INSTRUCTION FOR USE EIA-ANTI-HDV
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1 GWB-HDVIGG 96 INSTRUCTION FOR USE EIA-ANTI-HDV ENZYME IMMUNOASSAY FOR THE DETECTION OF TOTAL ANTIBODIES TO HEPATITIS DELTA VIRUS Page 1 of 10
2 Table of contents I. Intended use. 3 II. Contents of the EIA-ANTI-HDV III. Precautions.. 4 IV. Health and safety instructions... 4 V. Materials and equipment required but not provided with the kit 5 VI. Collection and handling of specimens... 5 VII. Preparation of the reagents.. 6 VIII. Test procedure.. 6 IX. Results X. Limits of the test 8 XI. Conditions of storage and transportation... 8 XII. Explanation of symbols Аnnex. 10 Page 2 of 10 Revision
3 This Package Insert provides information for Professional Use of the kit manufactured in 1 format. Format contains sufficient reagents for 96 (one breakable plate) assays including controls; the kit is intended for manual testing with a possibility of fractional (one strip) use of the kit or for simultaneous 96 assays using an open type automated analyzer for enzyme immunoassay. I. INTENDED USE EIA-ANTI-HDV is enzyme immunoassay kit intended for the detection of antibodies to Hepatitis Delta Virus in human blood serum (plasma). II. CONTENTS OF THE EIA-ANTI-HDV Table 1 LABEL NATURE OF THE REAGENTS PRESENTATION Polystyrene stripped plate with colorless transparent HDV Ag Coated wells coated with recombinant antigens of Hepatitis Strips Delta Virus. Store at 2-8 ºC until expiration date. 1 plate Conjugate 11-fold) Positive Control Inactivated Negative Control Inactivated Washing Solution 25-fold) Substrate Buffer TMB 11-fold) Stopping Reagent Human blood serum globulin fraction containing antibodies to Delta antigen (anti-hdv) labeled with horse radish peroxidase. Transparent or slightly opalescent liquid blue colored. Preserving agent: 0.01 % thimerosal, % gentamycin sulfate. Store at 2-8º C until expiration date in a tightly sealed vial. Human blood serum positive for HBsAg and antibodies to HDV, negative to anti-hiv-1,2 and anti- HCV; inactivated. Transparent or slightly opalescent liquid, crimson colored. Preserving agent: 0.05 % thimerosal, 0.01 % gentamycin sulfate, 0.02 % ProClin 300. Store at 2-8º C until expiration date in a tightly sealed vial. Human blood serum negative for HBsAg, antibodies to HDV, HIV-1,2 and HCV, inactivated. Transparent or slightly opalescent liquid, green colored. Preserving agent: 0.05 % thimerosal, 0.01 % gentamycin sulfate. Store at 2-8º C until expiration date in a tightly sealed vial. Transparent or slightly opalescent liquid, colorless, or pale yellow, sediment may form that dissolves at ºC and shaking. Store at 2-8 ºC until expiration date in a tightly sealed vial. Citric acid and sodium acetate solution, ph , containing H 2 O 2. Transparent colorless liquid. Preserving agent: 0.05 % ProClin 300. Store at 2-8 ºC until expiration date in a tightly sealed vial. Solution containing Tetramethylbenzidine (TMB). Transparent colorless or slightly yellow color liquid. Store at 2-8 ºC until expiration date in a tightly sealed vial M/L sulphuric acid solution. Transparent colorless liquid. Store at 2-8 ºC until expiration date in a tightly sealed vial ml 0.75 ml 1.5 ml 50.0 ml 15.0 ml 1.5 ml 25.0 ml Page 3 of 10 Revision
4 Bag with polystyrene breakable plate, vials with reagents, instruction for use are packed in a cardboard box or a plastic bag. Additionally the following may be included in the delivery set: - a lid for polystyrene 96-well plates or a protective film for EIA plates; - disposable tips; - a plastic dish for liquid reagents; - a plastic clip or plastic bag with zip lock. III. PRECAUTIONS The reliability of the results depends on correct implementation of the following Good Laboratory Practices: The temperature in the lab should be ºC. Do not use expired reagents. Do not mix reagents from different lots within a given test run. Before use, it is necessary to wait 30 minutes for the reagents to stabilize to room temperature (18-24 ºC). Carefully reconstitute the reagents avoiding any contamination. Do not carry out the test in the presence of reactive vapours (acid, alkaline, aldehyde vapours) or dust that could alter the enzyme activity of the conjugates. Use glassware thoroughly washed and rinsed with deionized water or preferably, disposable material. Wash the plastic dishes for liquid reagents with purified water prior the use. The glassware for Substrate mixture (dishes, vials and etc.) in case of repeated use should be washed immediately after the work with 70 % ethanol solution and then with purified water, or preferable, use disposable material. Do not allow the microplate to dry between the end of the washing operation and the reagent distribution. The enzyme reaction is very sensitive to metal ions. Consequently, do not allow any metal element to come into contact with the various conjugate or substrate solutions. Use a new distribution tip for each sample. Well washing is a critical step in this procedure: respect the recommended number of washing cycles and make sure that all wells are completely filled and then completely emptied. Incorrect washing may lead to inaccurate results. Never use the same container to distribute conjugate and development solution. Check the pipettes and other equipment for accuracy and correct operation. Do not change the assay procedure. Use high quality water. Avoid exposure of the reagents to excessive heat or sunlight during storage and incubation. IV. HEALTH AND SAFETY INSTRUCTIONS All reagents included in the kit are intended for in vitro diagnostic use. Human origin material used in the preparation of the Negative Control, has been tested and found non reactive for antibodies to hepatitis C and antibodies to human immunodeficiency virus (HIV-1 and HIV-2). Human origin material used in the preparation of the Positive Control has been tested and found non reactive for antibodies to hepatitis C and antibodies to human immunodeficiency virus (HIV-1 and HIV-2). Because no known test method can offer complete assurance that infections agents are absent, handle reagents and patients samples as if capable of transmitting infections disease. Page 4 of 10 Revision
5 Do not eat, drink, smoke, or apply cosmetics where immunodiagnostic materials are being handled. Do not pipette by mouth. Any equipment directly in contact with specimens and reagents as well as washing solutions should be considered as contaminated products and treated as such. Wear lab coats and disposable gloves when handling reagents and samples and thoroughly wash your hands after handling them. Avoid spilling samples or solutions containing samples. Avoid any contact of the Substrate Buffer, the ТМВ and the Stopping Reagent with the skin and mucosa. Provide adequate ventilation. Do not forget to neutralize and/or autoclave the washing wastes or any fluids containing biological samples before discarding them into the container. Solid wastes (used plates, tips, vials, glassware, etc.) should be disinfected by 6% peroxide of hydrogen with 0.5 % synthetic washing-up liquid or another resolved to application disinfectant. Total time of deactivation should be no less than one hour. Another approved disinfectant is possible to use. Also solid wastes should be disinfected by autoclaving for 1 hour at temperature ºС and pressure 1.5 khz/sm 2 (0.15 mpa). Liquid wastes (washing water) should be disinfected by resolved to application disinfectant. Also liquid wastes can be disinfected by boiling treatment for 30 min or by autoclaving for 1 hour at temperature ºС and pressure 1.5 khz/sm 2 (0.15 mpa). Tools and equipment should be wiped 2 times by 70 % ethanol before and after work. Some reagents contain ProClin 300 (0.05 %). Irritant. May cause sensitization by skin contact. After contact with skin, wash immediately with plenty of soap and water. V. MATERIALS AND EQUIPMENT REQUIRED BUT NOT PROVIDED WITH THE KIT: Microplate reader equipped with 450 nm or with and nm filters. Pipettes, single-channel and multi-channel. Disposable pipette tips. Microplate incubator. Automatic microplate washer. Purified water. Medical gloves. VI. COLLECTION AND HANDLING OF SPECIMENS Collection of blood samples should be implemented according to the current practices. Serum, plasma may be used as tested samples. For the exception of false results avoid thermo inactivation of tested samples, they should be collected and stored in the conditions preventing bacterial growing. Every sample should be collected with a new tip! The specimens can be stored at 2-8 ºC if screening is performed within 3 days or they may be deepfrozen at -20 ºC (Samples may be frozen and defrosted not more than 1 time). Separate the serum or plasma from the clot or red cells as soon as possible to avoid any haemolysis. Haemolysis may affect test performance. Samples with expressed bacterial growing, haemolysis, hyperlipidemia must not be analyzed. Specimens with observable particulate matter should be clarified by centrifugation prior to testing. Page 5 of 10 Revision
6 VII. PREPARATION OF THE REAGENTS 1. Ready for use reagents: Negative Control Positive Control Stopping Reagent 2. Reagents to prepare: HDV Ag Coated Strips. Each plate containing 12 strips is wrapped in a sealed foil-lined bag. Open the bag and remove the plate. Select the number of coated strips required for the assay. Unused strips should be placed back in the bag. After the bag has been opened, Immunosorbent is stable for 6 months at 2-8 ºC, provided that the foil-lined bag is resealed with the clip or the foil-lined bag is resealed in zip locked plastic bag. The silica gel bag should not be removed from the foil packaging. Working Washing Solution. Thoroughly mix the content of the vial with concentrated Washing Solution (x25). Dilute the required volume of concentrated Washing Solution with corresponding volume of purified water prior to use (See Table 2). Mix the solution thoroughly. The prepared Working Washing Solution is stable at least for 14 days at ºC or for 28 days at 2-8 ºC when used in GLP condition. Working Solution of Conjugate. Prepare before use. Dilute the contents of the vial with thoroughly mixed concentrate of Conjugate with the corresponding volume of Working Washing Solution (See Table 2). Mix thoroughly until diluted avoiding foaming. Do not apply intensive mixing. The Working Solution of Conjugate can be stored for not more than 12 hours at ºC in dark place. Substrate Mixture. The Substrate Mixture should be prepared before use. Dilute the required volume of TMB 11-fold) with the corresponding volume of Substrate Buffer (1:10 ratio) (See Table 2). Mix thoroughly until diluted. Prepared mixture can be stored for not more than 10 hours in a dark place at ºC in chemically neutral vial or in reagent container used in open automatic EIA analyzer. Substrate Mixture should be colorless! 3. Storage of unused reagents: After opening the vials with unused components of the kit: Negative Control, Positive Control, Washing Solution 25-fold), Substrate Buffer, Stopping Reagent, TMB 11-fold) can be stored in tightly sealed vials until the kit expiration date at 2-8 ºC. VIII. TEST PROCEDURE Note: Before use, allow reagents to reach room temperature (18 24 ºC) for 30 min. The required volumes of reagents depending on the number of used strips are represented in the table 2. Page 6 of 10 Revision
7 Number of used strips Table 2 The table of consumable components of the kit 1. Open the bag and remove the plate. Select the number of coated strips required for the assay. Unused strips should be placed back in the bag. After the bag has been opened, the strips are stable for 6 months at 2-8 ºC, provided that the foil-line bag is resealed with the clip or the foil-line bag is resealed in zip locked plastic bag. The silica gel bag should not be removed from the foil packaging. 2. Do not wash the coated strips before assay. 3. Use following algorithm to distribute Control Samples depending on a number of used strips: 1 strip 1 well of Positive Control and 2 wells of Negative Control; 2 strips 2 wells of Positive Control and 2 wells of Negative Control; 3 strips or more 2 wells of Positive Control and 3 wells of Negative Control. Add 50 µl of Positive Control into well(s) and Negative Control into other well(s) according to the algorithm. 4. Add 50 µl of tested specimens into the rest of the wells. 5. Add 50 µl of Working Solution of Conjugate into each well. 6. Mix the contents of the wells by careful tapping on the edge of the plate. Cover the plate by plate lid and incubate for 60 minutes at (37.0 ± 1.0) ºC. 7. Aspirate the content of the wells and wash the plate 4 times with the Working Washing Solution. Add into each well µl of Working Washing Solution. Allow a soak time at least 40 seconds and aspirate. Use of an automatic microplate washer is strongly recommended. Incomplete washing will adversely affect assay precision. 8. Add 100 µl of Substrate Mixture into each well. Keep covered by plate lid plates in the dark place during 20 minutes at ºC. 9. Add 50 µl of Stopping Reagent to each well and read the optical density at 450/ nm using a plate reader. Reading the absorbance at 450 nm only is possible. Scheme of the assay is represented in Annex. IX. RESULTS Working washing solution Washing Solution 25-fold) Purified water Working Solution of Conjugate Working Washing Solution Conjugate 11-fold) Substrate Mixture ТМB 11-fold) Substrate Buffer For the assay to be valid, average ОD values in wells with Negative Control must be not less than 0.8 and in wells with Positive Controls not greater than 0.2. The sample is considered as positive if the OD value is equal or less than the Cut-Off value. Page 7 of 10 Revision
8 Calculate Cut-Off value as: Cut-Off = 0.5 x average OD Negative Control 0.5 is a coefficient defined by manufacturer during statistical processing for each lot. X. LIMITS OF THE TEST The EIA-ANTI-HDV procedure and the interpretation of results must be followed when testing serum, plasma, for the presence of antibodies to Hepatitis Delta Virus. The user of this kit a advised to read the instruction for use carefully prior to conducting the test. In particular, the test procedure must be carefully followed for sample and reagent pipetting, plate washing, and timing of the incubation steps. All highly sensitive immunoassays have a potential for non-specific reactions which can lead to false positive results. The proportion of reactives that are false will depend on the sensitivity and specificity of the kit. Negative results can occur if the quantity of marker present in the sample is too low for the detection limit of the assay, or if the marker which is detected is not present during the stage of disease in which a sample is collected. XI. CONDITIONS OF STORAGE AND TRANSPORTATION Shelf life is 15 months. Keep in dark dry place at 2-8 C. Transportation may be done by all kinds of covered transport at temperature 9-20 C not more than during ten (10) days. Freezing is prohibited. GenWay Biotech, Inc Nancy Ridge Drive, San Diego, CA 92121, USA Tel:(858) , Fax: (858) sales@genwaybio.com, XII. EXPLANATION OF SYMBOLS Manufacturer Catalog number Sufficient for ] Lot code Storage temperature limitation Page 8 of 10 Revision
9 Expiry date ССYY-MM-DD Consult Instruction for use Contains irritant agent Page 9 of 10 Revision
10 Scheme of the assay 1 Add 50 µl of Positive Control, Negative Control Annex 2 Add 50 µl of tested samples 3 Add 50 µl of Working Solution of Conjugate 4 Incubate 60 min, (37.0 ± 1.0) ºС, microplate incubator 5 Wash the plate Working washing solution, not less than 380 µl, 4 times 6 Add 100 µl of Substrate Mixture 7 Incubate 20 min, ºС in the dark place 8 Add 50 µl of Stopping Reagent 9 Read the optical density 450 nm/ nm or 450 nm Page 10 of 10 Revision (8)
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