This reagent is for research use only Test principle: Bring your own material distilled water. Safety Avoid direct contact with the stop solution and substrates A and B. Once exposed to these liquids, rinse with water as soon as possible. Handling precautions Reagents should be stored according to label instructions, and returned to room temperature before use. The sparse standards should be discarded and cannot be stored. Sample collection, processing and storage methods Serum-avoid any cell stimulation during the operation. Use test tubes free of pyrogens and endotoxins. After collecting blood, centrifuge at 1000 × g for 10 minutes to separate the serum and red blood cells quickly and carefully. Reagent preparation standards: Serial dilutions of standards should be prepared during the experiment and cannot be stored. Before dilution, the standard was mixed by shaking. The dilution ratio is as follows: Dilution of washing buffer (50 ×): 50-fold dilution with distilled water. 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. Recommended experimental protocol Standard concentration (nmol / L) Kit performance 1. Sensitivity: The minimum 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. Judgment and analysis of results 2. Taking the absorbance OD value as the ordinate (Y) and the corresponding IAA standard concentration as the abscissa (X), make the corresponding curve. The IAA content of the sample can be converted from the standard curve according to its OD value to calculate the corresponding concentration. 3. Range of detection value: 0-40nmol / L 4. Sensitivity: 0.1 nmol / L
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The IAA kit is a solid-phase sandwich enzyme-linked immunosorbent assay (ELISA). Standards with known IAA concentrations and samples with unknown concentrations are added to microwell enzyme plates for detection. First, IAA and biotin-labeled antibodies are incubated simultaneously. 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 IAA in the sample.
Kit contents and preparation kit components (stored at 2-8 ° C) 96-well configuration 48-well configuration Preparation
96/48 serving microplate 1 plate (96T) half plate (48T) ready-to-use plastic membrane plate cover 1 half-plate ready-to-use standard: 40nmol / L 1 bottle (0.6ml) 1 bottle (0.3 ml) Carry out dilute blank control according to the instructions 1 bottle (1.0 ml) 1 bottle (0.5 ml) ready-to-use standard dilution buffer 1 bottle (5 ml) 1 bottle (2.5 ml) ready-to-use biotin-labeled anti-IAA antibody 1 bottle (6ml) 1 bottle (3.0ml) ready-to-use affinity streptavidin-HRP 1 bottle (10ml) 1 bottle (5.0ml) ready-to-use wash buffer 1 bottle (20ml) 1 bottle (10ml) according to the instructions Dilute Substrate A 1 bottle (6.0ml) 1 bottle (3.0ml) ready-to-use substrate B 1 bottle (6.0ml) 1 bottle (3.0ml) ready-to-use stop solution 1 bottle (6.0ml) 1 bottle (3.0 ml) ready-to-use specimen dilution 1 bottle (12ml) 1 bottle (6.0ml) ready-to-use
Sampler: 5ul, 10ul, 50ul, 100ul, 200ul, 500ul, 1000ul.
Oscillator and magnetic stirrer etc.
Do not eat, drink, smoke or use cosmetics during the experiment.
Do not use your mouth to absorb any ingredients in the kit.
The slats not used in the experiment should be immediately returned to the packaging bag, sealed and stored to avoid deterioration.
Unused other reagents should be packed or covered. Do not mix reagents of different batches. Use before warranty.
Use a disposable pipette tip to avoid cross-contamination, and avoid using a sampler with a metal part when drawing the stop solution and substrates A and B.
Use a clean plastic container to prepare the washing solution. Mix all components and samples in the kit thoroughly before use.
When washing the enzyme-labeled plate, it should be fully patted dry. Do not put the absorbent paper directly into the enzyme-labeled reaction well to absorb water.
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.
The order of adding reagents should be the same to ensure that all wells are incubated for the same time.
Perform the incubation operation according to the time, the amount and order of the liquid indicated in the instructions.
Plasma ----- EDTA, citrate, heparin plasma can be used for detection. Centrifuge at 1000 × g for 30 minutes to remove particles.
The cell supernatant was centrifuged at 1000 × g for 10 minutes to remove particles and polymers.
Tissue homogenate ----- Mash the tissue with appropriate amount of saline. Centrifuge at 1000 × g for 10 minutes, and take the supernatant for storage. If the sample is not used immediately, it should be divided into small parts and stored at -70 ℃ to avoid repeated freezing. If possible, do not use hemolysis or hyperlipidemia. If there are a large number of particles in the serum, centrifuge or filter before testing. Do not thaw at 37 ° C or higher. Thaw at room temperature and ensure that the sample is thawed evenly and adequately.
40 nmol / L (Standard No. 6) The original concentration is directly added to 50ul without dilution.
20 nmol / L (No. 5 standard) 100ul of the original standard is added to 100ul of standard dilution
10 nmol / L (No. 4 standard) 100ul of No. 5 standard is added with 100ul of standard diluent
5.0 nmol / L (Standard No. 3) Add 100ul of Standard No. 4 to 100ul of Standard Diluent
2.5 nmol / L (Standard No. 2) Add 100ul of No. 3 standard to 100ul of standard dilution
1.25 nmol / L (No. 1 standard) 100ul of No. 2 standard is added with 100ul of standard diluent
0 nmol / L (blank control) The original concentration is directly added to 50ul without dilution.
The number of slats required is determined by 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 as much as possible. The specimen was diluted 1: 1 with the specimen dilution solution and then added 50ul to the reaction well.
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, gently shake and mix, and incubate at 37 ° C for 1 hour.
Shake off the liquid in the wells, fill each well with washing liquid, shake for 30 seconds, shake off the washing liquid, pat dry with absorbent paper. Repeat this operation 3 times. If washing with a plate washer, the number of washes is increased once.
Add 80 ul of streptavidin-HRP to each well, gently shake and mix, and incubate at 37 ° C for 30 minutes.
Shake off the liquid in the wells, fill each well with washing liquid, shake for 30 seconds, shake off the washing liquid, pat dry with absorbent paper. Repeat this operation 3 times. If washing with a plate washer, the number of washes is increased once.
Add 50ul of substrate A and B to each well, mix gently by shaking, and incubate at 37 ° C for 10 minutes. Avoid light.
Remove the enzyme labeling plate and quickly add 50ul of stop solution. After adding the stop solution, the results should be measured immediately.
The OD value of each well was measured at a wavelength of 450 nm.
A 40 40 sample sample sample sample sample sample sample sample sample sample sample
B 20 20 sample sample sample sample sample sample sample sample sample sample sample
C 10 10 sample sample sample sample sample sample sample sample sample sample sample
D 5.0 ​​5.0 sample sample sample sample sample sample sample sample sample sample sample
E 2.5 2.5 sample sample sample sample sample sample sample sample sample sample sample
F 1.25 1.25 sample sample sample sample sample sample sample sample sample sample sample
G 0 0 sample sample sample sample sample sample sample sample sample sample sample
H sample sample sample sample sample sample sample sample sample sample sample sample sample
Limitation
The results of standard No. 6 and above are non-linear, and accurate results cannot be obtained based on this standard curve.
2. Specificity: Does not react with other cytokines.
3. Repeatability: The coefficients of variation within and between plates are less than 10%.
1. Instrument value: read the OD value of each well on a microplate reader with a wavelength of 450nm
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