Dengue Virus IgG ELISA Kit

Sample

Plasma, Serum

Species Reactivity

Human

Detection Method

iELISA

Intended Use

The ELISA test kit provides semiquantitative or quantitative in vitro determination of human antibodies of the immunoglobulin class IgG against dengue virus types 1-4 in serum or plasma to support the diagnosis of dengue virus infection.

Contents of Kit

1. Microplate wells coated with antigens
12 microplate strips each containing 8 individual break-off wells in a frame, ready for use.
--- 12 x 8 STRIPS.
2. Calibrator 1
200 RU/ml (IgG, human), ready for use, dark red, 1 x 2.0 ml CAL 1.
3. Calibrator 2
20 RU/ml (IgG, human), ready for use, red, 1 x 2.0 ml CAL 2.
4. Calibrator 3
2 RU/ml (IgG, human), ready for use, light red, 1 x 2.0 ml CAL 3.
5. Positive control
(IgG, human), ready for use, blue, 1 x 2.0 ml POS CONTROL.
6. Negative control
(IgG, human), ready for use, green, 1 x 2.0 ml NEG CONTROL.
7. Enzyme conjugate
peroxidase-labelled anti-human IgG (rabbit), ready for use
green, 1 x 12 ml, CONJUGATE.
8. Sample buffer
ready for use, light blue, 1 x 100 ml SAMPLE BUFFER.
9. Wash buffer
10x concentrate, colourless, 1 x 100 ml WASH BUFFER 10x.
10. Chromogen/substrate solution
TMB/H2O2, ready for use, colourless, 1 x 12 ml SUBSTRATE.
11. Stop solution
0.5 M sulphuric acid, ready for use colourless 1 x 12 ml STOP SOLUTION.
12. Protective foil --- 2 pieces FOIL.
13. Test instruction --- 1 booklet
14. Quality control certificate --- 1 protocol

Storage

The test kit has to be stored at a temperature between +2°C and +8°C. Do not freeze. Unopened, all test kit components are stable until the indicated expiry date.

Performance Characteristics

Calibration: As no international reference serum exists for antibodies against dengue viruses, the calibration is performed in relative units (RU).


For every group of tests performed, the extinction readings of the calibrators and the relative units and/or ratios determined for the positive and negative controls must lie within the limits stated for the relevant test kit lot. A quality control certificate containing these reference values is included. If the values specified for the controls are not achieved, the test results may be inaccurate and the test should be repeated.


The binding activity of the antibodies and the activity of the enzyme used are temperature-dependent. It is therefore recommended using a thermostat in all three incubation steps. The higher the room temperature (+18°C to +25°C) during the incubation steps, the greater will be the extinction. Corresponding variations apply also to the incubation times. However, the calibrators are subject to the same influences, with the result that such variations will be largely compensated in the calculation of the result.

Antigen: The microplate wells were coated with highly purified virus particles and recombinant glycoproteins E of types 1 to 4.


Linearity: The linearity of the Anti-Dengue Virus Type 1-4 ELISA (IgG) was determined by assaying at least 4 serial dilutions of different patient samples.The Anti-Dengue Virus Type 1-4 ELISA (IgG) is linear at least in the tested concentration range (2 RU/ml to 139 RU/ml).

Detection limit: The lower detection limit is defined as the mean value of an analyte-free sample plus three times the standard deviation and is the smallest detectable antibody titer. The lower detection limit of the Anti-Dengue Virus Type 1-4 ELISA (IgG) is 0.29 RU/ml.

Cross-reactivity: The quality of the antigen used in the ELISA guarantees a high specificity of the test. However, due to the significant homologies of the target antigens within the species of flaviviruses, crossreactions cannot be entirely excluded. For investigation of cross-reactivity, sera from patients after TBE vaccination or yellow fever vaccination, respectively, and patients with HCV-, West Nile virus, and Zika virus infections, respectively, were analysed. The most important cross-reactivities occur with anti-Zika virus IgG antibodies. However, it must be taken into account that double infections are possible, especially in endemic regions, or that an infection with another flavivirus may have taken place at a previous moment. In this case, positive results are not caused by a cross-reactivity of the corresponding antibodies.


Antibodies against n Anti-Dengue Virus Type 1-4 ELISA (IgG) positive
TBE virus 25 32.0%
Yellow fever virus 12 0.0%
Hepatitis C virus 6 0.0%
West Nile virus 40 50.0%
Zika virus 12 100.0%
Influenza virus 40 5.0% (2 positive)
Rheumatoid factor 35 0.0%
Diverse autoantibodies 34 5.9% (2 positive)
EBV 22 0.0% Treponema 50 8.0% (4 positive)

General Description

Due to the use of antigens from all dengue virus serotypes (1, 2, 3 and 4), the Anti-Dengue Virus Type 1-4 ELISA (IgG) is suitable for the serological detection of acute or past dengue virus infections and is an ideal supplement to direct pathogen detection, e.g. with the Dengue Virus NS1 ELISA. Seroconversion or an increase in the IgG antibody titer of at least 4-fold indicates an acute infection.

Reconstitution And Storage

All reagents must be brought to room temperature (+18°C to +25°C) approx. 30 minutes before use. The reagent can be kept unopened until the specified expiration date if stored at +2°C to +8°C. After opening for the first time, they must still be stored at +2°C to 8°C and protected against contamination.
The following list indicates the storage life of reagents after first opening. These limits apply unless a different period is specified on the reagent label:
Coated wells 4 month
Calibrators 12 month
Controls 12 month
Enzyme conjugate 12 month
Sample buffer 12 month
Diluted wash buffer 4 weeks
Chromogen/substrate solution 12 month
Stop solution 12 month
The thermostat adjusted ELISA incubator must be set at +37°C ± 1°C.


1. Coated wells: Ready for use. Tear open the resealable protective wrapping of the microplate at the recesses above the grip seam. Do not open until the microplate has reached room temperature to prevent the individual strips from moistening. Immediately replace the remaining wells of a partly used microplate in the protective wrapping and tightly seal with the integrated grip seam (Do not remove the desiccant bag).
2. Calibrators and controls: Ready for use. The reagents must be mixed thoroughly before use.
3. Enzyme conjugate: Ready for use. The enzyme conjugate must be mixed thoroughly before use.
4. Sample buffer: Ready for use.
5. Wash buffer: The wash buffer is a 10x concentrate. If crystallisation occurs in the concentrated buffer, warm it to +37°C and mix well before diluting. The quantity required should be removed from the bottle using a clean pipette and diluted with deionised or distilled water (1 part reagent plus 9 parts distilled water).
For example: For 1 microplate strip, 5 ml concentrate plus 45 ml water.
6. Chromogen/substrate solution: Ready for use. Close the bottle immediately after use, as the contents are sensitive to light. The chromogen/substrate solution must be clear on use. Do not use the solution if it is blue coloured.
7. Stop solution: Ready for use.
Waste disposal: Patient samples, calibrators, controls and incubated microplate strips should be handled as infectious waste. All reagents must be disposed of in accordance with local disposal regulations.
Warning: The calibrators and controls of human origin have tested negative for HBsAg, anti-HCV, anti-HIV-1 and anti-HIV-2. Nonetheless, all materials should be treated as being a potential infection hazard and should be handled with care. Some of the reagents contain sodium azide in a non-declarable concentration. Avoid skin contact.