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Dengue IgG ELISA Kit

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  • Product Name
  • Dengue IgG ELISA Kit

  • Cat.No.
  • DEIA508
  • Pkg#Size
  • 96T
  • Intended use
  • The CD Dengue virus IgG-ELISA is intended for the qualitative determination of IgG class antibodies against Dengue virus in human serum or plasma (citrate).
  • General Description
  • Dengue virus is a single-stranded RNA virus of about 50 nm in diameter belonging to the genus Flavivirus. Dengue and dengue hemorrhagic fever are caused by one of four closely related, but antigenically distinct, virus serotypes (DEN-1, DEN-2, DEN-3, and DEN-4). Infection with one of these serotypes does not provide crossprotective immunity, so persons living in a dengue-endemic area can have four dengue infections during their lifetimes. The viruses are transmitted by Aedes aegypti, a domestic, day-biting mosquito that prefers to feed on humans. Infection with dengue viruses produces a spectrum of clinical illness ranging from a nonspecific viral syndrome to severe and fatal hemorrhagic disease. It is primarily a disease of the tropics; its global distribution is comparable to that of malaria, and an estimated 2.5 billion people live in areas at risk for epidemic transmission. - Globally, there are an estimated 50 to 100 million cases of dengue fever and several hundred thousand cases of dengue hemorrhagic fever.
    § The case-fatality rate of DHF in most countries is about 5%; most fatal cases are among children and young adults.
    § Important risk factors for DHF include the strain and serotype of the infecting virus, as well as the age, immunestatus, and genetic predisposition of the patient.
    § Risk groups: residents of or visitors to tropical urban areas.
    The presence of virus resp. infection may be identified by Serology: Detection of antibodies by ELISA
    Infection produces lifelong immunity, but the antigenically distinct serotypes do not provide cross-protective immunity, so a person can theoretically experience four dengue infections; a dengue vaccine is not available
  • Principle Of The Test
  • The qualitative immunoenzymatic determination of IgG-class antibodies against Dengue Virus is based on the ELISA (Enzyme-linked Immunosorbent Assay) technique.
    Microtiter strip wells are precoated with Dengue Virus antigens type 2 to bind corresponding antibodies of the specimen. After washing the wells to remove all unbound sample material horseradish peroxidase (HRP) labelled anti-human IgG conjugate is added. This conjugate binds to the captured Dengue Virusspecific antibodies. The immune complex formed by the bound conjugate is visualized by adding Tetramethylbenzidine (TMB) substrate which gives a blue reaction product. The intensity of this product is proportional to the amount of Dengue Virus-specific IgG antibodies in the specimen. Sulphuric acid is added to stop the reaction. This produces a yellow endpoint colour. Absorbance at 450 nm is read using an ELISA microwell plate reader.
  • Reagents And Materials Provided
  • 1. Reagents supplied
    Dengue Virus Coated Wells (IgG): 12 breakapart 8-well snap-off strips coated with Dengue Virus Type 2 antigen; inresealable aluminium foil.
    IgG Sample Diluent ***: 1 bottle containing 100 ml of buffer for sample dilution; pH 7.2 ± 0.2; coloured yellow; ready to use; white cap.
    Stop Solution: 1 bottle containing 15 ml sulphuric acid, 0.2 mol/l; ready to use; red cap.
    Washing Solution (20x conc.)*: 1 bottle containing 50 ml of a 20-fold concentrated buffer (pH 7.2 ± 0.2) for washing the wells; white cap.
    Dengue Virus anti-IgG Conjugate**: 1 bottle containing 20 ml of peroxidase labelled rabbit antibody to human IgG; coloured blue, ready to use; black cap.
    TMB Substrate Solution: 1 bottle containing 15 ml 3,3'''',5,5''''-tetramethylbenzidine (TMB); ready to use; yellow cap.
    Dengue Virus IgG Positive Control***: 1 bottle containing 2 ml; coloured yellow; ready to use; red cap.
    Dengue Virus IgG Cut-off Control***: 1 bottle containing 3 ml; coloured yellow; ready to use; green cap.
    Dengue Virus IgG Negative Control***: 1 bottle containing 2 ml; coloured yellow; ready to use; blue cap.
    * contains 0.1 % Bronidox L after dilution
    ** contains 0.2 % Bronidox L
    *** contains 0.1 % Kathon
    2. Materials supplied
    Strip holder
    Cover foil
    Test protocol
  • Materials Required But Not Supplied
  • ELISA microwell plate reader, equipped for the measurement of absorbance at 450/620 nm
    Incubator 37°C
    Manual or automatic equipment for rinsing wells
    Pipettes to deliver volumes between 10 and 1000 μl
    Vortex tube mixer
    Deionised or (freshly) distilled water
    Disposable tubes
    Timer
  • Storage
  • The reagents are stable up to the expiry date stated on the label when stored at 2-8 °C.
  • Specimen Collection And Preparation
  • Use human serum or plasma (citrate) samples with this assay. If the assay is performed within 5 jours after sample collection, the specimen should be kept at 2...8°C; otherwise they should be aliquoted and stored deep-frozen (-20 to -70°C). If samples are stored frozen, mix thawed samples well before testing. Avoid
    repeated freezing and thawing. Heat inactivation of samples is not recommended.
    1. Sample Dilution
    Before assaying, all samples should be diluted 1+100 with IgG Sample Diluent. Dispense 10μl sample and 1 ml IgG Sample Diluent into tubes to obtain a 1+100 dilution and thoroughly mix with a Vortex.
  • Reagent Preparation
  • It is very important to bring all reagents, samples and controls to room temperature (20…25°C) before starting the test run!
    1. Coated snap-off Strips
    The ready to use breakapart snap-off strips are coated with Dengue Virus Type 2 antigen. Store at 2...8°C. Immediately after removal of strips, the remaining strips should be resealed in the aluminium foil along with the desiccant supplied and stored at 2...8 °C; stability until expiry date.
    2. Dengue Virus anti-IgG Conjugate
    The bottle contains 20 ml of a solution with anti-human-IgG horseradish peroxidase, buffer, stabilizers, preservatives and an inert blue dye. The solution is ready to use. Store at 2...8°C. After first opening stability until expiry date when stored at 2…8°C.
    3. Controls
    The bottles labelled with Positive, Cut-off and Negative Control contain a ready to use control solution. It contains 0.1% Kathon and has to be stored at 2...8°C. After first opening stability until expiry date when stored at 2…8°C.
    4. IgG Sample Diluent
    The bottle contains 100 ml phosphate buffer, stabilizers, preservatives and an inert yellow dye. It is used for the dilution of the patient specimen. This ready to use solution has to be stored at 2...8°C. After first opening stability until expiry date when stored at 2…8°C.
    5. Washing Solution (20xconc.)
    The bottle contains 50 ml of a concentrated buffer, detergents and preservatives. Dilute washing solution 1+19; e.g. 10 ml washing solution + 190 ml fresh and germ free redistilled water. The diluted buffer is stable for 5 days at room temperature. Crystals in the solution disappear by warming up to 37 °C in a water bath. After first opening the concentrate is stable until the expiry date.
    6. TMB Substrate Solution
    The bottle contains 15 ml of a tetramethylbenzidine/hydrogen peroxide system. The reagent is ready to use and has to be stored at 2...8°C, away from the light. The solution should be colourless or could have a slight blue tinge. If the substrate turns into blue, it may have become contaminated and should be thrown away. After first opening stability until expiry date when stored at 2…8°C.
    7. Stop Solution
    The bottle contains 15 ml 0.2 M sulphuric acid solution (R 36/38, S 26). This ready to use solution has to be stored at 2...8°C. After first opening stability until expiry date.
  • Assay Procedure
  • 1. Test Preparation
    Please read the test protocol carefully before performing the assay. Result reliability depends on strict adherence to the test protocol as described. The following test procedure is only validated for manual procedure. If performing the test on ELISA automatic systems we recommend to increase the washing steps from three to five and the volume of washing solution from 300μl to 350μl to avoid washing effects. Prior to commencing the assay, the distribution and identification plan for all specimens and controls should be carefully established on the result sheet supplied in the kit. Select the required number of microtiter strips or wells and insert them into the holder.
    Please allocate at least:
    1 well (e.g. A1) for the substrate blank,
    1 well (e.g. B1) for the negative control,
    2 wells (e.g. C1+D1) for the cut-off control and
    1 well (e.g. E1) for the positive control.
    It is recommended to determine controls and patient samples in duplicate, if necessary. Perform all assay steps in the order given and without any appreciable delays between the steps. A clean, disposable tip should be used for dispensing each control and sample. Adjust the incubator to 37° ± 1°C.
    1. Dispense 100μl controls and diluted samples into their respective wells. Leave well A1 for substrate blank.
    2. Cover wells with the foil supplied in the kit.
    3. Incubate for 1 hour ± 5 min at 37±1°C.
    4. When incubation has been completed, remove the foil, aspirate the content of the wells and wash each well three times with 300μl of Washing Solution. Avoid overflows from the reaction wells. The soak time between each wash cycle should be >5 sec. At the end carefully remove remaining fluid by tapping strips on tissue paper prior to the next step!
    Note: Washing is critical! Insufficient washing results in poor precision and falsely elevated absorbance values.
    5. Dispense 100μl Dengue Virus anti-IgG Conjugate into all wells except for the blank well (e.g. A1). Cover with foil.
    6. Incubate for 30 min 37±1°C.
    7. Repeat step 4.
    8. Dispense 100μl TMB Substrate Solution into all wells
    9. Incubate for exactly 30 min at room temperature in the dark.
    10. Dispense 100μl Stop Solution into all wells in the same order and at the same rate as for the TMB Substrate Solution.
    Any blue colour developed during the incubation turns into yellow.
    Note: Highly positive patient samples can cause dark precipitates of the chromogen! These precipitates have an influence when reading the optical density. Predilution of the sample with physiological sodium chloride solution, for example 1+1, is recommended. Then dilute the sample 1+100 with dilution buffer and multiply the results in U by 2.
    11. Measure the absorbance of the specimen at 450/620 nm within 30 min after addition of the Stop Solution.

    Measurement
    Adjust the ELISA Microwell Plate Reader to zero using the substrate blank in well A1. If - due to technical reasons - the ELISA reader cannot be adjusted to zero using the substrate blank in well A1, subtract the absorbance value of well A1 from all other absorbance values measured in order to obtain reliable results!
    Measure the absorbance of all wells at 450 nm and record the absorbance values for each control and patient sample in the distribution and identification plan.
    Dual wavelength reading using 620 nm as reference wavelength is recommended. Where applicable calculate the mean absorbance values of all duplicates.
  • Interpretation of Results
  • 1. Run Validation Criteria
    In order for an assay to be considered valid, the following criteria must be met:
    § Substrate blank in A1: Absorbance value < 0.100.
    § Negative control in B1: Absorbance value < 0.200 and < cut-off
    § Cut-off control in C1 and D1: Absorbance value 0.150 – 1.30.
    § Positive control in E1: Absorbance value > cut-off.
    If these criteria are not met, the test is not valid and must be repeated.
    2. Calculation of Results
    The cut-off is the mean absorbance value of the Cut-off control determinations.
    Example: Absorbance value Cut-off control 0.49 + absorbance value Cut-off control 0.47 =0.96 / 2 = 0.48
    Cut-off = 0.48
    3. Interpretation of Results
    Samples are considered POSITIVE if the absorbance value is higher than 10% over the cut-off.
    Samples with an absorbance value of 10% above or below the cut-off should not be considered as clearly positive or negative grey zone
    It is recommended to repeat the test again 2 - 4 weeks later with a fresh sample. If results in the second test are again in the grey zone the sample has to be considered NEGATIVE. Samples are considered NEGATIVE if the absorbance value is lower than 10% below the cut-off.
    3.1. Results in Units
    Patient (mean) absorbance value x 10 = Units [U]
    Cut-off
    Example:
    1.786 x 10 = 37 U
    0.48
    Cut-off: 10 U
    Grey zone: 9-11 U
    Negative: <9 U
    Positive: >11 U
  • Sensitivity
  • The diagnostic sensitivity is defined as the probability of the assay of scoring positive in the presence of the specific analyte. It is >90 %.
  • Specificity
  • The diagnostic specificity is defined as the probability of the assay of scoring negative in the absence of the specific analyte. It is 93 %.
  • Precision
  • Precision
  • Interferences
  • Interferences with hemolytic, lipemic or icteric sera are not observed up to a concentration of 10 mg/ml hemoglobin, 5 mg/ml triglycerides and 0.2 mg/ml bilirubin.
  • Precautions
  • 1. In compliance with article 1 paragraph 2b European directive 98/79/EC the use of the in vitro diagnostic medical devices is intended by the manufacturer to secure suitability, performances and safety of the product. Therefore the test procedure, the information, the precautions and warnings in the instructions for use have to be strictly followed. The use of the testkits with analyzers and similar equipment has to be validated. Any change in design, composition and test procedure as well as for any use in combination with other products not approved by the manufacturer is not authorized; the user himself is responsible for such changes. The manufacturer is not liable for false results and incidents for these reasons. The manufacturer is not liable for any results by visual analysis of the patient samples.
    2. Only for in-vitro diagnostic use.
    3. All components of human origin used for the production of these reagents have been tested for anti-HIV antibodies, anti-HCV antibodies and HBsAg and have been found to be non-reactive. Nevertheless, all materials should still be regarded and handled as potentially infectious.
    4. Do not interchange reagents or strips of different production lots.
    5. No reagents of other manufacturers should be used along with reagents of this test kit.
    6. Do not use reagents after expiry date stated on the label.
    7. Use only clean pipette tips, dispensers, and lab ware.
    8. Do not interchange screw caps of reagent vials to avoid cross-contamination.
    9. Close reagent vials tightly immediately after use to avoid evaporation and microbial contamination.
    10. After first opening and subsequent storage check conjugate and control vials for microbial contamination prior to further use.
    11. To avoid cross-contamination and falsely elevated results pipette patient samples and dispense conjugate without splashing accurately to the bottom of wells.
    12. The ELISA is only designed for qualified personnel who are familiar with good laboratory practice.
  • Limitations
  • The Flaviviridae family includes the serotypes of Dengue virus as well as the yellow fever, Japanese encephalitis viruses and Tick borne encephalitis (TBE). There is a cross reactivity among flaviviruses, due to the presence of common antigen determinants. Diagnosis of Dengue infection should be made in conjunction with other clinical signs and symptoms and laboratory findings. Known cross reactions among Dengue antigens must be considered during interpretation since epitopes are known to react with other flaviviruses. It is recommended to test a reactive sample witha second serological method. Bacterial contamination or repeated freeze-thaw cycles of the specimen may affect the absorbance values. Diagnosis of an infectious disease should not be established on the basis of a single test result. A precise diagnosis should take into consideration clinical history, symptomatology as well as serological data. In immunocompromised patients and newborns serological data only have restricted value.
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