Introduction:Helicobacter pylori is a spiral bacterium cultured from human gastric mucosa discovered by B.J. Marshall in 1982. Studies have indicated that the presence of H. pylori is associated with a variety of gastrointestinal diseases including gastritis, duodenal and gastric ulcers, non-ulcer dyspepsia, and gastric adenocarcinoma and lymphoma. The organism is present in 95-98% of patients with duodenal ulcers and 60-90% of patients with gastric ulcers. The studies have also demonstrated that removal of the organism by anti-microbial therapy is correlated with the resolution of symptoms and cure of diseases
Patients who present clinical symptoms relating to the gastrointestinal tract can be diagnosed for H. pylori infection by two methods:(1) Invasive techniques – include biopsy followed by culture or histologic examination of biopsy specimen or direct detection of urease activity
(2) Non-invasive techniques – include urea breath tests and serological methods. All of the testing performed on biopsy samples is subject to errors related to sampling and interference of contaminated bacteria. An ELISA test of the presence of H. pylori specific IgG antibody is the technique of choice for serologic tests because of its accuracy and simplicity.
Intended Use:The Helicobacter pylori IgG Test Kit is intended for the quantitative determination of IgG antibodies to Helicobacter pylori in human serum. This kit is for research use only and cannot be used for diagnostic procedures.
Principle of the Assay:Purified H. pylori antigen is coated on the surface of microwells. Diluted patients serum is added to the wells, and the H. pylori IgG-specific antibody, if present, binds to the antigen. All unbound materials are washed away. Enzyme conjugate is added, which binds to the antibody-antigen complex. Excess enzyme conjugate is washed off and a solution of TMB Reagent is added. The enzyme conjugate catalytic reaction is stopped at a specific time. The intensity of the color generated is proportional to the amount of IgG-specific antibody in the sample. The results are read by a microwell reader and compared in a parallel manner with calibrator and controls.
Kit Components:H1840-24H1: Purified H. pylori antigen-coated microtiter plate, 1x96 wellsH1840-24H2: Enzyme Conjugate Reagent (red color), 1x13 mlH1840-24H3: Sample Diluent (green color), 1x22 mlH1840-24H4: H. pylori IgG Negative Control (< 6.25U/ml), 1x150ulH1840-24H5: H. pylori IgG Positive Control (>100 U/ml), 1x150ulH1840-24H6: H. pylori IgG Standard 1 (0U/ml), 1x150ulH1840-24H7: H. pylori IgG Standard 2 (6.25U/ml), 1x150ulH1840-24H8: H. pylori IgG Standard 3(12.5U/ml), 1x150ulH1840-24H9: H. pylori IgG Standard 4 (25U/ml), 1x150ulH1840-24H10: H. pylori IgG Standard 5 (50U/ml), 1x150ulH1840-24H11: H. pylori IgG Standard 6 (100U/ml), 1x150ulH1840-24H12: Wash Buffer (20X), 1x50 mlH1840-24H13: TMB Reagent (One-Step), 1x11 mlH1840-24H14: Stop Solution (1N HCl), 1x11 ml
Storage and Stability:Unopened test kits should be stored at 4°C upon receipt and the microtiter plate should be kept in a sealed bag with desiccants to minimize exposure to damp air. Stable for 6 months after receipt. DO NOT FREEZE.
Procedure:Note: Before proceeding with the assay, bring all reagents, serum references and controls to room temperature (18-25°C). The wash procedure is critical. Insufficient washing will result in improper color development.
1. Secure the desired number of coated wells in the holder.
2. Prepare 1:40 dilution for test samples, all six H. pylori IgG standards, negative control, and positive control by adding 5ul of the sample to 200ul of sample diluent. Mix well.
3. Dispense 100ul of diluted sera, six standards, and controls into the appropriate wells. For the reagent blank, dispense 100ul sample diluent in the assigned well position. Tap the holder to remove air bubbles from the liquid and mix well for 10 seconds.
4. Incubate at room temperature for 30 minutes.
5. At the end of the incubation period, remove liquid from all wells. Rinse and flick the microtiter wells 4 times with diluted wash buffer (1X) and then one time with distilled water. (Please do not use tap water.)
6. Dispense 100ul of enzyme conjugate to each well. Mix gently for 10 seconds.
7. Incubate at room temperature for 30 minutes.
8. Remove enzyme conjugate from all wells. Rinse and flick the microtiter wells 4 times with diluted wash buffer (1x) and then one time with distilled water.
9. Add 100ul of TMB Reagent to each well. Mix gently for 10 seconds.
10. Incubate at room temperature for 20 minutes.
11. Add 100ul of Stop Solution to each well including the 2 blanks.
12. Mix gently for 30 seconds. It is important to make sure that all the blue color changes to yellow color completely.
13. Read the optical density at 450nm within 15 minutes with a microtiter plate reader.
