User Manual /96 tests

Transcription

1 Aspergillus Galactomannan Test Kit(ELISA) User Manual /96 tests Catalogue No.: YNQ CONTENTS 1. INTENDED USE PRINCIPLE SUMMARY AND EXPLANATION KIT COMPONENTS STORAGE AND STABILITY MATERIALS NEEDED BUT NOT SUPPLIED SAMPLE COLLECTION AND STORAGE FLOW CHART OF TESTING PROCEDURE SAMPLE TREATMENT PROCEDURE QUALITY CONTROL INTERPRETATION OF RESULTS PRECAUTIONS WARNINGS REFERENCE MANUFACTURER... 7

2 1. INTENDED USE Bio-Enoche Aspergillus Galactomannan Test Kit is based on Enzyme-linked Immunosorbent Assay (ELISA). It is for the quantitative detection of Aspergillus glactomannan antigen in human serum, offering a diagnostic reference for Aspergillus infection. The kit is intended for professional use. 2. PRINCIPLE Mix the treated serum with anti-galactomannan antibody and incubate. Pipette the mixture into wells coated with galactomannan antigen and then incubate. After removing the unbound material by washing, pipette the conjugate to wells and incubate. Again, after removing the unbound material by washing, the substrate solution is pipetted and incubated. Then the stopping solution is added to terminate the color development. The result is measured at 450 nm using an ELISA microplate reader. The intensity of color development is negatively proportional to the concentration of galactomanan antigen tested. 3. SUMMARY AND EXPLANATION With the wide application of broad-spectrum antibiotics, corticosteroid, immunosuppressant, anti-tumor drugs, as well as the prevalence of AIDS and the development of organ transplantation, the Invasive Fungal Infection, with a high mortality, is increased and complicated. The invasive Aspergillosis (IA) is rapidly increasing. The susceptible population is mainly people who receive immunosuppressive therapy, such as hematopoietic stem cell Transplantation patients, hematic malignant carcinoma patients, solid organ transplantation patients, bone marrow transplantation patients as well as long-term chemotherapy and corticosteroid therapy patients and severe AIDS patients. The clinical symptom of IA is non-specific. There are no identical features in CT scan and X-ray. The difficulty of early diagnosis and timely treatment results in a high mortality of 60%~100% [1]. The presence of galactomannan antigen against Aspergillus indicates a prior Aspergillus infection. 4. KIT COMPONENTS No. Component Content Quantity R1 Microtiter Strips 12 break apart strips with 8 wells each; coated with Aspergillus galactomannan antigen 1plate/12 8wells R2a R2b R2c R2d Standard a (0.25 μg/l) Standard b (0.5μg/L) Standard c (1 μg/l) Standard d (2.5 μg/l) 1

3 R2e R3 R4 R5 R6 R7 Standard e (5 μg/l) Anti-Galactomannan Antibody Conjugate Sample Treatment Solution Concentrated Washing Solution (20 ) Sample Dilution Anti-galactomannan antibodies, stabilized with protein stabilization solution Goat-anti-rabbit antibodies, conjugated with HRP; stabilized with protein stabilization solution EDTA Solution PBS with protein and Tween 20 PBS with protein and Tween mL 1 12mL 1 10mL 1 12mL 1 5mL R8 Substrate Solution Tetramethylbenzidine (TMB) 1 12mL R9 Stopping Solution 2M H 2 SO 4 1 8mL M1 Plate Sealer Adhesive membrane of microtiter plate 1 sheet 5. STORAGE AND STABILITY Item Storage Stability Microtiter Strips coated with Aspergillus galactomannan antigen after opening, stored in the sealed bag with desiccant at 2~8 2 4 weeks Standards (a, b, c, d and e) after opening, stored at 2~8 4 weeks Anti-Galactomannan after opening, stored at 2~8 Antibody Conjugate after opening, stored at 2~8 Sample Treatment Solution after opening, stored at 2~8 after opening, concentrated solution (20 ) stored at Concentrated Washing 2~8 Solution after dilution, washing solution stored at weeks Sample Dilution after opening, stored at 2~8 Substrate Solution after opening, stored at 2~8 in dark Stopping Solution after opening, stored at 2~8 or room temperature 6. MATERIALS NEEDED BUT NOT SUPPLIED 6.1. DD H 2 O: for the dilution of concentrated washing solution

4 6.2. Absorbent paper 6.3. Disposable gloves 6.4. Pipette tips (200μL, 300μL, 1000μL) 6.5. Polypropylene centrifugal tubes (0.6mL or 1.5mL, sealed and gas-tight) 6.6. Vortex mixer 6.7. Incubator 6.8. Semi-automatic plate washer (Recommended) 6.9. Microplate Reader 7. SAMPLE COLLECTION AND STORAGE Make sure the sample is not contaminated by fungal spores and bacteria. The sample must be placed inside sealed tubes to avoid exposure to air in the process of transfer and storage. Serum samples can be stored at 2-8 for up to 5 days. 8. FLOW CHART OF TESTING PROCEDURE Samples pretreatment: 300μL of serum+ 100μL of sample treatment 100 for 3min 10,000 g for 10min at 4 Collect 60μL of supernatants Addition of sample/standards: 60μL supernatants or standards +60μL anti-galactomannan antibody 37 for 30min Transfer: Pipette 100μL of above mixture into microtiter 37 for 30min Wash: Wash 3 300μL (1:19) Addition of conjugate: Conjugate: 100μL/well except substrate blank 37 for 30min Wash 3 300μL (1:19) 3

5 Addition of substrate solution: TMB solution: 100μL/well 37 for 15min Stop the reaction: Stopping solution: 50μL/well Measurement: Read OD at 450nm 9. SAMPLE TREATMENT 9.1. Add 300μL of serum into the centrifugal tube Add 100μL of sample treatment solution (R5) into the above centrifugal tube Vor tex the centrifugal tube for 10sec. Heat the tube at 100 for 3min in water bath Centrifuge the heated tube at 4 for 10min at 10,000 g Collect 60μL of supernatant for detection. 10. PROCEDURE Bring all reagents to room temperature for 30min before test Take the microtiter strips out of the sealed bag (R1). Return the unused strips and seal the bag tightly Prepare washing solution : Dilute the concentrated washing solution (20 ) at 1:19 ratio with DD H 2 O. The resultant washing solution is stored at 2~8 for up to 2 weeks. Adequate washing solution should be prepared for the entire test Prepare the polypropylene centrifugal tubes (0.6mL or 1.5mL) in advance Add 60μL of standards (a, b, c, d and e) and samples into each tube separately, and then add 60μL of anti-galactomannan antibody Vortex all tubes well. incubate all the tubes at 37 for 30 min Pipette 100μL of above mixture into wells as below. Add 100μL of sample dilution into one well as the substrate blank. Well A Substrate Blank Well B Standard a Well C Standard b Well D Standard c Well E Standard d Well F Standard e Well G Sample Seal the microtiter plate with a plate sealer and incubate it at 37 for 30min Remove the plate sealer and shake out the incubation solution. Wash the wells 3 times with 300μL/well washing solution each time. The soak time is 40sec. After the last wash, invert the microtiter plate and dry it by tapping on the absorbent paper. Add 100μL of conjugate into each well except the substrate blank Seal the microtiter plate with a plate sealer and incubate it at 37 for 30min. 4

6 Repeat step Add 100μL of substrate solution into each well including the substrate blank Incubate the microtiter plate at 37 for 15min without sealing Add 50μL of stopping solution into each well in the same order and at the same speed of the substrate solution addition. Shake the microtiter plate gently to mix Read OD at 450nm within 5min after addition of the stopping solution. 11. QUALITY CONTROL Substrate Blank: the OD must be <0.1; R2a: The OD must be within 0.7~1.3; R2e: The OD must be within 0.3~0.7; If these criteria are unmet, the test needs to be re-performed. 12. INTERPRETATION OF RESULTS Data analysis: The standard curve is displayed between concentration of galactomannan as X-axis (lg scale) and optical density as Y-axis (linear scale). The standard curve is estimated by a logarithmic regression. Determine the concentration of galactomannan in serum against the standard curve. Interpretation of the results: 1. Concentration of galactomannan < 0.75μg/L indicates a negative result. 2. Concentration of galactomannan > 0.95μg/L indicates a positive result μg/L Concentration of galactomannan 0.95μg/L indicates a grey-zone result. It is recommended to resampling within a week. Note: 1. When the concentration of galactomannan is beyond the range of the standard curve: OD sample > Standard R2a, it indicates a negative result. OD sample < Standard R2e, it indicates a positive result. The sample is recommended being diluted and retested. 2. If the square of correlation coefficient of the regression equation is lower than 0.98, it indicates the standard curve is unacceptable and a new test is needed. 13. PRECAUTIONS Prevent samples and reagents from contamination of fungi and bacteria Use a separate micropipette or individual disposable tips to avoid carry-over and cross-contaminations Use reagents with the same lot Chemical reagents (acid or alkaline) or dust may affect the activity of conjugate While washing, all the wells are filled with the same volume of washing solution. After the last wash, invert the microtiter plate to dry it by tapping against the absorbent paper to ensure no washing solution left and no foam existing Keep the substrate solution away from strong light and avoid contacting with oxidant. The substrate solution is invalid when turning from colorless to light blue. 5

7 14. WARNINGS Don t pipette by mouth Don t smoke, eat or drink in areas where samples or kit reagents are handled Wear disposable gloves, laboratory coat and safety glasses when handling the kit reagents and patients samples. Wash hands thoroughly after testing All the used samples or consumptive materials must be treated as infectious medical wastes The stopping solution is caustic and easy to induce an ambustion. Please wear safety glasses, disposable gloves and laboratory coat during the test. 15. REFERENCE [1] Up on invasive aspergillosis: clinical and diagnostic aspects. P. Munoz, J. Guinea and E. Bouza, Clin Microbiol Infect 2006,12 (7):

8 16. MANUFACTURER Company Name: Tianjin Bio-Enoche Engineering Co., Ltd. Registration Address: N1403, TJAB, No.220 Dongting Road, TEDA, Tianjin , P. R. China Tel: Fax: Manufacture Address: K , Hi-tech Green Industrial Base, No.6 Haitaifazhan 6 th Road, Huayuan Industrial Zone, Tianjin , P. R. China [ ] Company Name: Wellkang Ltd. Address: Suite B, 29 Harley Street, LONDON W1G 9QR, England, United Kingdom Tel: +44 (20) , Fax: +44(20) AuthRep@CE-marking.eu Website: [SYMBOLS USED] Symbol Description Use By Batch Code Manufacturer Keep Away from Sunlight Temperature Limitation In Vitro Diagnostic Medical Device Authorized Representative in the European Community CE Mark 7