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 (EDTA, heparin or citrate plasma).
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 × 8 STRIPS 2. Calibrator 1, 2 IU/ml (human IgG), ready for use, 1 × 1.5 ml CAL 1 3. Calibrator 2, 1 IU/ml (human IgG), ready for use, 1 × 1.5 ml CAL 2 4. Calibrator 3, 0.1 IU/ml (human IgG), ready for use, 1 × 1.5 ml CAL 3 5. Calibrator 4, 0.01 IU/ml (human IgG), ready for use, 1 × 1.5 ml CAL 4 6. Positive control (IgG, human), ready for use, 1 × 1.5 ml 7. Negative control (IgG, human), ready for use, 1 × 1.5 ml 8. Enzyme conjugate, peroxidase-labelled anti-human IgG, ready for use, 1 × 12 ml 9. Sample buffer, ready for use, 1 × 100 ml 10. Wash buffer, 20× concentrate, 1 × 50 ml 11. Substrate solution, TMB/H2O2, ready for use, 1 × 12 ml 12. Stop solution, 0.5 M sulfuric acid, ready for use, 1 × 12 ml 13. Protective foil --- 2 pieces of FOIL 14. Test instructions --- 1 booklet 15. Quality control certificate --- 1 protocol
Storage
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.
Performance Characteristics
Calibration: The controls of the Human Anti-Diphtheria Toxoid IgG ELISA Kit were calibrated using the international reference preparation NIBSC 10/262 (1st 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.
Standard Curve
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.
Citations
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Background
Diphtheria is an infectious and potentially fatal disease caused by Corynebacterium diphtheriae. It is generally accepted that the potent diphtheria toxin secreted by C. diphtheriae is the main virulence factor and that the symptoms of the disease are related to the action of the toxin. Diphtheria toxin (DT) is a single-chain 62kDa protein consisting of 535 amino acid residues produced by Corynebacterium diphtheriae. containing a lysogenic beta phage. DT mediates its cytolethal effects by inhibiting protein synthesis in susceptible cells. The DT molecule is Y-shaped and contains two functionally distinct regions: A and B. The A fragment located at the N-terminus contains a catalytic domain that prevents protein synthesis in the cell. The B fragment located at the C-terminus consists of a transmembrane domain that helps transfer the catalytic domain from the endosome to the cytoplasm and a receptor domain that helps bind the heparin-binding epidermal growth factor receptor on the surface of susceptible cells.
Figure 1. Schematic representation of diphtheria toxin (Source: Shafiee F, et al. 2019)
In the last century, some researchers found that diphtheria toxin lost its toxicity while maintaining immunogenicity after heat and formalin treatment. In this way, the treated diphtheria toxin was called diphtheria toxoid and was used in diphtheria vaccine preparation. Conventional vaccines are produced in several steps: cultivation of Corynebacterium diphtheriae and clarification of the toxin-containing medium, followed by concentration and inactivation of the toxin, purification of the toxoids by means of diafiltration and adsorption on aluminum salts. The quality of diphtheria toxoid depends mainly on the detoxification process, in which reaction conditions such as formaldehyde concentration, reaction time and temperature, and matrix composition are very important.
Traditionally used in combination with tetanus and pertussis antigens, the diphtheria–tetanus–pertussis (DTP) vaccine was one of the first vaccines used in humans to protect against three serious and potentially fatal diseases that affect children and adults. Diphtheria vaccines are now also frequently used in combination with other antigens, such as Hib component, hepatitis B and inactivated polio vaccines. As the epidemiology of the diphtheria pathogen has changed, researchers have identified the emergence of infections caused by non-producing strains of diphtheria that cannot be prevented by current vaccines containing only diphtheria toxoids, and there is a need to continue to develop prophylactic vaccines against non-producing strains of diphtheria.
Alternative Names
Human Anti-DT IgG ELISA
References
1. Shafiee F, et al. Targeted Diphtheria Toxin-Based Therapy: A Review Article. Front Microbiol. 2019 Oct 18;10:2340.
2. Prygiel M, et al. Diphtheria-tetanus-pertussis vaccine: past, current & future. Future Microbiol. 2022 Feb;17:185-197.
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