Chikungunya IgM ELISA Kit Instructions
Germany IBL imported original: RE58841)
Germany IBL China exclusive agent "Shenzhen Kerunda Biological Engineering Co., Ltd." dedicated to serve you
ã€expected usage】
The Chikungunya IgM antibody ELISA test kit is mainly used for qualitative detection of IgM antibodies against chikungunya virus in human serum and plasma.
[Experimental principle]
This kit is based on ELISA technology. The coated plate is coated with anti-human IgM antibody. If human serum or plasma contains IgM, it will specifically bind to it. The plate will wash away the unbound material, then add Chikungunya antigen solution and wash the plate. The unbound material is washed away, and then streptavidin and chikungunya antibody enzyme conjugate are added. After washing the plate, TMB substrate solution was added, the color turned blue, the reaction solution was terminated by adding a stop solution, the color changed from blue to yellow, and finally read at 450 nm with a microplate reader.
[reagent composition]
Coated plate : 12×8 detachable, coated with anti-human IgM antibody, sealed in resealable foil pouch
Chikungunya solution 1 : 1 bottle contains 6mL of Chikungunya antigen solution, ready to use, white cover
Chikungunya Solution 2 : 1 bottle contains 6 mL of biotinylated Chikungunya antibody, ready to use, blue, white cover
Chikungunya IgM positive property control : 1 bottle, 1.5mL, yellow, ready to use, red cover
Chikungunya IgM Critical Quality Control : 1 bottle, 2mL, yellow, ready to use, green cover
Chikungunya IgM Yin nature control : 1 bottle, 1.5mL, yellow, ready to use, blue cover
Sample Diluent: 1 bottle contains 100mL of ready-to-use buffer for dilution of sample, pH 7.2±0.2, yellow, white cover
Washing solution: 1 bottle containing 50mL of 20 times concentrated buffer, (pH 7.2±0.2) for washing, white cover
Streptavidin Binding Solution : 1 bottle contains 6 mL of peroxidase-conjugated streptavidin, ready-to-use, red, black cap
TMB substrate solution : 1 bottle contains 15mL TMB, ready to use, yellow cover
Stop solution: 1 bottle contains 15mL, ready to use, contains sulfuric acid, 0.2mol/l, red cover
[Required equipment and materials]
Fixed plate
Sealing plate
Microplate reader (450/620nm)
37 ° C incubator
Bottle washing or automatic washing machine
10~1000μL pipette
Whirl mixer
Distilled or deionized water
Disposable test tube
Timer
[Storage and Stability]
Reagents are stable at 2-8 °C during the period of validity
[Reagent preparation]
Preparation of washing liquid
The washing solution was diluted with double distilled water, for example: 10 ml of washing solution + 190 ml of double distilled water. The diluted wash solution is effective for 5 days at room temperature.
[Collection and preparation of samples]
The samples used in this experiment were human serum and plasma. If the experiment was performed within 5 days after sample collection, it should be stored at 2-8 ° C. Otherwise, it must be stored frozen at -20 ° C to -70 ° C. If the sample is cryopreserved, it needs to be thoroughly mixed before use to avoid repeated freezing and thawing.
Heat inactivated samples are not recommended
[Dilution of sample]
Mix 10 μL of sample with 1 ml of sample dilution and mix well with a vortex mixer.
[Experimental steps]
Please read the test instructions carefully before starting the test. The credibility of the results depends on strictly following the experimental instructions. At least one hole is left as a blank control (A1) and one negative control hole (B1). Two critical control holes (C1+D1). 1 positive control hole (E1). Allow all reagents to equilibrate to room temperature before starting the test
1. Pipette 50 μL of the control and the diluted sample into the corresponding wells, leaving the A1 well as a blank control well.
2. Sealing plate
3. Incubate for 1 hour ± 5 minutes at 37 ± 1 °C
4. When the incubation is complete, remove the sealing plate, discard the reaction solution, wash 300 μL of washing solution per well, and wash the plate 3 times to avoid spillage. The time for soaking in each well must be >5 seconds, and the final shot will take the remaining droplets.
5. Pipette 50 μL of Chikungunya solution 1 into each well except the blank control well, cover
6. Incubate for 30 minutes at room temperature
7. Repeat step 4
8. Mix Chikungunya Solution 2 with streptavidin conjugate for 10 minutes.
9. Pipette 50 μL of Chikungunya solution 2 with streptavidin into each well except the blank control well, cover.
10. Incubate for 30 minutes at room temperature
11. Repeat step 4
12. Pipette 100 μL of TMB substrate solution into each well
13. Incubate for 15 minutes in the dark (accurate)
14. Add 100 μL of Stop Solution to each well, and the interval and sequence must be the same as when adding TMB substrate solution.
15. Use a microplate reader to detect at 450/620 nm within 30 minutes after the addition of the stop solution.
ã€Detection】
Adjust the microplate reader, zero the blank control well, and measure the absorbance of all wells at 450 nm.
ã€result】
1. The condition for the test to take effect
The results of the test can only be considered valid if the following conditions are met.
Blank control well absorbance value <0.100
Negative control aperture absorbance value <critical quality control
Critical quality control hole absorbance value 0.150-1.300
Positive control hole absorbance value>critical quality control
If the above conditions are not met, the test results are invalid and need to be retested.
2. Calculation of results
Calculation of critical QC average absorbance values, examples : absorbance 1:0.39; absorbance 2:0.37
(0.39+0.37)/2=0.38
The average absorbance value is 0.38
3. Description of the results
If the sample is 10% higher than the critical value, it is considered positive.
If the sample is within 10% of the critical value, it is considered as a gray area (recommended to detect fresh samples after 2-4 weeks, if the sample is still gray, it can be considered as negative)
If the sample is 10% lower than the critical value, it is considered negative.
4. Unit of results
Patient sample average absorbance value × 10 = U
Threshold
Example: 1.216×10 =32U
0.38
Critical value: 10 U
Gray area: 9-11 U
Negative: <9 U
Positive: >11 U
This translation is for reference only, please refer to the original for details.
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