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RIDASCREEN® Campylobacter RIDASCREEN® FemtoLab H. pylori Article no: C2301 R-Biopharm AG, An der neuen Bergstraße 17, D-64297 Darmstadt, Germany Phone: +49 (0) 61 51 81 02-0 / Fax: +49 (0) 61 51 81 02-20 ) 1. Intended use For in vitro diagnostic use. The RIDASCREEN® FemtoLab H. pylori test is an enzyme immunoassay for the qualitative determination of Helicobacter pylori antigens in stool samples. 2. Summary and explanation of the test In 1984, Marshall and Warren found a campylobacter-like organism in the mucosa of the antrum and the corpus in patients with gastritis and peptic ulcers of the duodenum by using histology. Today, the causal relationship of Helicobacter pylori at the onset of gastrointestinal illnesses is now recognised. Infections with H. pylori lead to inflammations which have a causal relationship with chronic gastritis, ulcers in the stomach and small intestine, and stomach cancers. This is confirmed by the, in most cases, successful healing of gastritis and ulcers after eradication therapy. H. pylori has developed various protection mechanisms in order to survive in the acidic, bactericidal environment of the stomach. The enzyme, urease, splits urea into ammonia and carbon dioxide and neutralises the gastric acid. The production of catalase and superoxidedismutase protects against attacks from neutrophiles in the gastric mucosa. Many H. pylori positive patients develop gastritis and approx. 10 % of the patients develop ulcers. 90 % of patients with small intestine or stomach ulcers are H. pylori positive, whatever their age. The reasons for this are currently the subject of research all over the world, as are the transmission routes of the organism. There are two basic approaches to the diagnosis of H. pylori infections: the direct approach of determining the organism and the indirect approach of determining the antibodies against H. pylori which have been produced by the patient. Among the direct, but invasive methods of determining an infection are the urease- quick test, histology or cultivation of the organism from biopsy material. Culturing H. pylori from biopsy material is difficult and time consuming. The technical difficulties can lead to false negative results, i.e. low sensitivity. In addition, H. pylori is inclined to colonise the gastric mucosa in islands and can therefore be overlooked during endoscopic examinations. Another direct method of determining H. pylori is the breath test. In this case, the bacteria are determined from the carbon dioxide produced by the bacterial enzyme urease. The breath test is very sensitive and specific but requires special measuring instruments and the patient has to take isotope-labelled urea. One method which is frequently used is the serological determination of H. pylori specific antibodies. This is an indirect method where the antibodies produced by the patient are detected against H. pylori. The sensitivity and specificity vary greatly between the tests from different manufacturers. Furthermore, monitoring the success of an eradication therapy using serological methods is unsatisfactory since the antibody titer only drops slowly over a period of months. The ® RIDASCREEN FemtoLab H. pylori test is an enzyme immunoassay (EIA) in microwell-plate format for the direct, non-invasive determination of H. pylori antigens in human stools. The test is based on monoclonal antibodies, fluctuations between the individual lots being avoided. By determining the antigens directly, it is possible to support both the position of an initial diagnosis and to check the RIDASCREEN® FemtoLab H. pylori 10-08-10 2 success of the eradication therapy 4 to 6 weeks after its completion or detect the recurrence of an infection. 3. Test principle ® The RIDASCREEN FemtoLab H. pylori test uses an enzyme immunoassay with dual amplification technology for determining H. pylori antigens in stool samples. The wells in the microwell plate are coated with monoclonal antibodies against H. pylori antigens. The supernatant of a stool suspension and the peroxidase-labelled monoclonal antibodies (enzyme conjugate) are pipetted into the wells. During the incubation, H. pylori antigens from the stool suspension attach themselves both to the antibodies on the test plate and the peroxidase-labelled antibodies, thus forming a 'sandwich complex'. Enzyme conjugate which remains unattached is removed by washing the test plate. After adding the substrate, the attached enzyme changes the colour of the previously colourless solution in the wells of the microwell plate to blue if the test is positive. On adding the stop reagent, the colour changes from blue to yellow. The extinction is proportional to the concentration of H. pylori antigens present in the sample. 4. Reagents provided There are enough reagents in the pack for 96 determinations. Plate 96 det. Microwell plate coated with antibodies; 12 dividable microwell strips with 8 wells each; coated with monoclonal antibodies against H. pylori antigens; in sealable aluminium bag with desiccant; 1 piece cover film for microwell strips; 100 pieces sampling device (wooden splint) Diluent | 2 100 ml Sample dilution buffer; 75 mM phosphate buffer; pH 7.4; with antimicrobial additives; ready for use; green coloured Wash 100 ml Wash buffer concentrate (x10); 10 x concentrated 250 mM phosphate buffer; pH 7.4; with detergent and antimicrobial additives Control ⎮+ 2 ml Positive control; inactivated, fractionised H. pylori lysate in 75 mM phosphate buffer; pH 7.4; with antimicrobial additives; ready for use; red coloured Control ⎮- 2 ml Negative control; 75 mM phosphate buffer; pH 7.4; with antimicrobial additives; ready for use; blue coloured Conjugate 7 ml Enzyme conjugate; monoclonal antibodies against H. pylori antigens; peroxidase conjugate in 75 mM phosphate buffer; pH 7.4; with antimicrobial additives; ready for use; green coloured RIDASCREEN® FemtoLab H. pylori 10-08-10 3 Substrate 12 ml Urea peroxide/TMB; ready for use Stop 12 ml Stop solution; 1 N sulphuric acid; ready for use 5. Storage instructions All reagents must be stored at 2 – 8 °C and can be used until the date printed on the label. The diluted wash buffer can be used for up to three months, providing it is stored between 2 and 8 °C. Microbial contamination must be prevented. After the expiry date, the quality guarantee is no longer valid. The aluminium bag must be opened with scissors in such a way that the clip seal is not torn off. Any microwell strips which are not required must be placed in the aluminium bag immediately and stored at 2 – 8 °C. The colourless substrate must also be protected from direct light to prevent it from decomposing or turning blue due to auto-oxidation. Once the substrate has turned blue, it must not be used. 6. Additional necessary reagents – and necessary equipment 6.1. Reagents - Distilled or deionised water 6.2. Accessories - Test tubes - Washing unit for mictrotitration pipettes or multichannel pipettes (300 µl) - Photometer for microwell plates (450 nm and reference filter ≥ 600 nm where necessary) - Centrifuge (at least 5000 rpm) - Shaker, suitable for microwell plates - Vortex mixer - Stop clock - Measuring cylinder (1000 ml) - Micropipette for 50 - 100 µl and 1 ml volume - Filter paper (laboratory towels) - Waste container containing 0.5 % hypochlorite solution 7. Precautions for users For in vitro diagnostic use only. RIDASCREEN® FemtoLab H. pylori 10-08-10 4 This test must only be carried out by trained laboratory personnel. The guidelines for working in medical laboratories must be followed and the instructions for carrying out the test must be strictly adhered to. The positive controls in the kit contain inactivated helicobacter antigens. In spite of this, they and the patient samples must be treated as potentially infectious and handled in accordance with the national safety regulations. Samples or reagents must not be pipetted by mouth and contact with injured skin or mucous membranes must be prevented. When handling the samples, wear disposable gloves and when the test is finished, wash your hands. Do not smoke, eat or drink in areas where samples or test reagents are being used. Sample dilution buffer, wash buffer, positive and negative control and conjugate contain antimicrobial additives. This substance must not be allowed to come into contact with the skin or mucous membrane. Hydrogen peroxide can cause burns. Handle with care. The stop reagent contains 1 N sulphuric acid. Avoid contact with the skin and clothing. If the skin is contaminated with the stop reagent, rinse it off with water. All reagents and materials which have come into contact with potentially infectious samples must be treated with suitable disinfectants or autoclaved at 121 °C for at least 1 hour. CAUTION: To prevent the formation of poisonous gases, any liquid waste containing stop reagent must be neutralised before adding to hypochlorite solution. 8. Specimen collection and storage The test material must be stored at 2 – 8 °C until it is used in the test. If the material is not going to be used in the test within 3 days, we recommend storing it at –20 °C or colder. Multiple freezing and thawing of the sample must be avoided. Stool samples or rectal swabs should not be collected in transport containers which contain transport media with preservatives, animal sera, metal ions, oxidising agents or detergents since these may interfere with the RIDASCREEN® FemtoLab H. pylori test. If rectal swabs have to be used, make sure that sufficient stool material (approx. 200 mg) is collected to carry out the test. 9. Test procedure 9.1. General information RIDASCREEN® FemtoLab H. pylori 10-08-10 5 All reagents and the microwell plate Plate must be brought to room temperature (20 – 25 °C) before use. The microwell strips must not be removed from the aluminium bag until they have reached room temperature. The reagents must be thoroughly mixed immediately before use. After use, the microwell strips (in sealed bags) and the reagents must be stored at 2 – 8 °C.
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