This reagent is for research use only: This kit is used to determine the content of tumor necrosis factor alpha (TNF-α) in rabbit serum, plasma and related liquid samples.
Experimental principle:
The kit uses a double antibody sandwich assay to determine the level of tumor necrosis factor alpha (TNF-α) in rabbits. The microplate was coated with purified rabbit tumor necrosis factor alpha (TNF-α) antibody to prepare a solid phase antibody, and tumor necrosis factor alpha was sequentially added to the microcapsule of the coated monoclonal antibody, and then HRP-labeled goat anti-rabbit antibody was added. The antibody-antigen-enzyme-labeled antibody complex was formed by binding, and after thorough washing, the substrate TMB was added for color development. TMB is converted to blue under the catalysis of HRP enzyme and converted to the final yellow color by the action of an acid. The color depth is positively correlated with rabbit tumor necrosis factor alpha in the sample. The absorbance (OD value) was measured by a microplate reader at a wavelength of 450 nm, and the concentration of rabbit tumor necrosis factor alpha (TNF-α) in the sample was calculated by a standard curve.
Kit composition:
Kit composition
48-hole configuration
96-well configuration
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Instruction manual
1 serving
1 serving
Sealing film
2 pieces (48)
2 pieces (96)
sealed bag
1
1
Enzyme label coated plate
1×48
1×96
2-8 ° C preservation
Standard: 22.5ng/L
0.5ml × 1 bottle
0.5ml × 1 bottle
2-8 ° C preservation
Standard dilution
1.5ml × 1 bottle
1.5ml × 1 bottle
2-8 ° C preservation
Enzyme standard reagent
3 ml × 1 bottle
6 ml × 1 bottle
2-8 ° C preservation
Sample diluent
3 ml × 1 bottle
6 ml × 1 bottle
2-8 ° C preservation
Developer A solution
3 ml × 1 bottle
6 ml × 1 bottle
2-8 ° C preservation
Developer B solution
3 ml × 1 bottle
6 ml × 1 bottle
2-8 ° C preservation
Stop solution
3ml × 1 bottle
6ml × 1 bottle
2-8 ° C preservation
Concentrated washing solution
(20ml × 20 times) × 1 bottle
(20ml × 30 times) × 1 bottle
2-8 ° C preservation
Sample processing and requirements:
1. Serum: The blood is naturally coagulated at room temperature for 10-20 minutes and centrifuged for about 20 minutes (2000-3000 rpm). The supernatant is carefully collected, and if precipitation occurs during storage, it should be centrifuged again.
2. Plasma: EDTA or sodium citrate should be selected as an anticoagulant according to the requirements of the specimen. After mixing for 10-20 minutes, centrifuge for about 20 minutes (2000-3000 rpm). The supernatant is carefully collected, and if a precipitate forms during storage, it should be centrifuged again.
3. Urine: Collect with a sterile tube and centrifuge for about 20 minutes (2000-3000 rpm). The supernatant is carefully collected, and if a precipitate forms during storage, it should be centrifuged again. The chest and ascites and cerebrospinal fluid are referred to.
4. Cell culture supernatant: When detecting secreted components, collect them in a sterile tube. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. When the components in the cells were detected, the cell suspension was diluted with PBS (pH 7.2-7.4), and the cell concentration reached about 1 million/ml. By repeated freezing and thawing, the cells are destroyed and the intracellular components are released. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, it should be centrifuged again.
5. Tissue specimen: After cutting the specimen, weigh the weight. Add a certain amount of PBS, pH 7.4. It was quickly frozen and stored in liquid nitrogen for use. The specimen still maintains a temperature of 2-8 ° C after melting. A certain amount of PBS (pH 7.4) was added, and the specimen was homogenized by hand or homogenizer. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. One part of the package is to be tested, and the rest is frozen for use.
6. The specimens should be extracted as soon as possible after collection, and the extraction should be carried out according to the relevant literature. The experiment should be carried out as soon as possible after extraction. If the test cannot be performed immediately, the specimen can be stored at -20 °C, but repeated freezing and thawing should be avoided.
7. Samples containing NaN3 cannot be detected because NaN3 inhibits horseradish peroxidase (HRP) activity.
Steps
1. Dilution and loading of standard products: 10 holes of standard wells are placed on the enzyme label coating plate, 100 μl of standard is added to the first and second holes, and then standard is added to the first and second holes. 50 μl of the dilution solution, and mix; then, 100 μl of each of the first hole and the second hole are respectively added to the third hole and the fourth hole, and then 50 μl of the standard dilution solution is added to the third and fourth holes, respectively, and mixed; Then, 50 μl of each of the third hole and the fourth hole are discarded, and 50 μl of each is added to the fifth and sixth holes, respectively, and 50 ul of the standard dilution is added to the fifth and sixth holes, respectively, and mixed; After mixing, 50 μl from each of the fifth and sixth wells was added to the seventh and eighth wells respectively, and then 50 μl of the standard dilution was added to the seventh and eighth wells, and the mixture was mixed from the seventh and the seventh. 50 μl of each of the eight wells was added to the ninth and tenth holes, and 50 μl of the standard dilution was added to the ninth and tenth holes, respectively, and 50 μl of each of the ninth and tenth holes was discarded. (The amount of each well was 50 μl after dilution, and the concentrations were 15 ng/L, 10 ng/L, 5 ng/L, 2.5 ng/L, 1.25 ng/L, respectively).
2. Adding samples: Set blank holes separately (the blank control wells are not added with the sample and the enzyme standard reagent, the other steps are the same), and the sample holes to be tested. Add 40 μl of the sample dilution to the sample well to be tested on the enzyme-labeled plate, and then add 10 μl of the sample to be tested (the final dilution of the sample is 5 times). Add the sample to the bottom of the well of the microplate, try not to touch the wall of the well, and shake gently to mix.
3. Incubation: The plate was sealed with a sealing film and incubated at 37 ° C for 30 minutes.
4. Solution: 30 (48 times of 20T) concentrated washing solution was diluted with distilled water 30 (20 times of 48T) and used.
5. Washing: Carefully remove the sealing film, discard the liquid, dry it, fill each well with the washing solution, let stand for 30 seconds, then discard it, repeat 5 times, and pat dry.
6. Add enzyme: Add 50 μl of enzyme labeling reagent to each well, except for blank wells.
7. Incubation: The operation is the same as 3.
8. Washing: The operation is the same as 5.
9. Color development: Add 50 μl of the developer to each well, then add 50 μl of the developer B, gently shake and mix, and develop at 37 ° C for 15 minutes.
10. Termination: 50 μl of stop solution was added to each well to stop the reaction (the blue color turned yellow).
11. Measurement: The absorbance (OD value) of each well was measured sequentially with a blank air conditioner of zero and a wavelength of 450 nm. The measurement should be carried out within 15 minutes after the addition of the stop solution.
Precautions:
1. The kit should be taken out from the refrigerated environment and allowed to equilibrate for 15-30 minutes at room temperature. If the enzyme label is unsealed after opening, the slats should be stored in a sealed bag.
2. Concentrated washing liquid may crystallize. When diluted, it can be heated and dissolved in the water bath. The washing will not affect the result.
3. The sampler should be used for each step, and the accuracy should be corrected frequently to avoid test errors. It is best to control the loading time within 5 minutes. If the number of specimens is large, it is recommended to use a gun.
4. Please make a standard curve at the same time of each measurement, it is best to make a double hole. If the content of the substance to be tested in the specimen is too high (the OD value of the sample is larger than the OD value of the first hole of the standard well), please first dilute the sample dilution with a certain multiple (n times) and then measure it. When calculating, multiply the total dilution by the total dilution. Multiple (×n×5).
5. The sealing film is for one-time use only to avoid cross-contamination.
6. Keep the substrate away from light.
7. Strictly follow the instructions in the manual. The test results must be based on the reading of the microplate reader.
8. All samples, washings and various wastes should be treated as infectious materials.
9. The different batch components of this reagent must not be mixed.
10. In the case of an English manual, the English manual shall prevail.
The concentration of the standard is the abscissa and the OD is the ordinate.
Draw a standard curve on the coordinate paper, depending on the OD of the sample
The value is determined from the standard curve by the corresponding concentration; multiplied by the dilution
Multiples; or use the concentration of the standard and the OD value to calculate the standard
The linear regression equation of the quasi-curve, the OD value of the sample
Substituting the equation, calculating the sample concentration, multiplying by dilution
The multiple is the actual concentration of the sample.
(This picture is for reference only)
Kit performance:
1. The linear regression coefficient of the sample and the expected concentration correlation coefficient R value is 0.92 or more.
2. Within and within the batch should be less than 9% and 15% respectively
examination range:
1 ng/L -20 ng/L
Storage conditions and expiration date:
1. Kit storage: 2-8 °C.
2. Validity period: 6 months
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