Canine Luteinizing Hormone (LH) ELISA Kit is for research use only. This kit allows simultaneous detection of natural or recombinant canine lutein (LH) without cross-reactivity with other related proteins.
Validity: 6 months Expected application: ELISA method for quantitative determination of luteinizing hormone (LH) content in canine serum, plasma, cell culture supernatant or other related biological fluids.
Description
1. The kit is stored at -20 ° C (when not used for a long time); 2-8 ° C (when used frequently).
2. When the concentrated washing solution is stored at a low temperature, salt will be precipitated, and when diluted, it can be heated and dissolved in a water bath.
3. Chinese and English manuals may be inconsistent. Please refer to the English manual.
4. The newly opened enzyme-linked plate well may contain a little water-like substance, which is normal and will not have any effect on the experimental results.
Experimental principle
The microplate is coated with the purified antibody to prepare a solid phase carrier, and the sample or standard, the biotinylated anti-IL-9 antibody, and the HRP-labeled affinity are sequentially added to the microwell coated with the anti-IL-9 antibody. Su, after washing through thoroughly with the chromogenic substrate TMB. TMB is converted to blue under the catalysis of peroxidase and converted to the final yellow color by the action of an acid. The depth and color samples luteinizing hormone (LH) were positively correlated. The absorbance (OD value) was measured at a wavelength of 450 nm using a microplate reader to calculate the sample concentration.
Canine lutein (LH) ELISA kit composition and reagent preparation
1. Assay plate: one piece (96 holes).
2. Standard: 2 bottles (lyophilized product).
3. Sample Diluent: 1 x 20 ml / bottle.
4. Biotin-antibody Diluent: 1 x 10 ml/vial.
5. Horseradish peroxidase-labeled avidin dilution (HRP-avidin Diluent): 1 x 10 ml / bottle.
6. Biotin-antibody: 1 × 120 μl / bottle (1:100)
7. Horseradish peroxidase-labeled avidin (HRP-avidin): 1 × 120 μl / bottle (1: 100)
8. Substrate solution (TMB Substrate): 1 x 10 ml / bottle.
9. Wash Buffer: 1 × 20ml / bottle, each bottle is diluted 25 times with distilled water.
10. Stop Solution: 1 x 10 ml / bottle (2N H2SO4).
Collection and preservation of specimens
1. Serum: Whole blood samples should be placed at room temperature for 2 hours or 4 °C overnight, centrifuged at 1000 xg for 20 minutes, the supernatant can be detected, or the specimens should be stored at -20 ° C or -80 ° C, but should be avoided. Freezing and thawing.
2. Plasma: heparin or EDTA as an anticoagulant available, samples were collected after 30 min at 2 - 8 ° C 1000 xg centrifugation for 15 minutes or placed in the sample stored at -80 ℃ or -20 ℃, but should avoid repeated Freezing and thawing.
3. Cell culture supernatant or other biological specimens: centrifuge at 1000 xg for 20 minutes, take the supernatant for testing, or store the specimen at -20 °C or -80 °C, but avoid repeated freezing and thawing.
Note: Specimen hemolysis will affect the final test results, so hemolysis specimens should not be tested.
The dilution principle of the specimen:
First, the approximate content of the sample to be tested is known by means of literature search, and the appropriate dilution factor is determined. The results of the test are accurate only if they are diluted to within the range of the standard curve. Dilution process, should make detailed records. When the concentration is finally calculated, it is diluted "N" times, and the concentration of the sample should be multiplied by "N".
Standard dilution principle: 2 bottles, each bottle is diluted to 1ml with sample dilution before use, and then allowed to stand for more than 10 minutes, then repeatedly reversed/twisted to help dissolve, its concentration is 500 pg/ml, do After dilution, the series dilution was 500 pg/ml, 250 pg/ml, 125 pg/ml, 62.5 pg/ml, 31.2 pg/ml, 15.6 pg/ml, 7.8 pg/ml, and the sample dilution was directly used as the standard concentration. 0 pg/ml, prepared within 15 minutes before use.
The formulation 250 pg / ml standard: Take 0.5ml (not less than 0.5ml) 500 pg / ml was added to the above-described standard Eppendorf tube containing 0.5ml sample diluent, mixing can, the remaining concentration forth.
Dilution principle of biotinylated antibodies:
Dilute with biotin-labeled antibody dilution before use. Prepare according to the total amount of each experiment required before dilution (100μl per well). In actual preparation, 0.1-0.2ml should be prepared. Prepare the ratio of 10 μl biotinylated antibody plus 990 μl biotinylated antibody dilution, mix gently, and prepare within one hour before use.
Dilution principle of horseradish peroxidase-labeled avidin:
Before use diluted horseradish peroxidase labeled avidin-biotin dilutions, prior to dilution in accordance with a precalculated amount of preparation required for each experiment (100 l per well), should be formulated in the actual formulation plurality 0.1- 0.2ml . For example, 10 μl of horseradish peroxidase-labeled avidin plus 990 μl of horseradish peroxidase-labeled avidin dilution was prepared, gently mixed, and prepared within one hour before use.
Steps
Before starting the experiment, please configure all the reagents in advance. When the reagents or samples are diluted, mix them well. Avoid mixing when mixing. A standard curve should be used for each test. If the sample concentration is too high , dilute with the sample diluent to make the sample meet the detection range of the kit.
1. Adding samples: blank holes, standard holes, and sample holes to be tested. Add 100 μl of the sample dilution to the blank well, add 100 μl of the standard or the sample to be tested, and note that there should be no air bubbles. Add the sample to the bottom of the well of the microplate. Try not to touch the wall of the well. Gently shake and mix. The plate was coated or covered with a lid and reacted at 37 ° C for 120 minutes. To ensure the validity of the results, use a new standard solution for each experiment.
2. Discard the liquid, dry it, and do not wash it. Each well of biotin-labeled antibody solution 100 l (ratio of biotinylated antibody to take 1μl Add 99μl of biotin-labeled antibody dilution formulation, mix gently, formulated in one hour before use), 37 ℃, 60 min.
3. After 60 minutes of incubation, discard the liquid in the well, dry and wash the plate 3 times, soak for 1-2 minutes each time, 350 μl/well, and dry.
4. Add per 100 mg of horseradish peroxidase-labeled avidin working solution (with biotinylated antibody working solution) to each well at 37 ° C for 60 minutes.
5. After 60 minutes of incubation, discard the liquid in the well, dry it, wash the plate 5 times, soak for 1-2 minutes each time, 350 μl/well, and dry.
6. Add 90 μl of substrate solution to each well in order, and avoid coloration at 37 ° C. (In 30 minutes, the first 3-4 wells of the standard can be seen by the naked eye. There is a clear gradient blue, and the gradient of the 3-4 holes is not obvious. , you can terminate) .
7. Add 50 μl of the stop solution to each well in sequence to terminate the reaction (in this case, the blue color turns yellow). The order of addition of the stop liquid should be as close as possible to the order in which the substrate liquid is added. To ensure the accuracy of the experimental results, substrate should be added after the reaction time to quickly stop solution.
8. The optical density (OD value) of each well was measured sequentially at 450 nm using an enzyme-linked instrument. The test was carried out within 15 minutes after the addition of the stop solution.
Note:
1. When using the kit for the first time, the user should centrifuge the various reagent tubes for a few minutes to concentrate the reagents on the bottom of the tube.
2. Leave one hole for each experiment as a blanking zero hole. This well is not added with any reagents, only the final substrate solution and 2N H2SO4. Use this hole to adjust the OD value to zero when measuring.
3. To prevent evaporation of the sample, place the reaction plate in a closed box covered with a damp cloth during the test, and add or cover the plate.
4. Unused plate or reagent, please store at 2-8 °C. Standard, biotinylated antibody solution, horseradish peroxidase labeled avidin using configure the working fluid depending on the desired amount. Do not reuse diluted standards, biotinylated antibody working solutions, or horseradish peroxidase-labeled avidin working solutions.
5. It is recommended to set the double hole measurement when testing the sample to ensure the accuracy of the test results.
Washing method
Manual washing method: suck (not touch the wall) or pry off the liquid in the microplate; pour a few layers of absorbent paper on the test bench, and take a few times with the microplate; use the recommended washing buffer at least 0.3 Mol was injected into the well and soaked for 1-2 minutes. Repeat this process several times as needed.
Automatic washing: If there is an automatic washing machine, it should be used in the formal experiment after skilled use.
Calculation
The standard concentration for the abscissa (logarithmic scale), OD value for the vertical (normal coordinates), the standard curve drawn on semi-logarithmic graph paper, the concentration of the corresponding OD values of samples isolated according to the standard curve Multiply by the dilution factor; or calculate the linear regression equation of the standard curve using the concentration of the standard and the OD value, substitute the OD value of the sample into the equation, calculate the sample concentration, and multiply by the dilution factor, which is the actual concentration of the sample. .
Precautions
1. When mixing protein solutions, try to be gentle and avoid foaming.
2. The washing process is very important, and insufficient washing can cause false positives.
3. 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.
5. If the content of the substance to be tested in the specimen is too high, please dilute it before measuring it. When calculating, multiply by the dilution factor.
6. When preparing the standard and test solution working solution, please prepare with the corresponding dilution solution, not to be confused.
7. Keep the substrate away from light.
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