This reagent is for research use only. Specimen: Serum
Test principle:
The AT-III kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA). Standards of known AT-III concentration and samples of unknown concentration are added to the microplates for detection. AT-III and biotinylated antibodies were first incubated simultaneously. After washing, avidin-labeled HRP was added. After incubation and washing, the unbound enzyme conjugate is removed, and the human anti-thrombin-III ELISA assay kit is then added to substrate A, B, and the enzyme conjugate simultaneously. Produce color. The depth of the color is proportional to the concentration of AT-III in the sample.
Dilution of wash buffer (50 x): 50-fold dilution of distilled water.
Kit composition:
Steps
Mix all reagents thoroughly before use. Do not allow the liquid to generate a large amount of foam, so as to avoid adding a large amount of air bubbles during the loading, resulting in errors in the loading.
The number of slats required is determined by the number of samples to be tested plus the number of standards. Multiple holes are recommended for each standard and blank hole. Each sample is determined according to its own quantity, and it is possible to use the double hole as much as possible to make a double hole.
50 ul of the diluted standard was added to the reaction well, and 50 ul of the sample to be tested was added to the reaction well. Immediately add 50 ul of biotinylated antibody. Cover the membrane, mix gently by shaking, and incubate for 1 hour at 37 °C.
Remove the liquid from the well, fill each well with the washing solution, shake for 30 seconds, remove the washing solution, and pat dry with absorbent paper. Repeat this operation 3 times. If washing with a washer, the number of washings is increased once.
80 ul of affinity streptavidin-HRP was added to each well, and the mixture was gently shaken and incubated at 37 ° C for 30 minutes.
Remove the liquid from the well, fill each well with the washing solution, shake for 30 seconds, remove the washing solution, and pat dry with absorbent paper. Repeat this operation 3 times. If washing with a washer, the number of washings is increased once.
Add 50 μl of each of the substrates A and B to each well, mix gently by shaking, and incubate at 37 ° C for 10 minutes. Avoid lighting.
Remove the microplate and quickly add 50 ul of stop solution. Immediately after adding the stop solution, the results should be determined.
The OD value of each well was measured at a wavelength of 450 nm.
Limit
The results above the No. 6 standard are non-linear and no accurate results can be obtained from this standard curve.
Kit performance
1. Sensitivity: The minimum detection concentration is less than the No. 1 standard. The linearity of the dilution of the human antithrombin-III ELISA kit . The linear regression coefficient of the sample and the expected concentration correlation coefficient R value was 0.990.
2. Specificity: Does not react with other cytokines.
3. Repeatability: The coefficient of variation between the plate and the plate is less than 10%.
Result judgment and analysis
1. Instrument value: read the OD value of each hole on a microplate reader with a wavelength of 450 nm.
2. The absorbance OD value is the ordinate (Y), and the corresponding AT-III standard concentration is the abscissa (X). The corresponding curve is obtained. The AT-III content of the sample can be converted from the standard curve according to the OD value. The corresponding concentration.
3. Detection range: 0-40IU/ml
4. Sensitivity: 0.1 IU/ml
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