This kit is for research use only
Specificity: This kit can detect human anti-tox IgG, and does not cross-react with other related antibodies.
Validity: 6 months Expected application: ELISA method for quantitative determination of anti-tox IgG content in human serum, plasma 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.
Kit composition and reagent preparation
1. Assay plate: one piece (96 wells).
2. Conjugate: 2 × 6 ml / bottle.
3. Sample Diluent: 2 × 6 ml / bottle.
4. Positive Control: 1 × 0.5ml / bottle.
5. Negative Control: 1 × 0.5ml / bottle.
6. Substrate A: 1 × 7 ml / vial.
7. Substrate B: 1 × 7 ml / vial.
8. Wash Buffer: 1 × 15ml / bottle, each bottle is diluted 20 times with distilled water.
9. Stop Solution (Stop Solution): 1 × 7ml / bottle.
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 left at room temperature for 2 hours or overnight at room temperature and centrifuged at 1000 xg for 20 minutes. Take the supernatant for detection, or store the samples at -20 ℃ or -80 ℃, but avoid repeated freezing melt.
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.
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 to make the sample meet the detection range of the kit.
1. Add samples: set blank wells without any solution; set 2 negative control wells and add 100 μl of negative control; set 2 positive control wells and add 100 μl of positive control; add 100 μl of sample dilution in the remaining wells, and then add directly 5μl of sample to be tested. Note that there are no bubbles, 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, gently shake and mix well, add the cap or film to the microplate, and place it at 37 ℃ for 30 minutes.
2. Discard the liquid in the well, spin dry, wash the plate 5 times, soak for 1-2 minutes each time, 200μl / well, spin dry.
3. Add 100μl of enzyme conjugate to each well (except the blank well), mix well, add the enzyme label to the cover or cover the film, and place at 37 ℃ for 20 minutes.
4. Discard the liquid in the well, spin dry, wash the plate 5 times, soak for 1-2 minutes each time, 200μl / well, spin dry.
5. Add 50μl of substrate solution A and B to each well in sequence, and develop at 37 ° C in the dark for 10 minutes.
6. 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.
7. Measure the optical density (OD value) of each well in sequence at 450nm wavelength using an enzyme-linked instrument. 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.
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 (do not touch the wall) or shake off the liquid in the microplate; place a few layers of absorbent paper on the experimental table, and force the microplate down several times; pat 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.
Calculation
1. Visual method: samples with darker color than the critical control are positive, and those with the same or lighter color than the critical control are negative.
2. Determination by microplate reader: judgment value (OD value) = average OD value of negative control × 2.1, sample OD value is greater than the judgment value is positive, less than or equal to negative. When the average OD value of the negative control is less than 0.05, it is calculated as 0.05.
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. When preparing the working solution of the test solution, please prepare it with the corresponding diluent, not to be confused.
6. Please keep the substrate away from light.
7. Do not replace the reagents in the kit with reagents from other manufacturers.
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