Rat matrix metalloproteinase-2 (MMP-2) enzyme-linked immunoassay (ELISA)
About Our Wardrobe
A wardrobe is a piece of furniture used for storing clothes and other personal items. It typically consists of a large cabinet with doors or drawers, providing ample space for hanging garments, folded clothes, shoes, accessories, and even linens. Wardrobes come in various sizes, styles, and materials, allowing individuals to choose one that suits their needs and complements their interior decor. They are a practical and essential storage solution for organizing and preserving clothing, making it easily accessible and protecting it from dust, sunlight, and other elements. Wardrobes can be found in bedrooms, dressing rooms, or even in common areas of a home or apartment.
Home Furniture Wardrobe,Single Door Clothing Cabinet,Metal Clothes Cabinet Steel Locker,Bedroom Hanging Clothing Storage Cabinet Henan Toda Technology Co., Ltd. , https://www.httofficefurniture.com
Kit Instructions for Use This reagent is for research use only Purpose: This kit is used to determine rat serum, plasma,
The content of matrix metalloproteinase-2 (MMP-2) in the supernatant and related liquid samples.
Experimental principle:
This kit uses the double antibody sandwich method to determine the level of rat matrix metalloproteinase 2 (MMP-2) in the specimen. Microporous plates were coated with pure rat matrix metalloproteinase 2 (MMP-2) antibody to make solid-phase antibodies. Matrix metalloproteinase 2 (MMP-2) was added to the microwells coated with monoclonal antibody and then labeled with HRP MMP-2 antibodies bind to form antibodies-
Enzyme-labeled antibody complexes were washed thoroughly and added with substrate TMB for color development. TMB is catalyzed by HRP enzyme and converted into the final yellow color under the action of acid. Color depth and matrix metalloproteinase 2 (MMP
Positive correlation. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the concentration of human matrix metalloproteinase 2 (MMP-2) in the sample was calculated by a standard curve.
Kit composition:
Kit composition 48-well configuration 96-well configuration warranty manual 1 part 1 part sealing film 2 pieces (48) 2 pieces (96)
One sealed bag and one enzyme label coated plate 1 × 48 1 × 96 2-8 ℃ standard product: 450μg / L 0.5ml × 1 bottle 0.5ml × 1 bottle 2-8 ℃ standard product dilution 1.5ml × 1 bottle of 1.5ml × 1 bottle of 2-8 ℃ retention enzyme label reagent 3 ml × 1 bottle of 6 ml × 1 bottle of sample dilution at 2-8 ℃ 3 ml × 1 bottle of 6 ml × 1 bottle of color preservation at 2-8 ℃ Reagent A solution 3 ml × 1 bottle 6 ml × 1 bottle 2-8 ℃ color retention agent B solution 3 ml × 1 bottle 6 ml × 1 bottle 2-8 ℃ retention solution 3ml × 1 bottle 6ml × 1 bottle 2- 8 ℃ preservation concentrated washing solution (20ml × 20 times) × 1 bottle (20ml × 30 times) × 1 bottle 2-8 ℃ preservation sample processing and requirements:
1. Serum: room temperature blood coagulates naturally for 10-20 minutes, centrifuged for about 20 minutes (2000-3000 rpm). Carefully clean up, if there is a precipitate during storage, it should be centrifuged again.
2. Plasma: EDTA or sodium citrate should be selected as the anticoagulant according to the requirements of the specimen. After mixing for 10-20 minutes,
About 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, it should be centrifuged.
3. Urine: collected in sterile tubes and centrifuged for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, centrifuge 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 (2000-30
Minute). Collect the supernatant carefully. When detecting the 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 20
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 save with liquid nitrogen for later use. After the specimen melts, it still maintains a temperature of 2-8 ° C. Add a certain amount of PBS (PH7.4) and homogenize the specimen with a manual or homogenizer. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. After aliquoting, a portion is to be tested, and the rest is frozen for future 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 the test cannot be performed immediately, 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 loading of standard products: set 10 standard wells on the enzyme-coated plate, add 100 μl of the standard products in the first and second wells, and then add the standard products in the first and second wells. 50μl of diluent, mix well; then add 100μl from the first well and the second well respectively to the third well and the fourth well, then add 50μl of the standard dilution solution to the third well
Mix well; then take 50μl each in the third and fourth wells and discard them, then add 50μl in the fifth and sixth wells respectively, and then add 50ul of standard dilution in the fifth and sixth wells Mix; after mixing, take 50μl from the fifth and sixth wells and add them to the seventh and eighth wells respectively. Then add 50μl of the standard dilution solution to the seventh and eighth wells respectively. 7. Take 50μl from the eighth and eighth wells respectively and add it to the ninth and tenth wells. Then add 50μl of the standard dilution solution to the ninth and tenth wells respectively. After mixing, take 50μl from the ninth and tenth wells and discard. (After dilution, the volume of each well is 50μl,
The concentrations were 300μg / L, 200μg / L, 100μg / L, 50μg / L, 25μg / L).
2. Add samples: set up blank wells (the blank control wells do not add samples and enzyme reagents, the rest of the steps are the same) and the sample wells to be tested. Add 40μl of sample diluent to the test sample well of the enzyme-coated plate, and then add 10μl of the test sample (the final dilution of the sample 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.
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 the washing solution, let it stand for 30 seconds and discard, 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. The measurement should be carried out within 15 minutes after adding the stop solution.
Precautions:
1. The kit should be equilibrated at room temperature for 15-30 minutes before being taken out of the refrigerated environment. If the enzyme label coated plate is unopened, the strip should be stored in a sealed bag.
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. It is best to control the sampling time within 5 minutes. If there are many specimens, 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 substance to be tested in the specimen is too high (the OD value of the sample is greater than the OD value of the first well of the standard well), please dilute it with a certain multiple (n times) of the sample diluent and then determine it. Multiple (× 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.
(This picture is for reference only)
Kit performance:
1. The correlation coefficient R between the linear regression of the sample and the expected concentration is above 0.95.
2. The batch and approval shall be less than 9% and 11% respectively
examination range:
20μg / L-400μg / L
Storage conditions and validity period:
1. Store the kit: 2-8 ℃.
2. Validity: 6 months