The guinea pig interleukin 10 (IL-10) ELISA detection kit is a solid-phase sandwich enzyme-linked immunosorbent assay (ELISA). Standards with known IL-10 concentrations and samples with unknown concentrations are added to microwell enzyme plates for detection. First, IL-10 and biotin-labeled antibody were incubated at the same time. After washing, HRP labeled with avidin was added. After incubation and washing, the unbound enzyme conjugate is removed, and then substrates A and B are added to act simultaneously with the enzyme conjugate. Produce colors. The color depth is proportional to the concentration of IL-10 in the sample.
Bring your own materials:
1. Distilled water.
2. Sampler: 5ul, 10ul, 50ul, 100ul, 200ul, 500ul, 1000ul.
3. Oscillator and magnetic stirrer, etc.
Guinea pig interleukin 10 (IL-10) ELISA detection kit operation notes:
â— Reagents should be stored according to the label instructions and returned to room temperature before use. The sparse standards should be discarded and cannot be stored.
â— Slats not used in the experiment should be immediately returned to the packaging bag, sealed and stored to avoid deterioration.
â— Any unused reagents should be packed or covered. Do not mix reagents of different batches. Use before warranty.
â— Use a disposable pipette tip to avoid cross-contamination, and avoid using a sampler with a metal part when drawing the stop solution and the substrates A and B.
â— Use a clean plastic container to prepare the washing solution. Mix all components and samples in the kit thoroughly before use.
â— Substrate A should evaporate to avoid opening the lid for a long time. Substrate B is sensitive to light and avoid prolonged exposure to light. Avoid contact with hands, it is toxic. The OD value should be read immediately after the experiment is completed.
â— The order of adding reagents should be consistent to ensure the same incubation time of all reaction wells.
â— Perform the incubation operation in accordance with the time, amount and sequence of the liquid indicated in the manual.
1. Avoid direct contact with the stop solution and substrates A and B. Once contact with these liquids, please rinse with water as soon as possible.
2. Don't eat, drink, smoke or use cosmetics in real difficulties.
3. Do not use your mouth to absorb any components in the kit.
1. Before use, mix all reagents thoroughly. Don't make the liquid generate a lot of foam, so as to avoid adding a large number of bubbles during sample addition, which will cause errors in sample addition.
2. Determine the number of slats required based on the number of samples to be tested plus the number of standard products. It is recommended to make multiple holes for each standard and blank hole. Each sample is determined according to its own quantity, and those that can use multiple holes can be used for multiple holes.
3. Add 50ul of the diluted standard to the reaction well and 50ul of the sample to be tested in the reaction well. Immediately add 50ul of biotin-labeled antibody. Cover the membrane plate, mix gently by shaking, and incubate at 37 Â° C for 45 minutes.
4. Shake off the liquid in the hole, fill the hole with washing liquid, shake for 30 seconds, shake off the washing liquid, pat dry with absorbent paper. Repeat this operation 4 times. If washing with a plate washer, the number of washes is increased once.
5. Add 100ul of streptavidin-HRP to each well, gently shake and mix, and incubate at 37 Â° C for 30 minutes.
6. Shake off the liquid in the hole, fill each hole with washing liquid, shake for 30 seconds, shake off the washing liquid, pat dry with absorbent paper. Repeat this operation 4 times. If washing with a plate washer, the number of washes is increased once.
7. Add 50ul of substrate A and B to each well, gently shake and mix, and incubate at 37 Â° C for 5 minutes. Avoid light.
8. Remove the enzyme labeling plate and quickly add 50ul of stop solution. After adding the stop solution, the results should be measured immediately.
9. Measure the OD value of each well at 450nm wavelength.
Guinea pig interleukin 10 (IL-10) ELISA test kit performance:
1. Sensitivity: The smallest detection concentration is less than No. 1 standard. Linearity of dilution. The correlation coefficient R between the linear regression of the sample and the expected concentration is 0.990.
2. Specificity: Does not react with other cytokines.
3. Repeatability: The coefficients of variation within and between plates are less than 10%.
Judgment and analysis of results:
1. Instrument value: read the OD value of each well on a microplate reader with a wavelength of 450nm
2. Taking the absorbance OD value as the ordinate (Y) and the corresponding IL-10 standard concentration as the abscissa (X), make the corresponding curve. The IL-10 content of the sample can be calculated from the standard curve according to its OD Corresponding concentration, then multiply by the dilution factor; or use the standard concentration and OD value to calculate the regression equation of the standard curve, substitute the OD value of the sample into the equation, calculate the sample concentration, and multiply by the dilution factor, which is the sample â€™s Actual concentration.
3. Range of detection value: 0-10ng / ml
4. Sensitivity: 0.1ng / ml
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