ELISA/assay

Human Tetanus Toxoid IgG ELISA Kit

DEIA378

96T

Human Tetanus Toxoid IgG ELISA Kit

C. tetani Tetanus Toxoid

Human Tetanus Toxoid IgG ELISA Kit

iELISA

C. tetani Tetanus Toxoid

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.

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 Anti-Tetanus Toxoid ELISA (IgG).

Tetanus Toxoid

Human

The ELISA test kit provides a quantitative in vitro assay for human antibodies of the immunoglobulin class IgG against tetanus toxoid in serum or plasma for the clarification of an unclear immune status.

The Anti-Tetanus Toxoid ELISA (IgG) is based on inactivated tetanus toxin and is designed for the quantitative determination of human IgG antibodies against tetanus toxoid in serum or plasma. This test is suited for both investigation the immune status and for vaccination control.

The test kit contains microtiter strips each with 8 break-off reagent wells coated with tetanus 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 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, 5 IU/ml (human IgG), ready for use, 1 x 2.0 ml CAL 1 3. Calibrator 2, 2 IU/ml (human IgG), ready for use, 1 x 2.0 ml CAL 2 4. Calibrator 3, 1 IU/ml (human IgG), ready for use, 1 x 2.0 ml CAL 3 5. Calibrator 4, 0.1 IU/ml (human IgG), ready for use, 1 x 2.0 ml CAL 4 6. Calibrator 5, 0.01 IU/ml (human IgG), ready for use, 1 x 2.0 ml CAL 5 7. Positive control, (IgG, human), ready for use, 1 x 2.0 ml POS CONTROL 8. Negative control, (IgG, human), ready for use, 1 x 2.0 ml NEG CONTROL 9. Enzyme conjugate, peroxidase-labelled anti-human IgG (rabbit), ready for use, 1 x 12 ml CONJUGATE 10. Sample buffer, ready for use, 1 x 100 ml SAMPLE BUFFER 11. Wash buffer, 10x concentrate, 1 x 100 ml WASH BUFFER 10x 12. Chromogen/substrate solution, TMB/H 2 O 2 , ready for use, 1 x 12 ml SUBSTRATE 13. Stop solution, 0.5 M sulphuric acid, ready for use, 1 x 12 ml STOP SOLUTION 14. Protective foil --- 2 pieces FOIL 15. Test instruction --- 1 booklet 16. Quality control certificate --- 1 protocol

Samples: 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 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 above pipetting protocol is an example for the quantitative analysis of 24 patient samples (P 1 to P 24). The calibrators (C 1 to C 5), 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. 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.

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. 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 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. 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. 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 the agent sodium azide in a nondeclarable concentration. Avoid skin contact.

Sample incubation: (1 st 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. 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. 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 (program setting: e.g. TECAN Columbus Washer "Overflow Mode"). Leave the wash buffer in each well for 30 to 60 seconds per washing cycle, then empty the wells. After washing, 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. Conjugate incubation: (2 nd 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 rd 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: 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 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.

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 5 calibration sera against the corresponding units (linear/linear). Use "point-to-point" plotting for calculation of the standard curve by computer. If the extinction for a patient sample lies above the value of calibrator 1 (5 IU/ml), the result should be reported as ">5 IU/ml". It is recommended that the sample be retested 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.

Based on the literature references [4,8,9,13] the following evaluation of the test results is recommended: 0.5-1.1 IU/ml Sufficient immunity, booster vaccination in 2 to 5 years >1.1-5.0 IU/ml Sufficient immunity, booster vaccination in 5 to 10 years >5.0 IU/ml Sufficient immunity, booster vaccination in approx. 10 years For the interpretation of antibody titers, country-specific recommendations should be considered. For duplicate determinations the mean of the two values should be taken. If the two values deviate substantially from one another, CDrecommends retesting the samples. Alongside the serological finding, the vaccination history of the patient must always be taken into account for diagnosis.

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.

The levels of anti-tetanus toxoid antibodies (IgG) were analysed with this CD ELISA in a panel of 500 healthy blood donors. With a cut-off of 0.1 IU/ml, 97% of the blood donors showed an immunisation protection. 3% of the blood donors required a basic immunisation or a booster.

Calibration: The Anti-Tetanus Toxoid ELISA (IgG) was calibrated using the first international WHO standard preparation TE-3 (1 st International Standard for Tetanus Immunoglobulin, Human NIBSC Code TE-3). For every group of tests performed, the extinction values of the calibrators 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 the 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. Antigen: The microtiter wells were coated with inactivated Tetanus toxoid antigen.

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-Tetanus Toxoid ELISA (IgG) is 0.001 IU/ml.

The linearity of the Anti-Tetanus Toxoid ELISA (IgG) was determined by assaying at least 4 serial dilutions of different patient samples. The Anti-Tetanus Toxoid ELISA (IgG) is linear at least in the tested concentration range (0.01 IU/ml to 4.2 IU/ml).

The reproducibility of the test was investigated by determining the intra- and interassay 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. Intra-assay variation, n = 20, CV 1.1-2.7% Inter-assay variation, n = 4 x 6 , CV 3.6-9.7%

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. Ahluwalia IB, Ding H, D'Angelo D, Shealy KH, Singleton JA, Liang J, Rosenberg KD; Centers for Disease Control and Prevention (CDC). Tetanus, diphtheria, pertussis vaccination coverage before, during, and after pregnancy – 16 States and New York City, 2011. MMWR Morb Mortal Wkly Rep 64 (2015) 522-526. 2. Brook I. Current concepts in the management of Clostridium tetani infection. Expert Rev Anti Infect Ther 6 (2008) 327-336. 3. Fowkes FJ, McGready R, Johnstone-Robertson S, Nosten F, Beeson JG. Antibody boosting and longevity following tetanus immunization during pregnancy. Clin Infect Dis 55 (2013) 749-750. 4. Kuhlmann WD. Tetanus. Impfung, Impftiter und Impfreaktion Zentrales Institut des Sanitätsdienstes der Bundeswehr Koblenz. (1991). 5. Lassi ZS, Mansoor T, Salam RA, Das JK, Bhutta ZA. Essential pre-pregnancy and pregnancy interventions for improved maternal, newborn and child health. Reprod Health 11 (2014) 1-19. Epub 2014 Aug 21. 6. McCormack PL. Reduced-antigen, combined diphtheria, tetanus and acellular pertussis vaccine, adsorbed (Boostrix®): a review of its properties and use as a single-dose booster immunization. Drugs 72 (2012) 1765-1791. 7. McVicar J. Should We test for tetanus immunity in all emergency department patients with wounds. Emerg Med J 30 (2013) 177-189. 8. Müller HE, Müller M, Schick W. Tetanus-Schutzimpfung-Indikation und Kontraindikation Dtsch. med. Wschr. 113 (1988) 1326-8. 9. Pietsch M. Indikationsbeispiele für die Tetanus-Antikörper-Bestimmung Fortschritte der Diagnostik 1 (1991) 30-1. 10. RKI. Tetanus: Zwei Fallberichte zu Erkrankungen. Epidemiologisches Bulletin 24 (2008). 11. Orsi GB, Modini C, Principe MA, Di Muzio M, Moriconi A, Amato MG, Calderale SM. Assessment of tetanus immunity status by tetanus quick stick and anamnesis: a prospective double blind study. Ann Ig 27 (2015) 467-474. 12. Schlumberger M, Yvonnet B, Que HV, Chhem DB, Saliou P, Le Tu TC, Glaziou P. Serological study carried out in Cambodia during a tetanus vaccination in adults. [Article in French] Bull Soc Pathol Exot 101 (2008) 36-42. 13. Schröder JP, Kuhlmann WD. Tetanusimmunität bei Männern und Frauen in der Bundes-republik Deutschland Immun. Infekt. 19 (1991) 14-7. 14. Shohat T, Marva E, Sivan Y, Lerman I, Mates A, Cohen A. Immunologic response to a single dose of tetanus toxoid in older people. J Am Geriatr Soc 48 (2000) 949-951.

The ELISA test kit provides a quantitative in vitro assay for human antibodies of the immunoglobulin class IgG against tetanus toxoid in serum or plasma for the clarification of an unclear immune status.

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, 5 IU/ml (human IgG), ready for use, 1 x 2.0 ml CAL 1 3. Calibrator 2, 2 IU/ml (human IgG), ready for use, 1 x 2.0 ml CAL 2 4. Calibrator 3, 1 IU/ml (human IgG), ready for use, 1 x 2.0 ml CAL 3 5. Calibrator 4, 0.1 IU/ml (human IgG), ready for use, 1 x 2.0 ml CAL 4 6. Calibrator 5, 0.01 IU/ml (human IgG), ready for use, 1 x 2.0 ml CAL 5 7. Positive control, (IgG, human), ready for use, 1 x 2.0 ml POS CONTROL 8. Negative control, (IgG, human), ready for use, 1 x 2.0 ml NEG CONTROL 9. Enzyme conjugate, peroxidase-labelled anti-human IgG (rabbit), ready for use, 1 x 12 ml CONJUGATE 10. Sample buffer, ready for use, 1 x 100 ml SAMPLE BUFFER 11. Wash buffer, 10x concentrate, 1 x 100 ml WASH BUFFER 10x 12. Chromogen/substrate solution, TMB/H 2 O 2 , ready for use, 1 x 12 ml SUBSTRATE 13. Stop solution, 0.5 M sulphuric acid, ready for use, 1 x 12 ml STOP SOLUTION 14. Protective foil --- 2 pieces FOIL 15. Test instruction --- 1 booklet 15. Quality control certificate --- 1 protocol

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.