ELISA/assay

Human Anti-Adenovirus IgG ELISA Kit

DEIA309

96T

Human Anti-Adenovirus IgG ELISA Kit

ADV

Human Anti-Adenovirus IgG ELISA Kit

iELISA

ADV

Human

Quantitative

Serum, plasma

96T

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

ADV

Human

The ELISA test kit provides a semiquantitative or quantitative in vitro assay for human antibodies of the IgG class against adenovirus in serum or plasma.

The test kit contains microtiter strips each with 8 break-off reagent wells coated with adenovirus antigens. In the first reaction step, diluted patient samples are incubated in the wells. In the case of positive samples, specific IgG antibodies (also IgA and IgM) will bind to the antigens. To detect the bound antibodies, a second incubation is carried out using an enzyme-labelled anti-human IgG (enzyme conjugate) catalysing a colour reaction.

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/H 2 O 2 , 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. Test instruction --- 1 booklet 13. Protocol with target values --- 1 protocol

Sample material: Human serum or EDTA, heparin or citrate plasma. Stability: Patient samples to be investigated can generally be stored at +2°C to +8°C for up to 14 days. Diluted samples should be incubated within one working day. Sample dilution: Patient samples are diluted 1:101 in sample buffer. For example: dilute 10 μl serum in 1.0 ml sample buffer and mix well by vortexing (sample pipettes are not suitable for mixing). Note: Calibrators and controls are prediluted and ready for use, do not dilute them.

The pipetting protocol for microtiter strips 1-4 is an example for the semiquantitative analysis of 24 patient samples (P 1 to P 24). The pipetting protocol for microtiter strips 7-10 is an example for the quantitative analysis of 24 patient samples (P 1 to P 24). The calibrators (C 1 to C 3), the positive (pos.) and negative (neg.) controls, and the patient samples have each been incubated in one well. The reliability of the ELISA test can be improved by duplicate determinations for each sample. The wells can be broken off individually from the strips. This makes it possible to adjust the number of test substrates used to the number of samples to be examined and minimises reagent wastage. Both positive and negative controls serve as internal controls for the reliability of the test procedure. They should be assayed with each test run.

Note: All reagents must be brought to room temperature (+18°C to +25°C) approx. 30 minutes before use. After first use, the reagents are stable until the indicated expiry date if stored at +2°C to +8°C and protected from contamination, unless stated otherwise below. - 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). Once the protective wrapping has been opened for the first time, the wells coated with antigens can be stored in a dry place and at a temperature between +2°C and +8°C for a minimum of 4 months. - Calibrators and controls: Ready for use. The reagents must be mixed thoroughly before use. - Enzyme conjugate: Ready for use. The enzyme conjugate must be mixed thoroughly before use. - Sample buffer: Ready for use. - 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 microplatestrip: 5 ml concentrate plus 45 ml water. The ready-to-use diluted wash buffer is stable for 1 month when stored at +2°C to +8°C and handled properly. - 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. - Stop solution: Ready for use. Warning: The control sera used have been tested negative for HBsAg, anti-HCV, anti-HIV-1 and anti-HIV-2 using enzyme immunoassays and indirect immunofluorescence methods. Nonetheless, all materials should be treated as being a potential infection hazard and should be handled with care. Some of the reagents contain the toxic agent sodium azide. Avoid skin contact.

For semiquantative analysis incubate calibrator 2 along with the positive and negative controls and patient samples. For quantitative analysis incubate calibrators 1, 2 and 3 along with the positive and negative controls and patient samples. Sample incubation: (1. step) Transfer 100 μl of the calibrators, positive and negative controls or diluted patient samples into the individual microplate wells according to the pipetting protocol. Incubate for 30 minutes at room temperature (+18°C to +25°C). Washing: Manual: Empty the wells and subsequently wash 3 times using 300 μl of working strength wash buffer for each wash. Automatic: Wash reagent wells 3 times with 450 μl working strength wash buffer (program setting: e.g. TECAN Columbus Washer "Overflow Modus"). Leave the wash buffer in each well for 30 to 60 seconds per washing cycle, then empty the wells. After washing (manual and automated tests), thoroughly dispose of all liquid from the microplate by tapping it on absorbent paper with the openings facing downwards to remove all residual wash buffer. Note: Residual liquid (> 10 μl) in the reagent wells after washing can interfere with the substrate and lead to false low extinction values. Insufficient washing (e.g., less than 3 wash cycles, too small wash buffer volumes, or too short reaction times) can lead to false high extinction values. Free positions on the microplate strip should be filled with blank wells of the same plate format as that of the parameter to be investigated. Conjugate incubation: (2. step) Pipette 100 μl of enzyme conjugate (peroxidase-labelled anti-human IgG) into each of the microplate wells. Incubate for 30 minutes at room temperature (+18°C to +25°C). Washing: Empty the wells. Wash as described above. Substrate incubation: (3. step) Pipette 100 μl of chromogen/substrate solution into each of the microplate wells. Incubate for 15 minutes at room temperature (+18°C to +25°C) (protect from direct sunlight). Stopping the reaction: Pipette 100 μl of stop solution into each of the microplate wells in the same order and at the same speed as the chromogen/substrate solution was introduced. Measurement: Photometric measurement of the colour intensity should be made at a wavelength of 450 nm and a reference wavelength of between 620 nm and 650 nm within 30 minutes of adding the stop solution. Prior to measuring, slightly shake the micro-plate to ensure a homogeneous distribution of the solution.

Semiquantitative: Results can be evaluated semiquantitatively by calculating a ratio of the extinction of the control or patient sample over the extinction of the calibrator 2. Calculate the ratio according to the following formula: Extinction of the control or patient sample/Extinction of calibrator 2 = Ratio We recommend interpreting results as follows: Ratio 0.8: negative Ratio ≥0.8 to 1.1: borderline Ratio ≥1.1: positive In cases of borderline test results, an additional patient sample should be taken 7 days later and retested in parallel with the first patient sample. The results of both samples allow proper evaluation of titer changes. Quantitative: The standard curve from which the concentration of antibodies in the patient samples can be taken is obtained by point-to-point plotting of the extinction readings measured for the 3 calibration sera against the corresponding units (linear/linear). Use "point-to-point" plotting for calculation of the standard curve by computer. The following plot is an example of a typical calibration curve. Please do not use this curve for the determination of antibody concentrations in patient samples. If the extinction for a patient sample lies above the extinction of calibrator 1 (corresponding to 200 RU/ml), the result should be reported as ">200 RU/ml". It is recommended that the sample be retested at a dilution of e.g. 1:400. The result in RU/ml read from the calibration curve for this sample must then be multiplied by factor 4. The upper limit of the normal range of non-infected persons (cut-off value) recommended by Creative Diagnostics is 20 relative units (RU)/ml. We recommend interpreting results as follows: 16 RU/ml: negative ≥16 to 22 RU/ml: borderline ≥22 RU/ml: positive For duplicate determinations the mean of the two values should be taken. If the two values deviate substantially from one another, Creative Diagnostics recommends retesting the samples.

The levels of the anti-Adenovirus antibodies (IgG) were analysed with this ELISA in a panel of 300 healthy blood donors. With a cut-off of 20 RU/ml, 64.3% of the blood donors were anti-Adenovirus positive (IgG) which reflects the known percentage of infections in adults.

Calibration: As no international reference serum exists for antibodies against adenovirus, the calibration is performed in relative units (RU). For every group of tests performed, the extinction values of the calibrators and the relative units and/or the ratios determined for the positive and negative controls must lie within the limits stated for the relevant test kit lot. A protocol containing these target 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 activity of the enzyme used is temperature-dependent and the extinction values may vary if a thermostat is not used. The higher the room temperature during substrate incubation, the greater will be the extinction values. Corresponding variations apply also to the incubation times. However, the calibration sera are subject to the same influences, with the result that such variations will be largely compensated in the calculation of the result. Antigen: The antigen source is provided by cell lysates of MRC-5 cells infected with the "Adenoid 6" strain of adenovirus.

The detection limit is defined as a value of three times the standard deviation of an analyte-free sample and is the smallest detectable antibody titer. The lower detection limit of the Anti-Adenovirus ELISA (IgG) is 1 RU/ml.

The linearity of the test was investigated using series dilutions of patient sera with high antibody concentrations. The Anti-Adenovirus ELISA (IgG) is linear in the measurement range 2-200 RU/ml.

The reproducibility of the test was investigated by determining the intra- and inter-assay coefficients of variation using 3 sera. The intra-assay CVs are based on 20 determinations and the inter-assay CVs on 4 determinations performed in 6 different test runs.

Haemolytic, lipaemic and icteric samples showed no influence at the result up to a concentration of 10 mg/ml for haemoglobin, 20 mg/ml for triglycerides and 0.4 mg/ml for bilirubin in this ELISA.

The ELISA test kit provides a semiquantitative or quantitative in vitro assay for human antibodies of the IgG class against adenovirus in serum or plasma.

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/H 2 O 2 , 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. Test instruction --- 1 booklet 13. Protocol with target values --- 1 protocol

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0.5M

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