This kit is for research use only
Specificity: This kit can detect natural or recombinant rat CS3B3 at the same time, and has no cross-reactivity with other related proteins.
Validity: 6 months Expected application: ELISA method for quantitative determination of CS3B3 in rat serum, plasma, cell culture supernatant or other related biological fluids.
Explanation
1. Kit storage: -20 ℃ (when not in use for a long time); 2-8 ℃ (when used frequently).
2. The concentrated washing liquid will be salted out at low temperature, and it can be heated and dissolved in the water bath when diluted.
3. There may be inconsistencies between the Chinese and English instructions. Please refer to the English instructions.
4. The well of the ELISA plate just opened may contain a little water-like substance. This is normal and will not have any impact on the experimental results.
Experimental principle The purified CS3B3 protein antigen is used to coat the microwell plate to make a solid phase carrier. The samples to be tested are added to the microwells, and then the horseradish peroxidase-labeled anti-human antibody is added for reaction, after thorough washing After adding substrate TMB color. TMB is converted into blue under the catalysis of peroxidase, and into the final yellow under the action of acid. The color depth is positively correlated with CS3B3 in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the titer of the antibody in the sample was calculated (the highest dilution factor that the antiserum can eventually develop as the titer).
Kit composition and reagent preparation
1. Assay plate: one piece (96 wells).
2. Sample Diluent: 2 × 20ml / bottle.
3. Horseradish peroxidase labeled secondary antibody (HRP-anti-antibody): 1 × 120μl / bottle (1: 100)
4. Substrate solution (TMB Substrate): 1 × 10ml / bottle.
5. Wash Buffer: 1 × 20ml / bottle, each bottle is diluted 25 times with distilled water.
6. Stop solution (Stop Solution): 1 × 10ml / bottle (2N H2SO4).
Reagents and equipment needed but not provided
1. Standard Specification Microplate Reader
2. High-speed centrifuge
3. Electric heating thermostat incubator
4. Clean test tubes and Eppendof tubes
5. Series adjustable pipettes and tips. When testing more samples at one time, it is best to use multi-channel pipettes
6. Collection and preservation of specimens such as distilled water and volumetric flasks
1. Serum: Whole blood samples should be placed at 37 ° C for 1 hour or 4 ° C overnight and then centrifuged at 1000 xg for 20 minutes. The supernatant can be taken for detection, or the specimen can be stored at -20 ° C or -80 ° C Avoid repeated freezing and thawing.
2. Plasma: EDTA or heparin can be used as anticoagulant. Centrifuge the sample at 2-8 ° C 1000 xg for 15 minutes within 30 minutes after collection, or store the specimen at -20 ° C or -80 ° C, but avoid repeated freezing melt.
3. Cell culture supernatant or other biological specimens: centrifuge at 1000 xg for 20 minutes, take the supernatant for detection, or store the specimen at -20 ℃ or -80 ℃, but avoid repeated freezing and thawing.
Note: Hemolysis of specimens will affect the final test results, so hemolysis specimens should not be tested.
Dilution principle of specimens:
First of all, we should know the approximate content of the sample to be tested through literature search, and determine the appropriate dilution factor. Detailed records should be made during the dilution process. When calculating the concentration at the end, it was diluted "N" times, and the concentration of the specimen should be multiplied by "N".
Dilution principle of horseradish peroxidase labeled secondary antibody:
Before use, dilute with the sample diluent. Before dilution, prepare according to the pre-calculated total amount required for each experiment (100μl per well). In actual preparation, 0.1-0.2ml should be prepared. For example, 10μl horseradish peroxidase labeled secondary antibody plus 990μl sample diluent is prepared, mixed gently, and prepared within one hour before use.
Before starting the experiment, please configure all reagents in advance. When diluting the reagents or samples, they should be mixed evenly. Try to avoid foaming when mixing. If the sample concentration is too high, dilute with sample diluent.
1. Add sample: set blank hole and sample hole to be tested respectively. Add 100μl of sample diluent to the blank well and 100μl of the sample to be tested in the remaining well. Be careful not to have air bubbles. Add the sample to the bottom of the well of the microtiter plate. Do not touch the wall of the well as much as possible. Cover or cover the membrane and react at 37 ° C for 60 minutes.
2. Discard the liquid, spin dry, wash the plate 3 times, soak for 1-2 minutes each time, 200μl / well. Add 100 μl of horseradish peroxidase-labeled secondary antibody working solution to each well (prepared at a ratio of 1 μl HRP-labeled antibody plus 99 μl sample dilution, and mix gently).
3. After incubating for 60 minutes, discard the liquid in the well, spin dry, wash the plate 5 times, soak for 1-2 minutes each time, 200μl / per well, spin dry.
4. Add 90 μl of substrate solution to each well in sequence, and develop color at 37 ° C in the dark (within 30 minutes, when the visible wells are clearly blue in the positive wells, it can be terminated).
5. Add 50μl of stop solution to each well in sequence to stop the reaction (in this case, the blue color turns to yellow). The order of adding the stop solution should be the same as that of the substrate solution. In order to ensure the accuracy of the experimental results, the termination solution should be added as soon as possible after the substrate reaction time expires.
6. Measure the optical density (OD value) of each well in sequence using an enzyme-linked instrument at a wavelength of 450 nm. Test within 15 minutes after adding stop solution.
Note:
1. When using the reagent kit for the first time, the user should centrifuge various reagent tubes for several minutes so that the reagents are concentrated to the bottom of the tube.
2. Leave one well for each experiment as a blank zero-adjusting well. No reagents are added to this well, only the substrate solution and 2N H2SO4 are added at the end. Use this hole to adjust the OD value to zero when measuring.
3. To prevent the sample from evaporating, place the reaction plate in a closed box covered with a damp cloth during the test, and add a cover or film to the enzyme label plate.
4. Store unused microplates or reagents at 2-8 ° C. The working solution of horseradish peroxidase labeled secondary antibody should be used according to the required amount. Do not reuse the diluted horseradish peroxidase labeled secondary antibody working solution.
5. It is recommended to set double-hole measurement when testing samples to ensure the accuracy of the test results.
Plate washing method Manual plate washing method: suck (not touch the wall) or shake off the liquid in the microplate; put a few layers of absorbent paper on the experimental table, and tap the microplate down several times with force; the recommended wash buffer Inject at least 0.3ml of solution into the hole and soak for 1-2 minutes. Repeat this process several times as needed.
Automatic plate washing: If there is an automatic plate washing machine, it should be used in the formal experiment process after being used skillfully.
Calculated to detect the antibody titer of MBP protein in the sample, the customer can take the sample 2 times the dilution of the sample to calculate the antibody titer, generally take 1:40 as the starting dilution factor (use the sample diluent for dilution). The maximum dilution factor is determined by the customer's own test requirements. The final color development is compared with the negative control well. The maximum dilution with obvious difference is determined as the final titer of the antibody.
Precautions
1. When mixing protein solutions, try to be as gentle as possible to avoid foaming.
2. The washing process is very important. Insufficient washing can easily cause false positives.
3. 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. If the content of the substance to be tested in the specimen is too high, please dilute it and then determine it. When calculating, please multiply by the dilution factor.
5. Please keep the substrate away from light.
6. Do not replace the reagents in the kit with reagents from other manufacturers.
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