user's Guide
This reagent is for research use only Purpose: This kit is used to determine bovine serum, plasma and related
The content of FMD-A-Ab in liquid samples.
Experimental principle:
This kit uses the double antigen sandwich method to determine the level of FMD-A-Ab in specimens. With purification
The antigen is coated on the microwell plate to make a solid phase antigen, and then the foot and mouth disease type A antibody is added to the microwell coated with the monoclonal antibody in sequence.
(FMD-A-Ab), then combined with HRP-labeled antigen to form an antigen-antibody-enzyme-labeled antigen complex, after thorough washing
After polyester washing, add substrate TMB to develop color. TMB is converted into blue under the catalysis of HRP enzyme, and into the final under the action of acid
Yellow. The color depth is positively correlated with the FMD type A antibody (FMD-A-Ab) in the sample. Use a microplate reader at 450nm
Measure the absorbance (OD value) at the wavelength and calculate the FMD-A antibody (FMD-A-Ab) in the sample through the standard curve
concentration.
Kit composition:
Sample processing and requirements:
1. Serum: room temperature blood coagulates naturally for 10-20 minutes, centrifuged for about 20 minutes (2000-3000 rpm). Carefully collected
If there is precipitation during storage, centrifuge again.
2. Plasma: EDTA or sodium citrate should be selected as anticoagulant according to the requirements of the specimen, mixed for 10-20 minutes, and centrifuged
About 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, it should be
Centrifuge.
3. Urine: collected in sterile tubes and centrifuged for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully and save the process
If a precipitate forms, it should be centrifuged again. Pleural and ascites, cerebrospinal fluid reference implementation.
4. Cell culture supernatant: When detecting secreted components, collect with a sterile tube. Centrifuge for about 20 minutes (2000-3000 revolutions /
Minute). Collect the supernatant carefully. When detecting intracellular components, dilute the cell suspension with PBS (PH7.2-7.4).
The concentration reaches about 1 million / ml. Through repeated freezing and thawing, the cells are destroyed and the intracellular components are released. Centrifuge for 20 minutes
Around Zhong (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, it should be centrifuged again.
5. Organize the specimen: after cutting the specimen, weigh it. Add a certain amount of PBS, PH7.4. Quickly freeze and prepare with liquid nitrogen
use. After the specimen melts, it still maintains a temperature of 2-8 ° C. Add a certain amount of PBS (PH7.4), use hand or homogenizer
Thoroughly homogenize the specimen. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. Wait until after packing
Test, the rest is frozen for use.
6. The specimen should be extracted as soon as possible after collection. The extraction should be carried out according to relevant literature. The experiment should be carried out as soon as possible after extraction. If not immediately
For testing, the specimen can be stored at -20 ℃, but repeated freezing and thawing should be avoided.
7. The sample containing NaN3 cannot be detected because NaN3 inhibits the activity of horseradish peroxidase (HRP).
Steps
1. Dilution and sample addition of standard products: 10 standard wells are provided on the enzyme-coated plate, and the standard is added in the first and second wells respectively
100μl of quasi-standard, then add 50μl of standard diluent to the first and second wells, mix well; then from the first well and second
Take 100μl of each well and add them to the third and fourth wells respectively, then add 50μl of standard dilution solution to the third and fourth wells.
Mix well; then take 50μl each in the third and fourth wells and discard, then add 50μl each to the fifth and sixth wells
In the middle, add 50ul of the standard dilution solution to the fifth and sixth wells respectively, mix well;
Add 50μl to the seventh and eighth wells respectively, then add 50μl of the standard dilution solution to the seventh and eighth wells respectively, mix
After smoothing, take 50μl from the seventh and eighth holes respectively and add them to the ninth and tenth holes, then add the standards to the ninth and tenth holes respectively
50μl of the product dilution, after mixing, take 50μl from the ninth and tenth wells and discard. (After dilution, the volume of each well is 50μl,
The concentrations were 90 ng / L, 60 ng / L, 30 ng / L, 15 ng / L, 7.5 ng / L).
2. Add sample: set blank wells separately (the blank control wells do not add samples and enzyme reagents, the rest of the steps are the same), the sample to be tested
Pinhole. Add 40μl of sample diluent to the test sample well of the enzyme-coated plate, and then add 10μl of the test sample (sample
The final dilution of the product is 5 times). Add the sample and add the sample to the bottom of the well of the microplate, try not to touch the wall of the well, shake gently to mix
uniform.
3. Incubation: seal the plate with the sealing film and incubate at 37 ° C for 30 minutes.
4. Mixing solution: Dilute 30 times (20 times of 48T) concentrated washing liquid with distilled water 30 times (20 times of 48T) and then use.
5. Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing liquid, let stand for 30 seconds and then discard, so
Repeat 5 times and pat dry.
6. Add enzyme: add 50μl of enzyme label reagent to each well, except blank well.
7. Incubation: The operation is the same as 3.
8. Washing: The operation is the same as 5.
9. Color development: add 50μl of developer A to each well, and then add 50μl of developer B, mix gently, and develop color at 37 ℃ in the dark
15 minutes.
10. Termination: Add 50μl of stop solution to each well to stop the reaction (at this time the blue will turn to yellow).
11. Determination: Measure the absorbance (OD value) of each well in sequence with the blank air conditioner at zero and 450 nm wavelength. Determination should be terminated
Within 15 minutes after the solution.
Precautions:
1. The kit should be equilibrated at room temperature for 15-30 minutes before it is taken out from the refrigerated environment.
After use, the slats should be stored in sealed bags.
2. Crystals may be precipitated in the concentrated washing liquid, which can be heated and dissolved in a water bath during dilution, and the results will not be affected during washing.
3. The sampler should be used at each step of sample addition, and the accuracy should be regularly checked to avoid test errors. One sample loading time is best
Control within 5 minutes, if the number of specimens is large, it is recommended to use a volley gun to add samples.
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 test substance in the specimen is too high (sample OD value
Greater than the OD value of the first well of the standard product), please dilute it with a certain multiple (n times) of the sample diluent before measuring.
When calculating, please multiply the total dilution factor (× n × 5).
5. The sealing film is limited to one-time use to avoid cross-contamination.
6. Please keep the substrate away from light.
7. Strictly follow the instructions, and the test results must be determined by the microplate reader.
8. All samples, washing liquids and various wastes should be treated as infectious agents.
9. The components of different batches of this reagent shall not be mixed.
10. If there is any difference with the English manual, the English manual shall prevail.
Calculation:
Taking the concentration of the standard as the abscissa and the OD value as the ordinate,
Draw a standard curve on coordinate paper, according to the OD of the sample
The value is determined by the standard curve; then multiplied by the dilution
Multiple; or calculate the standard using the concentration and OD value of the standard
The linear regression equation of the quasi-curve, the OD value of the sample
Substitute into the equation, calculate the sample concentration, and multiply by the dilution
The multiple is the actual concentration of the sample.
Kit performance:
1. The correlation coefficient R between the linear regression of the sample and the expected concentration is more than 0.990.
2. The batch and approval shall be less than 9% and 11% respectively
examination range:
5ng / L -120 ng / L
Storage conditions and validity period:
1. Store the kit: 2-8 ℃.
2. Validity: 6 months
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