|Year : 2005 | Volume
| Issue : 2 | Page : 117-119
Efficacy of Sonicated and Acid-Extractable Antigens in the Serodiagnosis of H. pylori infection in Peptic Ulcer Patients
N Parimala, M Ishaq
Department of Genetics, Osmania University, Hyderabad, India
Department of Genetics, Osmania University, Hyderabad - 500 007, AP
Source of Support: None, Conflict of Interest: None
Helicobacter pylori is implicated in causation of peptic ulcers and gastric cancer and plays a pivotal role in gastric pathophysiology. In the present study we evaluated the relative efficacy of sonicated and acid-extractable antigens in the serodiagnosis of H. pylori infection in peptic ulcer patients by ELISA. In the present study we evaluated the relative efficacy of sonicated and acid-extractable antigens in the serodiagnosis of H. pylori infection in peptic ulcer patients by ELISA. The two types of antigens mentioned above were prepared from H. pylori subcultures following appropriate procedures. Sera were collected from 13 subjects of whom eight were diagnosed to be suffering from duodenal ulcer (DU) and five from non-ulcer dyspepsia (NUD) and screened for the presence of anti H. pylori antibodies by ELISA. A case was considered seropositive, if the OD value was more than or equivalent to twice the mean OD value of blank. Analysis of our results showed that, with acid extractable antigen at a concentration of 2 mg/mL, 12 cases were seropositive. Contrastingly, with sonicated antigen, at a concentration of 2 mg/mL only eight cases were positive. It is concluded from this study, that the use of relatively purified antigens like acid extractable antigens enhances the sensitivity and specificity of this serodiagnostic test, indicative of its relatively higher efficacy over sonicated lysate containing multiple antigens.
Keywords: Helicobacter pylori, sonicated antigen, acid extractable antigen, ELISA
|How to cite this article:|
Parimala N, Ishaq M. Efficacy of Sonicated and Acid-Extractable Antigens in the Serodiagnosis of H. pylori infection in Peptic Ulcer Patients. Indian J Med Microbiol 2005;23:117-9
|How to cite this URL:|
Parimala N, Ishaq M. Efficacy of Sonicated and Acid-Extractable Antigens in the Serodiagnosis of H. pylori infection in Peptic Ulcer Patients. Indian J Med Microbiol [serial online] 2005 [cited 2019 Jun 15];23:117-9. Available from: http://www.ijmm.org/text.asp?2005/23/2/117/16052
The discovery of Helicobacter pylori has brought a fundamental change in understanding the aetiopathogenesis of peptic ulcer disease. It is known to play a vital role in gastric pathophysiology. H. pylori causes chronic active gastritis and is implicated as one of the prime pathogenic factors in peptic ulcer disease and is surmised to have a role in pathogenesis of gastric carcinoma.
Discovery of H. pylori as gastric pathogen has provided much impetus for researchers to understand its complicity in causation of disease. H. pylori infection causes chronic active gastritis in 70-80% of infected subjects and in substantial proportion of these subjects, H. pylori induced inflammatory response contributes immensely to development of atrophic gastritis eventually leading over a period of time to gastric cancer and has also been classified as group I carcinogen.
The most crucial observation that ulcers stay healed for longer periods after eradication of H. pylori and that its eradication alleviates the recurrence of ulcers convincingly indicates the essential role of H. pylori in the development of gastroduodenal diseases., The importance of H. pylori as an aetiological agent in gastroduodenal disease necessitates its treatment as the main target for elimination of infection. However, it is observed that, despite the availability of various antibiotic regimens, ulcer recurs and response to treatment is precarious, in that it does not give desired results as expected in a considerable proportion of cases.,
Several invasive and non-invasive tests for diagnosing H. pylori infection have been developed. With increasing availability of different invasive and non-invasive tests there is a continuous improvement in diagnosis of H.pylori infection. Invasive biopsy based tests include rapid urease test, histology, culture and molecular diagnostic techniques.
Non invasive tests compromise on serology and urea breath test and are more pertinent, in that they appear to circumvent the problems surfacing in invasive tests, thereby obviating the need for endoscopy. Serology is now a highly sensitive and specific test (85%-95%) for current or past H. pylori infection. Serological methods like ELISA have proven especially valuable in screening large number of individuals in epidemiological studies., The serological test ELISA is used as a global method of diagnosis as infected subjects develop elevated levels of IgG antibodies to H. pylori. Infection of gastric mucosa with H. pylori results in systemic as well as local immune responses, including elevation of specific IgG and IgA levels in serum and elevated levels of secretory IgA and IgM in stomach. Screening of anti H. pylori antibodies is recommended as pre-endoscopy or as a pre-treatment test. Thus, serological tests are more compliant in that they show positive result in a patient with gastric atrophy in whom the number of H. pylori organisms is so small as to be undetectable by biopsy / breath test based methods. Detection of antibodies is regarded as more sensitive as it also reveals the intracellular survival of H. pylori after treatment.
In the present study, we examined the diagnostic value of detecting systemic (IgG) antibodies, against H. pylori using ELISA technique employing sonicated and acid extractable antigens from H. pylori suspension and also assessed the relative efficacy of these antigens in the serodiagnosis of H. pylori infection.
| ~ Subjects and Methods|| |
H. pylori cultures obtained by inoculating biopsy extracts from different patients were subcultured on blood-agar medium (containing 5% sheep blood) and incubated at 37oC for 5 days under strict microaerophilic conditions. Characteristic translucent tiny colonies were subjected to different morphological and biochemical tests for establishing identification of H. pylori .
Preparation of acid extractable antigen
Acid extractable antigens were prepared from H. pylori subcultures by the method described by Goodwin et al .
H. pylori cells from the subcultures were harvested in sterile distilled water. Cells were washed twice, centrifuged and the pelleted cells were suspended in 0.2M glycine hydrochloride buffer (pH 2.2) at a concentration of 0.1 gm w/w of cells to 1.5 mL of buffer. The suspension was stirred at 25oC for 15 minutes and then centrifuged at 11000 g for 15 minutes at 4oC. The supernatant was carefully removed and its pH was made neutral with sodium hydroxide. It was then dialysed against sterile distilled water for 24 hours. Subsequently, this supernatant was concentrated and stored at -20oC until further use as an antigen for ELISA.
Preparation of sonicated antigen
H. pylori cells were harvested in sterile distilled water, washed twice in sterile distilled water and subjected to sonication for 6 minutes.
Collection of blood
Subjects visiting gastroenterology unit of Gandhi hospital were selected for present study. The test group comprised of 15 cases of which eight were diagnosed to be suffering from duodenal ulcer (DU) and five from non-ulcer dyspepsia (NUD) based on upper gastrointestinal endoscopy. Blood (5 mL) was collected from these patients. Sera obtained were stored at - 70oC, which were later used for serodiagnosis using ELISA.
Antigens extracted from H. pylori using sonicated and acid extractable methods were diluted to a final concentration of 2 mg/mL and 5 mg/mL in the coating buffer (0.1 M carbonate buffer, pH 9.6) respectively. Microtitre tray wells were coated with 100 ml of each of these antigens and the plates were incubated overnight at 4oC. Antigen solution was then flicked from the plates and the plates washed thrice with phosphate buffer saline tween (PBST). The non-specific binding sites were then saturated by addition of 150 mL of 1% bovine serum albumin (BSA) in PBST and the plates incubated for one hour at 37oC followed by washing with PBST thrice.
Patient's sera were subjected to 1:100 dilution in PBST - BSA and 100 mL of these patients sera were added to the wells separately. The plates were incubated for 2 hours at room temperature and washed with PBST thrice. 100 mL of antihuman IgG-HRP conjugate diluted 1:500 with PBST-BSA was dispensed in each well and incubated for 2 hours at 37oC and washed with PBST thrice. 100 mL of substrate solution (58 mg of tri-methyl benzedine was dissolved in 10 mL of dimethyl sulfoxide containing 0.015% of hydrogen peroxide) was added to each well and incubated at 37oC for 30 minutes for development of colour. The reaction was stopped by adding 50 mL of stopping solution (IN H2SO4) and OD values recorded at 405nm in an ELISA reader.
| ~ Results|| |
The principal focus in this study was on assessment of relative efficacy of sonicated and acid - extractable Helicobacter pylori antigens in the serodiagnosis of H. pylori infection in peptic ulcer patients by ELISA.
The test group comprised of 13 cases of which 8 were diagnosed to be suffering from DU and the remaining were NUD cases. Anti H. pylori systemic (IgG) antibodies were screened in sera from 13 H. pylori infected subjects. OD values representing antibody titres in each case are given in [Table - 1]. A case was considered seropositive for H. pylori if its OD value was more than twice the mean OD value of blank (lacking patient's sera). The corresponding mean OD values of blank were 0.192, 0.252 for sonicated antigen at a concentration of 2 mg/mL and 5 mg/mL respectively and 0.083, 0.103 for acid extractable antigen at a concentration of 2 mg/mL and 5 mg/mL respectively.
| ~ Discussion|| |
ELISA is characterized by its sensitivity and is widely used as a global method of diagnosis as infected subjects develop elevated levels of IgG antibodies to H. pylori. In the present study, sonicated and acid extractable antigens were employed after adjusting the protein concentration to 3 mg/mL. Two different concentrations of antigen viz, 2 mg/mL and 5 mg/mL were used to determine optimum concentration to be employed for regular screening.
Considering acid-extractable antigen at a concentration of 2 mg/mL, a perusal of individual OD values of H. pylori infected subjects reveals that, excepting one case (case no. 3), individual OD values of the remaining 12 cases are more than twice the mean OD value of blank. Contrastingly, with sonicated antigen, at a concentration of 2 mg/mL, there is a marked deviation, in that only eight cases display OD values greater than twice the mean OD value of blank. Thus it is concluded that acid - extractable antigen is relatively more effective and at a concentration of 2 mg/mL, can be routinely employed in serodiagnosis of H. pylori infection.
Conclusively, we recommend the use of relatively purified antigens like acid- extractable antigen at a concentration of 2 mg/mL for serodiagnosis of H. pylori infection by ELISA owing to its relatively higher efficacy over sonicated lysate containing multiple antigens.
| ~ References|| |
|1.||Kuipers EJ, Perez- Perez GI, Blaser MJ. Helicobacter pylori and atrophic gastritis: Importance of CagA status. J Natl Cancer Inst 1995;87 :1777-80. |
|2.||NIH Consensus Development panel. Helicobacter pylori in peptic ulcer disease. JAMA 1994;272 :65-9. |
|3.||Borody TJ, Cole P, Noonan S, et al . Recurrence of duodenal ulcer and Campylobacter pylori infection after eradication. Med J Aust 1989;151 :431-5. |
|4.||Rauws EA, Tytgat GN. Cure of duodenal ulcer associated with eradication of Helicobacter pylori. Lancet 1990;335 :1233-5. [PUBMED] |
|5.||Lam SK. Current strategies in ulcer management with special reference to the use of antibiotics. Yale J Biol Med 1997;70 :27-31. |
|6.||Wang WH, Wong BC, Lam SK, et al . High prevalence of H. pylori infection with dual resistance to Metronidazole and Clarithromycin. Aliment Pharmacol Ther 2000;14 :901-10. |
|7.||Perez-Perez GI, Dworkin BM, Chodos JE, et al . Campylobacter pylori antibodies in humans. Ann Intern Med 1988;109 :11-7. |
|8.||Taylor NS, Fox JG, Akopyants NS, et al . Long term colonization with single and multiple strains of Helicobacter pylori assessed by DNA finger printing. J Clin Microbiol 1995;33 :918-23. |
|9.||Dunn BE, Cohen H, Blaser MJ. Helicobacter pylori. Clin Microbiol Rev 1997;10 :720-41. [PUBMED] [FULLTEXT]|
|10.||Karnes WE Jr, Samloff IM, Siurala M. Positive serum antibody and negative tissue staining for Helicobacter pylori in subjects with atrophic body gastritis. Gastroenterology 1991;101 :167-74. |
|11.||Glupezynski Y. Microbiology and Serological diagnostic tests for H. pylori: An overview. Br Med Bull 1998;54 :175-86. |
|12.||Goodwin CS, Blincow E, Peterson G. Enzyme Linked Immunosorbent Assay for Campylobacter pyloridis: Correlation with presence of C.pylori in gastric mucosa. J Infect Dis 1987;155 :488-94. |
[Table - 1]