Instruction Manual of. Reagent for determination of anti-helicobacter pylori antibody Thoroughly read this instruction manual before use of this kit

Transcription

1 Instruction Manual of IMMUNIS anti-pylori EIA Reagent for determination of anti-helicobacter pylori antibody Thoroughly read this instruction manual before use of this kit [Background of development] Helicobacter pylori (H. pylori) has shown its implication with chronic gastritis and peptic ulcer and its association with gastric cancer is also suspected. For the diagnosis of the infection with H. pylori, endoscopy biopsies of gastric tissue are often subjected to the microscopic examination, cultural technique or rapid urease test. However, besides they are all invasive, sometimes they fail to detect H. pylori due to patchy distribution of the microorganism in the stomach. There are other diagnostic techniques that do not require the endoscopy such as 13 C-urea breath test and antibody determination method but the former is costly and time consuming in spite of its high sensitivity and noninvasiveness. On the other hand, the antibody determination method is easy to collect samples and can determine a large number of samples in a short time. Presence of the antibody against H. pylori in serum closely correlates to infection with the microorganism in the stomach and its titer decreases with reduction or eradication of the microorganism by antibiotic therapy 1). This kit is a reagent for the detection of the antibody against H. pylori based on the enzyme immuno assay (EIA) and is considered to be useful for screening patients to identify H. pylori infection, as a supplementary test to the diagnosis by the endoscopy, or for the determination of the effect of the eradication therapy of the microorganism. [Kit configuration] 1. Microplate coated with H. pylori antigen (96 wells)... 1 ea. (H. pylori antigen) 2. Sample diluent ml 1 vial 3. Positive control ml 1 vial 4. Positive control ml 1 vial 5. Positive control ml 1 vial 6. Positive control ml 1 vial 7. Enzyme labeled monoclonal antibody ml 1 vial (Peroxidase labeled anti-human IgG mouse monoclonal antibody) 8. Enzyme substrate ml 1 vial (H 2 O 2 and Tetramethylbenzidine) 9. Reaction stopper ml 1 vial 10. Washing solution (20 times concentrated) ml 2 vials 11. Plate seal... 5 ea. [Effect] The detection of the anti-h. pylori IgG class antibody in plasma and serum

2 [Principle of determination] The detection system of this kit is based on the enzyme immuno assay (EIA) and is made up of 2 steps of the antigen-antibody reaction and the enzyme coloring reaction. The first antigen-antibody reaction takes place between the H. pylori antigen coated on the microplate and the anti-h. pylori antibody and the second reaction between the IgG class antibody bound to the antigen coated on the microplate and the antibody labeled with enzyme (peroxidase labeled antibody). When the anti-h. pylori antibody is present in samples, the first and second reactions take place and absorbance by color proportional to the amount of the IgG class anti-h. pylori antibody in samples developed by the enzyme reaction is measured after a given reaction time. H. pylori antigen H. pylori antibody Enzyme labeled antibody Solid Plate (1st Reaction) (2nd Reaction) [Operation] 1. Materials required but not supplied with this kit. Absorbance Measurement (1) Micropipettes, 10 µl, 100 µl and 200 µl. (2) A measuring cylinder, 1 L. (3) A microplate mixer (4) An aspirator and a polypropylene washing bottle or a microplate washer. (5) A dark box (a light tight cupboard or a drawer will do). (6) A dual-wavelength microplate reader (main wavelength 450 nm, sub wavelength 630 nm). (7) Semilogarithmic scale plotting paper or a computer for data processing. 2. Preparation of reagent Washing solution Dilute Washing solution 20 times with purified water. Store it at 2 ~ 8 o C after preparation and use. This diluted washing solution can be used for one month. 3. Operation. Make sure that room temperature is in the range of 15 ~ 25 o C and return the kit to room temperature before use. In each assay, determine absorbance of the Sample diluent and the Positive controls using wells as illustrated. 1 A D B D C P1 D P1 E P2 F P2 G P3 H P3 2 P4 P4 S1 S2 S3 S4 S5 S6 3 P7 P8 P9 D P1 P2 S1 S2-2 - Enzyme Reaction : Sample diluent P3 P4 : Positive controls S3 S4... : Samples

3 IMMUNIS anti-pylori EIA (1) Addition of samples. Add 200 µl of Sample diluent in the wells for the sample diluent. Add 200 µl each of Positive controls in the wells for the positive controls. Add 200 µl of Sample diluent in the wells for samples, then add 10 µl of samples (serum or plasma) and thoroughly mix using pipettes. After adding samples, thoroughly mix the contents on a microplate mixer for 30 sec. Samples which are expected to overshoot the calibration curve should be properly diluted with Sample diluent before hand. Note To minimize difference in the reaction time, add Sample diluent, Positive controls and samples within 10 min. Each of the 96 wells can be detached from their holder. Use only required number of wells and keep at 2 ~ 8 o C remaining wells in an air tight aluminum pouch with desiccant. (2) 1st reaction. Cover the microplate wells with a plate seal provided and leave them to stand at 15 ~ 25 o C for 30 min. (3) Washing. Remove the plate seal and suck out the well contents using an aspirator. Fill each well with the washing solution prepared in 2. Preparation of reagent using a washing bottle and shake out the solution by holding the microplate wells up side down. Repeat this washing 3 times. When the microplate is washed using a microplate washer, wash 5 times. Note While washing the microplate wells, care should be taken not to dry inside the wells. After washing, follow the next step below. (4) Addition of Enzyme labeled monoclonal antibody. Add 100 µl of Enzyme labeled monoclonal antibody in all wells. (5) 2nd reaction. Cover the microplate wells with a plate seal provided and leave it to stand at 15 ~ 25 o C for 10 min. (6) Washing. Wash the microplate wells as in (3) above. (7) Addition of Enzyme substrate. Add 100 µl of Enzyme substrate in all wells. (8) Enzyme reaction. Cover the microplate wells with a plate seal provided and leave it to stand in a dark box (light tight cupboard or drawer will do) at 15 ~ 25 o C for 10 min. (9) Addition of Reaction stopper. Remove the plate seal and add 100 µl of Reaction stopper in all wells and thoroughly mix. (10) Absorbance measurement. Measure absorbance of each well (main wavelength 450 nm, sub wavelength 630 nm). Note Measure absorbance within 30 min after stopping the reaction

4 [Sampling, Storage and Interference Substances] 1. Collection of samples and their storage. (1) When collecting blood, avoid hemolysis and separate plasma or serum immediately. (2) When plasma is determined, add EDTA, heparin, citric acid or oxalic acid to prevent coagulation. (3) It is recommended to determine samples on the day collected. Keep frozen if they are stored and avoid frequent freeze-thaw cycles. 2. Interference substances. Up to 2000 degree of chylomicron (formazine turbidity), 20 mg/dl of bilirubin C, 20 mg/dl of bilirubin F, 500 mg/dl of hemolytic hemoglobin, and/or 500 IU/mL of rheumatoid factor do not interfere with the determination. [Interpretation of results] 1. Calculation of the antibody titer. (1) Confirm that absorbance of the sample diluent in both wells is less than (2) Calculate the mean absorbance of the sample diluent in the 2 wells. If absorbance of either well is or higher, use absorbance of the other as the mean rather than calculating the mean absorbance of the 2 wells. If absorbance of both wells is or higher, the assay is void. Repeat assay again. (3) Calculate the Net OD of the positive controls ([the mean absorbance of each the positive control - the mean absorbance of the sample diluent]). (4) Calculate the Net OD of each sample ([absorbance of each sample - the mean absorbance of the sample diluent]). (5) Determine antibody titer from the Net OD. 1) When semilogarithmic plotting paper is used. Plot on the X axis (logarithmic scale) the antibody titer of each the positive control, and the Net OD on the Y axis (real number scale) and draw a calibration curve using an adjustable ruler. Antibody titers of the positive control are as follows. Positive control 1 10 U/mL Positive control 2 35 U/mL Positive control U/mL Positive control U/mL Apply the Net OD of each sample to this calibration curve and read its antibody titer. 2.0 Calibration Curve 1.5 Net OD Antibody Titer (U/mL)

5 [Note] This graph is shown only as an example. Prepare in each assay a calibration curve using the positive controls for determination of the antibody titer. 2) When a computer is used. As in 1), prepare a calibration curve from the antibody titer of each positive control and the Net OD and calculate the antibody titer of samples from their Net OD. When the Net OD of a sample exceeds that of the positive control 4, dilute it properly with Sample diluent and repeat the assay. 2. Interpretation of the results. The following criteria of the antibody titers to the interprepation of the results of the assay. Antibody titer of samples Less than 10 U/mL Interpretation Negative 10 U/mL or higher Positive Summary of the test procedure and arrangement of wells. 1 Wells 1A, 1B Sample Diluent Positive Controls 1C ~ 1H 2A ~ 2B Samples* 2C ~ 12H 2 Addition of Sample diluent µl 3 Addition of Sample diluent, Positive controls and samples* 200 µl 200 µl 4 First reaction 30 min. at 15 ~ 25 o C (left to stand) 10 µl (thoroughly mix) 5 Washing 3 times by hand or 5 times by washer 6 Addition of Enzyme labeled monoclonal antibody. 100 µl 7 Second reaction 10 min. at 15 ~ 25 o C (left to stand) 8 Washing 3 times by hand or 5 times by washer 9 Addition of Enzyme substrate 100 µl 10 Enzyme reaction 10 min. at 15 ~ 25 o C (left to stand in the dark) 11 Addition of Reaction stopper 100 µl 12 Absorbance measurement Main wavelength 450 nm, sub wavelength 630 nm 13 Interpretation of results *Samples or samples diluted with Sample diluent

6 [Performance] 1. Sensitivity test. When the sample diluent and the positive control 1 are assayed 4 times, the following equation applies. (The means value - Standard deviation x 2) of the positive control 1 > (The means value + Standard deviation x 2) of the sample diluent. 2. Specificity test. When the panel sera negative for the Helicobacter pylori IgG antibody are determined, this kit can test them negative for the Helicobacter pylori IgG antibody. When the panel sera positive for the Helicobacter pylori IgG antibody are determined, this kit can test them positive for the Helicobacter pylori IgG antibody. 3. Simultaneous reproducibility. When the Helicobacter pylori positive panel sera are tested 10 times simultaneously, the CV value of absorbance is 15% or less. [Correlation] When the results by this kit were compared with those by a kit of the same determination principle manufactured by company A, their correlation was 92.5%. This kit Kit by A Company Negative Inconclusive Positive Total Negative Positive Total This discrepancy of the results is considered to derive from different sources of antigens. Now that the 15 samples showing different results were tested positive for anti-h. pylori by the rapid urease test, the hosts of those samples are considered to carry H. pylori in their stomach. [Operational and Handling Warning and Precautions] Use this kit strictly according to the instructions given in this manual. There is no assurance given to any effect other than that stated in this manual or to the results obtained not according to the instructions given in this manual. 1. General precautions. (1) Make sure to return the kit to 15 ~ 25 o C before use. (2) Do not combine and use reagents of different manufacturing lots. Microplate wells can not be reused. (3) Strictly observe the operational instructions. (4) Avoid contamination of the reagents with microorganisms. (5) All materials to be used for the assay must be thoroughly washed and rinsed with purified water. (6) Micropipette tips must be replaced for each sample and reagent. (7) Do not use expired reagents

7 2. Operational precautions. (1) Absorbance of Sample diluent and Positive controls must be measured for each assay. (2) When samples are added, thoroughly stir by pipetting to assure a uniform concentration. (3) Once assay is started, all operation must be finished promptly within specified time. Care should be taken to allow the same reaction time for all samples (maximum 10 min for addition of samples). (4) Enzyme substrate contains volatile organic solvent and should be sealed tightly and stored in the dark when not in use. (5) The first and second antigen-antibody and the enzyme reaction should take place in the temperature range from 15 ~ 25 o C. (6) Absorbance should be measured within 30 min after stopping the enzyme reaction. (7) During assay, do not touch or scrape the bottom of the wells, and do not dry inside the wells. 3. Handling precautions. (1) Although Positive controls in this kit are tested negative for HBsAg, HCV antibody and HIV antibody, handle them carefully as if they were capable of transmitting these diseases. (2) Avoid contact of Enzyme substrate or Reaction stopper with skin or mucous membrane. If they may come in contact with skin, wash it off with a sufficient volume of water. (They are toxic and irritable and burn skin or mucous membrane.) Note that Enzyme substrate contains volatile organic solvent and should be handled with due care. (3) Samples should be handled as if they were capable of transmitting HBV, HCV and HIV and the samples, reagents and the materials used for the assay should be treated with either of the followings. 1) Immerse in 0.05% formalin solution at 37 o C for over 72 hrs. 2) Immerse in 2% glutaraldehyde solution for over 1 hr. 3) Immerse in sodium hypochlorite solution (available chlorine density 1000ppm or more) for over 1 hr. 4) Autoclave at 121 o C for 20 min. (4) Sample diluent and Positive controls contain sodium azide. Flush drains with a sufficient volume of water to prevent forming of explosive metal azide after disposing of them. 4. Others (1) Do not use the containers and other materials in this kit for purposes other than specified. (2) The clinical diagnosis should not be made by the results of this test alone but should be made in combination with supportive clinical information and other tests. [Storage and shelf life] This kit should be stored at 2 ~ 8 o C in the dark avoiding freezing. The shelf life of this kit is 1 year after the date of manufacture (use up this kit before the expiry date shown on the package)

8 [Package] 1 kit for 96 tests. Code No. 1HC1 [Reference] (1) Alexander M. H., Manfred L. R.: Serological tests for monitoring Helicobacter pylori eradication treatment. J. Gastroenterol. 31 [Suppl IX] : 33-36, , Koraku, Bunkyo-Ku, Tokyo , JAPAN Tel: Fax: IMC19601X07E