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Indian Journal of Medical Microbiology, Vol. 23, No. 2, April-June, 2005, pp. 117-119 Original Article Efficacy of Sonicated and Acid-Extractable Antigens in the Serodiagnosis of H. pylori infection in Peptic Ulcer Patients Parimala N., Ishaq M. Department of Genetics, Osmania University, Hyderabad Correspondence Address:Department of Genetics, Osmania University, Hyderabad - 500 007, AP, parimala_narne@yahoo.co.in Code Number: mb05031 Abstract 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 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. 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.[7],[8] 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.[9] 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.[10] Detection of antibodies is regarded as more sensitive as it also reveals the intracellular survival of H. pylori after treatment.[11] 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 cells were harvested in sterile distilled water, washed twice
in sterile distilled water and subjected to sonication for 6 minutes. Patient′s sera were subjected to 1:100 dilution in PBST - BSA and 100 μL 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 μL of antihuman IgG-HRP conjugate diluted 1:500 with PBST-BSA was dispensed in each well and incubated for 2 hours at 37°C and washed with PBST thrice. 100 μL 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 37°C for 30 minutes for development of colour. The reaction was stopped by adding 50 μL 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.
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