ELISA/assay

Chikungunya IgM ELISA Kit

DEIA2163

96T

Chikungunya IgM ELISA Kit

CHIKV

Chikungunya IgM ELISA Kit

ELISA

CHIKV

Human

Qualitative

Serum or plasma (citrate, heparin).

96T

Store the kit at 2. 8 °C. The opened reagents are stable up to the expiry date stated on the label when stored at 2. 8 °C.

Cross reactivity with antibodies against Borrelia, CMV and Toxoplasma cannot be excluded. Interference with polyclonal stimulation of EBV infections is likely. In the presence of infectious Mononucleosis (Pfeiffer´s Disease, EBV infection) polyclonal stimulation of B lymphocytes can occur. This may result in non-specific reactions in the detection of antibodies of the IgM class. Therefore it is recommended to exclude an EBV infection by differential diagnosis. Cross reactivity with antibodies against other alpha viruses cannot be excluded.

CHIKV

Human

The Chikungunya Virus IgM μ-capture ELISA is intended for the qualitative determination of IgM class antibodies against Chikungunya Virus in human serum or plasma (citrate, heparin).

The qualitative immunoenzymatic determination of specific IgM-class antibodies is based on the ELISA (Enzyme-linked Immunosorbent Assay) μ-capture technique. Microtiterplates are coated with anti-human IgM antibodies to bind the corresponding antibodies of the sample. After washing the wells to remove all unbound sample material, antigen is added. This antigen binds to the captured specific IgM antibodies. After a further washing step biotinylated antibody is pipetted into the wells. After washing horseradish peroxidase (HRP) labelled streptavidin is added that binds to the captured specific immune complex. After a further washing step the immune complexes are visualized by adding Tetramethylbenzidine (TMB) substrate which gives a blue reaction product. The intensity of this product is proportional to the amount of specific IgM antibodies in the sample. Sulphuric acid is added to stop the reaction. This produces a yellow endpoint colour. Absorbance at 450/620 nm is read using an ELISA Microtiterplate reader.

1. Microtiterplate: 12 break-apart 8-well snap-off strips coated with anti-human IgM; in resealable aluminium foil. 2. Sample Dilution Buffer: 1 bottle containing 100 mL of phosphate buffer (10 mM) for sample dilution; pH 7.2 ± 0.2; coloured yellow; ready to use; white cap; ≤ 0.0015% (v/v) CMIT/ MIT (3:1). 3. Stop Solution: 1 bottle containing 15 mL sulphuric acid, 0.2 mol/L; ready to use; red cap. 4. Washing Buffer (20x conc.): 1 bottle containing 50 mL of a 20-fold concentrated phosphate buffer (0.2 M), pH 7.2 ± 0.2, for washing the wells; white cap. 5. Antigen Solution, lyophilized: 6 bottles containing lyophilized Chikungunya virus Antigen Solution; red cap; ≤ 0.02% (v/v) MIT. 6. Antibody Solution: 1 bottle containing 6 mL of biotinylated Chikungunya virus antibody, ready to use; coloured blue; white cap; ≤ 0.02% (v/v) MIT. 7. Conjugate: 1 bottle containing 6 mL Streptavidin conjugated with peroxidase, ready to use; coloured red; black cap. 8. TMB Substrate Solution: 1 bottle containing 15 mL 3,3',5,5'-tetramethylbenzidine (TMB), 9. Positive Control: 1 vial containing 1.5 mL control; coloured yellow; ready to use; red cap; ≤ 0.02% (v/v) MIT. 10. Cut-off Control: 1 vial containing 2 mL control; coloured yellow; ready to use; green cap; ≤ 0.02% (v/v) MIT. 11. Negative Control: 1 vial containing 1.5 mL control; coloured yellow; ready to use; blue cap; ≤ 0.0015% (v/v) CMIT/ MIT (3:1).

1. 1 Cover foil 2. 1 Instruction for use (IFU) 3. 1 Plate layout 4. ELISA Microtiterplate reader, equipped for the measurement of absorbance at 450/620 nm 5. Incubator 37 °C 6. Manual or automatic equipment for rinsing Microtiterplates 7. Pipettes to deliver volumes between 10 and 1000 μL 8. Vortex tube mixer 9. Distilled water 10. Disposable tubes

Use human serum or plasma (citrate, heparin) samples with this assay. If the assay is performed within 5 days after sample collection, the samples should be kept at 2. 8 °C; otherwise they should be aliquoted and stored deep-frozen (-70…-20 °C). If samples are stored frozen, mix thawed samples well before testing. Avoid repeated freezing and thawing. Heat inactivation of samples is not recommended. Sample Dilution Before assaying, all samples should be diluted 1+100 with Sample Dilution Buffer. Dispense 10 μL sample and 1 mL Sample Dilution Buffer into tubes to obtain a 1+100 dilution and thoroughly mix with a Vortex.

It is very important to bring all reagents and samples to room temperature (20…25 °C) and mix them before starting the test run! 1. Microtiterplate The break-apart snap-off strips are coated with anti-human IgM. Immediately after removal of the strips, the remaining strips should be resealed in the aluminium foil along with the desiccant supplied and stored at 2. 8 °C. 2. Antigen Solution The bottles contain lyophilized Chikungunya virus Antigen Solution. The content of each vial has to be resolved in 1 mL diluted Washing Buffer by turning it slowly (no vortex) and 15 min incubation at room temperature (20…25 °C). The reconstituted solution is stable for 1 day at 2. 8 °C. 3. Washing Buffer (20x conc.) Dilute Washing Buffer 1 + 19; e. g. 10 mL Washing Buffer + 190 mL distilled water. The diluted buffer is stable for 5 days at room temperature (20…25 °C). In case crystals appear in the concentrate, warm up the solution to 37°C e.g. in a water bath. Mix well before dilution. 4. TMB Substrate Solution The reagent is ready to use and has to be stored at 2. 8 °C, away from the light. The solution should be colourless or could have a slight blue tinge. If the substrate turns into blue, it may have become contaminated and should be thrown away.

Please read the instruction for use carefully before performing the assay. Result reliability depends on strict adherence to the instruction for use as described. The following test procedure is only validated for manual procedure. If performing the test on ELISA automatic systems we recommend increasing the washing steps from three up to five and the volume of Washing Buffer from 300 μL to 350 μL to avoid washing effects. Pay attention to chapter 12. Prior to commencing the assay, the distribution and identification plan for all samples and standards/controls (duplicates recommended) should be carefully established on the plate layout supplied in the kit. Select the required number of microtiter strips or wells and insert them into the holder. Perform all assay steps in the order given and without any delays. A clean, disposable tip should be used for dispensing each standard/control and sample. Adjust the incubator to 37 ± 1 °C. 1. Dispense 50 μL standards/controls and diluted samples into their respective wells. Leave well A1 for substrate blank. 2. Cover wells with the foil supplied in the kit. 3. Incubate for 1 hour ± 5 min at 37 ± 1 °C. 4. When incubation has been completed, remove the foil, aspirate the content of the wells and wash each well three times with 300 μL of Washing Buffer. Avoid overflows from the reaction wells. The interval between washing and aspiration should be > 5 sec. At the end carefully remove remaining fluid by tapping strips on tissue paper prior to the next step! Note: Washing is important! Insufficient washing results in poor precision and false results. 5. Dispense 50 μL reconstituted Chikungunya virus Antigen into all wells except for the Substrate Blank well A1. 6. Incubate for 30 min at room temperature (20. 25 °C). 7. Repeat step 4. 8. Dispense 50 μL Chikungunya virus Antibody Solution into all wells except for the Blank well A1. 9. Incubate for 30 min at room temperature (20. 25 °C). 10. Repeat step 4. 11. Dispense 50 μL Streptavidin peroxidase conjugate into all wells except for the Blank well A1. 12. Incubate for 30 min at room temperature (20. 25 °C). Do not expose to direct sunlight. 13. Repeat step 4. 14. Dispense 100 μL TMB solution into all wells 15. Incubate for exact 15 min. at room temperature (20. 25 °C) in the dark. A blue colour occurs due to an enzymatic reaction. 16. Dispense 100 μL Stop Solution into all wells in the same order and at the same rate as for the TMB Substrate Solution, thereby a colour change from blue to yellow occurs. 17. Measure the absorbance at 450/620nm within 30 min after addition of the Stop Solution. Measurement Adjust the ELISA Microtiterplate reader to zero using the Substrate Blank. If - due to technical reasons - the ELISA Microtiterplate reader cannot be adjusted to zero using the Substrate Blank, subtract its absorbance value from all other absorbance values measured in order to obtain reliable results! Measure the absorbance of all wells at 450 nm and record the absorbance values for each standard/control and sample in the plate layout. Bichromatic measurement using a reference wavelength of 620 nm is recommended. Where applicable calculate the mean absorbance values of all duplicates.

In order for an assay run to be considered valid, these Instructions for Use have to be strictly followed and the following c riteria must be met: ▪ Substrate Blank: Absorbance value ▪ Negative Control: Absorbance value ▪ Cut-off Control: Absorbance value 0.150 – 1.300 ▪ Positive Control: Absorbance value > Cut-off If these criteria are not met, the test is not valid and must be repeated.

The Cut-off is the mean absorbance value of the Cut-off Control determinations. Example: Absorbance value Cut-off control 0.44 + absorbance value Cut-off control 0.42 =0.86 / 2 = 0.43 Cut-off = 0.43 Sample (mean) absorbance value x 10 / Cut-off = [CU] Example: 1.591 x 10 / 0.43 = 37 CU

1. Cut-off: 10 CU. 2. Positive: > 11 CU. Antibodies against the pathogen are present. There has been a contact with the antigen (pathogen resp. vaccine). 3. Equivocal: 9 – 11 CU. Antibodies against the pathogen could not be detected clearly. It is recommended to repeat the test with a fresh sample in 2 to 4 weeks. If the result is equivocal again the sample is judged as negative. 4. Negative: < 9 CU. The sample contains no antibodies against the pathogen. A previous contact with the antigen (pathogen resp. vaccine) is unlikely.

The diagnostic specificity is defined as the probability of the assay of scoring negative in the absence of the specific anal yte. It is 100% (95% confidence interval: 95.01% - 100%). The diagnostic sensitivity is defined as the probability of the assay of scoring positive in the presence of the specific analyte. It is 100% (95% confidence interval: 96.19% - 100%).

Interferences with hemolytic, lipemic or icteric samples are not observed up to a concentration of 10 mg/mL hemoglobin, 5 mg/mL triglycerides and 0.5 mg/mL bilirubin.

The physical and chemical properties of the reagents contained in this kit have been tested individually. Reagents do not contain ingredients which have been determined to be health hazards and which comprise greater than 1% of the mixture or which could be released from the mixture in concentrations that would exceed OSHA permissible exposure limits. Hazardous Ingredients: The lyophilized standard, antiserum and biotinylated tracer contain thimerosal or ProClin™ 150 as preservative. Physical and Chemical Data : Components are stable in closed containers under normal temperatures and pressures. No hazardous polymerization is known. Fire and Explosion Data: Components are non-combustible with negligible fire hazard when exposed to heat or flame. Fire fighting media should be appropriate to burning material. Health Hazards: Components may be harmful by inhalation, ingestion, or skin absorption and may cause skin irritation or eye irritation. In case of eye contact, flush eye with water and contact a physician. In case of skin contact, wash skin with soap and water. Reactivity Data: Components are stable in closed containers under normal temperatures and pressures. Spill and Disposal Procedures: For spills, ventilate area and wash spill site. For disposal, please dispose in accordance with local regulations. Handling and Storage Information: Safety glasses, gloves, and a full-length lab coat should be worn to prevent unnecessary contact. The above information is believed to be correct but does not purport to be all-inclusive and shall be used only as a guide. It is the user's responsibility to determine the suitability of this information for the adoption of safety precautions as may be necessary.

Bacterial contamination or repeated freeze-thaw cycles of the sample may affect the absorbance values.

The Chikungunya Virus IgM μ-capture ELISA is intended for the qualitative determination of IgM class antibodies against Chikungunya Virus in human serum or plasma (citrate, heparin).

1. Microtiterplate: 12 break-apart 8-well snap-off strips coated with anti-human IgM; in resealable aluminium foil. 2. Sample Dilution Buffer: 1 bottle containing 100 mL of phosphate buffer (10 mM) for sample dilution; pH 7.2 ± 0.2; coloured yellow; ready to use; white cap; ≤ 0.0015% (v/v) CMIT/ MIT (3:1). 3. Stop Solution: 1 bottle containing 15 mL sulphuric acid, 0.2 mol/L; ready to use; red cap. 4. Washing Buffer (20x conc.): 1 bottle containing 50 mL of a 20-fold concentrated phosphate buffer (0.2 M), pH 7.2 ± 0.2, for washing the wells; white cap. 5. Antigen Solution, lyophilized: 6 bottles containing lyophilized Chikungunya virus Antigen Solution; red cap; ≤ 0.02% (v/v) MIT. 6. Antibody Solution: 1 bottle containing 6 mL of biotinylated Chikungunya virus antibody, ready to use; coloured blue; white cap; ≤ 0.02% (v/v) MIT. 7. Conjugate: 1 bottle containing 6 mL Streptavidin conjugated with peroxidase, ready to use; coloured red; black cap. 8. TMB Substrate Solution: 1 bottle containing 15 mL 3,3',5,5'-tetramethylbenzidine (TMB), 9. Positive Control: 1 vial containing 1.5 mL control; coloured yellow; ready to use; red cap; ≤ 0.02% (v/v) MIT. 10. Cut-off Control: 1 vial containing 2 mL control; coloured yellow; ready to use; green cap; ≤ 0.02% (v/v) MIT. 11. Negative Control: 1 vial containing 1.5 mL control; coloured yellow; ready to use; blue cap; ≤ 0.0015% (v/v) CMIT/ MIT (3:1).

See the individual product datasheet

See the individual product datasheet

See the individual product datasheet

See the individual product datasheet

See the individual product datasheet

The Chikungunya Virus IgM μ-capture ELISA is intended for the qualitative determination of IgM class antibodies against Chikungunya Virus in human serum or plasma (citrate, heparin).