ELISA/assay

Human Anti-Diphtheria Toxoid IgG ELISA Kit

DEIA1776

96T

Human Anti-Diphtheria Toxoid IgG ELISA Kit

C. diphtheriae Toxin

Human Anti-Diphtheria Toxoid IgG ELISA Kit

iELISA

C. diphtheriae Toxin

Human

Quantitative

Serum or 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.

The quality of the antigen used ensures a high specificity of the ELISA. Sera from patients with infections caused by various agents were investigated with the Human Anti-Diphtheria Toxoid IgG ELISA Kit.

Diphtheria toxin

Human

The Human Anti-Diphtheria Toxoid IgG ELISA Kit is based on inactivated diphtheria toxin and is designed for the quantitative determination of human IgG antibodies against diphtheria toxoid in serum or plasma. This test is for research use only.

The test kit contains microtiter strips each with 8 break-off reagent wells coated with Diphtheria toxoid. 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 reader 2. 37°C incubator 3. Automated plate washer 4. Precision single and multi-channel pipette and disposable tips 5. Clean tubes and Eppendorf tubes 6. Deionized or distilled water

Samples: Human serum or EDTA, heparin or citrate plasma. Stability: 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: Samples are diluted 1:101 sample buffer. For example: dilute 10 μl sample in 1.0 ml sample buffer and mix well by vortexing (sample pipettes are not suitable for mixing). NOTE: The calibrators and controls are prediluted and ready for use, do not dilute them.

The Pipetting protocol is an example for the quantitative analysis of 24 samples (P 1 to P 24). The calibrators (C 1 to C 4), the positive (pos.) and negative (neg.) controls, and the 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. Therefore, the number of tests performed can be matched to the number of samples, minimising 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.

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. The thermostat adjusted ELISA incubator must be set at +37°C ± 1°C. 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 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 10× 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. The working strength wash buffer is stable for 4 weeks 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.

Washing: Manual: Remove the protective foil, empty the wells and subsequently wash 3 times using 300 μl of working strength wash buffer for each wash. Automatic: Remove the protective foil and wash the reagent wells 3 times with 450 μl of working strength wash buffer. 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 residence 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. Procedure: 1. Sample incubation: Transfer 100 μl of the calibrators, positive and negative controls or diluted samples into the individual microplate wells according to the pipetting protocol. For manual processing of microplate wells, cover the finished test plate with the protective foil. When using an automated microplate processor for incubation follow the recommendations of the instrument manufacturer. Incubate 60 minutes at +37°C ± 1°C. 2. Washing: Empty the wells. Wash as described above. 3. Conjugate incubation: 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). 4. Washing: Empty the wells. Wash as described above. 5. Substrate incubation: 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). 6. Stopping: 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 intro-duced. 7. Measurement: Photometric measurement of the colour intensity should be made at a wavelength of 450 nm and a reference wavelength between 620 nm and 650 nm within 30 minutes of adding the stop solution. Prior to measuring, slightly shake the microplate to ensure a homogeneous distribution of the solution.

Quantitative: The standard curve from which the concentration of antibodies in the serum samples can be taken is obtained by point-to-point plotting of the extinction values measured for the 4 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 samples. If the extinction for a sample lies above the value of calibrator 1 (2 IU/ml), the result should be reported as ">2 IU/ml". It is recommended that the sample be remeasured in a new test run at a dilution of e.g. 1:400. The result in IU/ml read from the calibration curve for this sample must then be multiplied by a factor of 4.

Recommendations taken from literature for the evaluation of test results (1,2,3) and subsequent procedures (4) are summarised below: 0.01 IU/ml: No protection (individuals without immunisation should receive two vaccinations with a time interval of 4 to 8 weeks and a third vaccination 6 to 12 months after the second one) 0.01-0.099 IU/ml: Uncertain protection ≥0.1 IU/ml: Immunisation protection present >1.0 IU/ml: Long-term immunisation protection According to the Standing Committee on Vaccination (STIKO) at the Robert Koch Institute recommendations (4), the determination of the anti-diphtheria toxoid titer is recommended in order to ascertain whether there is sufficient evidence of immunity or whether basic vaccination or a booster shot is required. Alongside the serological finding, the vaccination history of the patient must always be taken into account for diagnosis.

Calibration: The controls of the Human Anti-Diphtheria Toxoid IgG ELISA Kit were calibrated using the international reference preparation NIBSC 10/262 (1 st International Standard for Diphtheria Antitoxin Human, National Institute for Biological Standards and Control, Hertfordshire, England). For every group of tests performed, the extinction values of the calibration sera and the international units 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 incubation steps, the greater will be the extinction values. 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.

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 Human Anti-Diphtheria Toxoid IgG ELISA Kit is 0.001 IU/ml.

The linearity of the Human Anti-Diphtheria Toxoid IgG ELISA Kit was determined by assaying at least 4 serial dilutions of different patient samples. The Human Anti-Diphtheria Toxoid IgG ELISA Kit is linear at least in the tested concentration range (0.06 IU/ml to 1.17 IU/ml).

The reproducibility of the test was investigated by determining the intra- and inter-assay coefficients of variation (CV) using 3 samples. 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 on 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.

1. Samples, calibrators, controls and incubated microplate strips should be handled as infectious waste. All reagents are to be disposed of according to official regulations. 2. 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 the agent sodium azide in a non-declarable concentration. Avoid skin contact.

1. Efstratiou A, George RC. Laboratory guidelines for the diagnosis of infections caused by Corynebacterium diphtheriae and C. ulcerans. World Health Organization. Commun Dis Public Health 2 (1999) 250-257. 2. di Giovine P, Kafatos G, Nardone A, Andrews N, Ölander RM, Alfarone G, Broughton K, Cohen D, Kriz B, Mikova I, O'Flanagan D, Schneider F, Selga I, Valinsky L, Velicko I, Karacs I, Pebody R, von Hunolstein C. Comparative seroepidemiology of diphtheria in six European countries and Israel. Epidemiol. Infect. 141 (2013) 132-142. 3. WHO: The Immunological Basis for Immunization Series, Modul 2: Diphtheria, Update 2009. ISBN 978 92 4 159786 9. 4. RKI. Empfehlungen der Ständigen Impfkommission (STIKO) am Robert Koch-Institut. Epidemiologisches Bulletin Nr. 34 (2015).

The Human Anti-Diphtheria Toxoid IgG ELISA Kit is based on inactivated diphtheria toxin and is designed for the quantitative determination of human IgG antibodies against diphtheria toxoid in serum or plasma. This test is for research use only.

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0.5M

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See the individual product datasheet