [PMC]

3. Momtaz, H., Dabiri, H., Souod, N. and Gholami, M. (2014) Study of Helicobacter pylori genotype status in cows, sheep, goats and human beings. BMC Gastroenterol. 14(61): 1-7. [Crossref] [PubMed] [PMC]

4. Safaei, H.G., Rahimi, E., Zandi, A. and Rashidipour, A. (2011) Helicobacter pylori as a zoonotic infection: The detection of H. pylori antigens in the milk and feces of cows. J. Res. Med. Sci., 16(2): 184-187.

5. IARC Working Group.(1994) Helicobacter pylori. IARC Monographs 100B. Pp. 385-435.

6. Tokudome, S., Soeripto, S.W.D., Triningsih, F.X., Suzuki, S., Hosono, A., Triono, T., Sarjadi, I.W., Miranti, I.P., Ghadimi, R. and Moore, M.A. (2005) Helicobacter pylori infection appears essential for stomach carcinogenesis: Observations in Semarang, Indonesia. Cancer Sci., 96(12): 873-875. [Crossref] [PubMed]

7. Wroblewski, L.E., Peek, J.R.M. and Wilson, K.T. (2010) Helicobacter pylori and gastric cancer: Factors that modulate disease risk. Clin. Microbiol. Rev., 23(4): 713-739. [Crossref] [PubMed] [PMC]

8. Azuma, T., Yamazaki, S., Yamakawa, A., Ohtani, M., Muramatsu, A., Suto, H., Ito, Y., Dojo, M., Yamazaki, Y., Kuriyama, M., Keida, Y., Higashi, H. and Hatakeyama, M. (2004) Association between diversity in the Src homology 2 domain containing tyrosine phosphatase binding site of Helicobacter pylori CagA protein and gastric atrophy and cancer. J. Infect. Dis., 189(5): 820-827. [Crossref] [PubMed]

9. Satomi, S., Yamakawa, A., Matsunaga, S. and Masaki, R. (2006) Relationship between the diversity of the cagA gene of Helicobacter pylori and gastric cancer in Okinawa, Japan. J. Gastroenterol., 41(7): 668-673. [Crossref] [PubMed]

10. Batista, S.A., Rocha, G.A., Rocha, A.M., Saraiva, I.E., Cabral, M.M., Oliveira, R.C. and Queiroz, D.M. (2011) Higher number of Helicobacter pylori CagA EPIYA C phosphorylation sites increases the risk of gastric cancer, but not duodenal ulcer. BMC Microbiol., 11(61): 1-7. [Crossref] [PubMed] [PMC]

11. Johnson, E.M., Gaddy, J.A., Voss, B.J., Hennig, E.E. and Cover, T.L. (2014) Genes required for assembly of pili associated with the Helicobacter pylori cag Type IV secretion system. Infect. Immun., 82(8): 3457-3470. [Crossref] [PubMed] [PMC]

12. Salyer, A.A. and Whitt, D.D. (2002) Bacterial Pathogenesis a Molecular Approach. ASM Press, Washington DC. p115-127.

13. Suharsono, H., Hendrayana, M.A., Astawa, I.N.M. and Prawiro, S.R. (2014) Confirmation of adherence protein hemagglutinin sub-unit pili with MW 49.6 kDa Helicobacter pylori on mice gastric epithelial cell. IOSR J. Pharm. Biol. Sci., 9(2): 59-66. [Crossref]

14. Alzahrani, S., Lina, T.T., Gonzalez, J., Pinchuk, I.V., Beswick, E.J. and Reyes, V.E. (2014) Effect of Helicobacter pylori on gastric epithelial cells. World J. Gastroenterol., 20(36): 12767-12780. [Crossref] [PubMed] [PMC]

15. Roesler, B.M., Rabelo-Goncalves, E.M.A. and Zeitune, J.M.R. (2014) Virulence factors of Helicobacter pylori: A review. Clin. Med. Insights Gastroenterol., 2014(7): 9-17. [Crossref] [PubMed] [PMC]

16. Huang, Y., Wang, Q., Cheng, D., Xu, W. and Lu, N. (2016) Adhesion and invasion of gastric mucosa epithelial cells by Helicobacter pylori. Front. Cell. Infect. Microbiol., 6(159): 1-11. [Crossref] [PubMed] [PMC]

17. Weatherall, D., Greenwood, B., Chee, H.L. and Wasi, P. (2006) Science and technology for disease control: Past, present, and future. In: Jamison, D.T., Breman, J.G., Measham, A.R., Alleyne, G., Claeson, M. and Evans, D.B., editors. Disease Control Priorities in Developing Countries. 2nd ed. Oxford University Press, New York.

18. Khalifehgholi, M., Shamsipour, F., Ajhdarkosh, H., Daryani, N.E. and Pourmand, M.R., Hosseini, M., Ghasemi, A. and Shirazi, M.H. (2013) Comparison of five diagnostic methods for Helicobacter pylori. Iran. J. Microbiol., 5(4): 396-401.

19. Garza-Gonzalez, E., Perez-Perez, G.I., Maldonado-Garza, H.I. and Bosques-Padilla, F.J. (2014) A review of Helicobacter pylori diagnosis, treatment, and methods to detect eradication. World J. Gastroenterol., 20(6): 1438-1449. [Crossref] [PubMed] [PMC]

20. Herbrink, P. and van Doorn, L.J. (2000) Serological methods for diagnosis of Helicobacter pylori infection and monitoring of eradication therapy. Eur. J. Clin. Microbiol. Infect. Dis., 19(3): 164-173. [Crossref]

21. Shukla, S., Pujani, M., Agarwal, A., Pujani, M. and Rohtagi, A. (2012) Correlation of serology with morphological changes in gastric biopsy in Helicobacter pylori infection and evaluation of immunohistochemistry for H. pylori identification. Saudi J. Gastroenterol., 18(6): 369-374. [Crossref] [PubMed] [PMC]

22. Daivasikamai, P. (2014) Advantages of serological testing for Helicobacter pylori infection as a screening test. Int. J. Adv. Med., 1(3): 217-221. [Crossref]

23. She, R.C., Wilson, A.R. and Litwin, C.M. (2009) Evaluation of Helicobacter pylori immunoglobulin G (IgG), IgA, and IgM serologic testing compared to stool antigen testing. Clin. Vaccine Immunol., 16(8): 1253-1255. [Crossref] [PubMed] [PMC]

24. Wang, Y., Kuo, F., Liu, C., Wu, M., Shih, H., Wang, S.S., Wu, J.Y., Kuo, C.H., Huang, Y.K. and Wu, D.C. (2015) Diagnosis of Helicobacter pylori infection: Current options and developments. World J. Gastroenterol., 21(40): 11221-11235. [Crossref] [PubMed] [PMC]

25. Miftahussurur, M. and Yamaoka, Y. (2016) Diagnostic methods of Helicobacter pylori infection for epidemiological studies: Critical importance of indirect test validation. Biomed. Res. Int., 2016(4819423): 1-14. [Crossref] [PubMed] [PMC]

26. Sumarno, R.P., Uun, Y., Winarsih, S., Islam, S. and Sanarto, S. (2012) Detection of molecule adhesion sub-unit pili 48 kDa Salmonella Typhi by immunochemistry method using sera patients suffering from typhoid fever. J. Basic Appl. Sci. Res., 2(9): 8527-8532.

27. Marchetti, M., Arico, B., Burroni, D., Figura, N., Rappuoli, R. and Ghiara, P. (1995) Development of a mouse model of Helicobacter pylori infection that mimics human disease. Science, 267(5204): 1655-1658. [Crossref]

28. EMD Millipore. (2013) Rapid Lateral Flow Test Strips. EMD Millipore Corporation, Billerica, MA, Darmstadt, Germany.

29. Li, H., Yang, J., Bao, D., Hou, J., Zhi, Y., Yang, Y., Ji, P., Zhou, E., Qiao, S. and Zhang, G. (2017) Development of an immunochromatographic strip for detection of antibodies against porcine reproductive and respiratory syndrome virus. J. Vet. Sci., 18(3): 307-316. [Crossref] [PubMed] [PMC]

30. Locatelli, A., Catapani, W.R., Junior, C.R.G., Silva, C.B.P. and Waisberg, J. (2004) Detection of anti-Helicobacter pylori antibodies in serum and duodenal fluid in peptic gastroduodenal disease. World J. Gastroenterol., 10(20): 2997-3000. [Crossref] [PubMed] [PMC]

31. Muttaqin, Z., Suharjono, Aulanni'am, A. (2016) Phylogenetic analysis of 3' region of Helicobacter pylori cagA gene of Lombok isolates and the association with gastric pathology. IOSR J. Pharm. Biol. Sci., 11(1): 1-6.

32. Kumagai, T., Yan, J., Graham, D.Y., Tozuka, M., Okimura, Y., Ikeno, T., Sugiyama, A., Katsuyama, T. and Ota, H. (2001) Serum immunoglobulin G immune response to Helicobacter pylori antigens in mongolian gerbils. J. Clin. Microbiol., 39(4): 1283-1288. [Crossref] [PubMed] [PMC]

33. Abadi, A.T.B. (2018) Diagnosis of Helicobacter pylori using invasive and noninvasive approaches. J. Pathog., 2018(9064952): 1-13. [Crossref] [PubMed] [PMC]

34. Gatta, L., Ricci, C., Tampieri, A. and Vaira, D. (2003) Non-invasive techniques for the diagnosis of Helicobacter pylori infection. Clin. Microbiol. Infect., 9(6): 489-496. [Crossref]