Before the start of the experiment, all reagents should be balanced to room temperature (reagent can not be directly dissolved in 37 ℃); reagent or sample dilution, the required mixing, mixing time to avoid foaming. Content of the sample to be predicted before the experiment, such as the sample concentration is too high, the sample should be diluted in order to meet the diluted sample kit for the detection range, the calculation multiplied by the corresponding dilution.
1. Adding sample: blank holes are located, standard hole, tested the sample hole. Blank wells plus sample diluent 100μl, and the remaining holes were standard or sample was 100μl, careful not have bubbles, add sample to sample additional hole in the bottom of microtiter plates, trying not to touch the wall hole. After that, gently shake mix, enzyme The panels covered or coated with, 37 ℃ 120 minutes reaction. To ensure the validity of experimental results, each experiment uses the new standard solution.
2. Discard the liquid, drying, do not wash. A test solution per well plus working fluid 100μl (one hour in preparation prior to use), microtiter plate coated with, 37 ℃ reaction 60 minutes.
3. After 60 minutes incubation, discard the liquid to the hole, drying, plate washer 3 times, soaked for 1-2 minutes, about 400μl / per hole, drying (which can also tap the hole liquid and pat dry) .
4. Every test solution B plus hole working fluid (working fluid with the test A) 100μl, microtiter plates coated with 60 minutes at 37 ℃.
5. After 60 minutes incubation, discard the liquid to the hole, drying, washer five times, each time soak for 1-2 minutes, 350μl / per hole, drying (which can also tap the hole liquid and pat dry).
6. Order of substrate solution per well plus 90μl, microtiter plate coated with dark color 37 ℃ (30 minutes, this time visible standard was the first 3-4 Kongyou Ming blue gradient, then gradient 3-4 hole was not obvious, you can terminate).
7. Order of termination of the solution per well plus 50μl, terminate the reaction, then turn yellow and blue stand.Termination order of adding liquid substrate solution should be to join with the same order. In order to ensure the accuracy of results, the substrate reaction time to the solution should be terminated as soon as possible.
8. With enzyme-linked instrument in the order of 450nm wavelength measurement of optical density of each hole (OD). In Canada immediately after the termination of fluid for testing.
1. Adding sample: blank holes are located, standard hole, tested the sample hole. Blank wells plus sample diluent 100μl, and the remaining holes were standard or sample was 100μl, careful not have bubbles, add sample to sample additional hole in the bottom of microtiter plates, trying not to touch the wall hole. After that, gently shake mix, enzyme The panels covered or coated with, 37 ℃ 120 minutes reaction. To ensure the validity of experimental results, each experiment uses the new standard solution.
2. Discard the liquid, drying, do not wash. A test solution per well plus working fluid 100μl (one hour in preparation prior to use), microtiter plate coated with, 37 ℃ reaction 60 minutes.
3. After 60 minutes incubation, discard the liquid to the hole, drying, plate washer 3 times, soaked for 1-2 minutes, about 400μl / per hole, drying (which can also tap the hole liquid and pat dry) .
4. Every test solution B plus hole working fluid (working fluid with the test A) 100μl, microtiter plates coated with 60 minutes at 37 ℃.
5. After 60 minutes incubation, discard the liquid to the hole, drying, washer five times, each time soak for 1-2 minutes, 350μl / per hole, drying (which can also tap the hole liquid and pat dry).
6. Order of substrate solution per well plus 90μl, microtiter plate coated with dark color 37 ℃ (30 minutes, this time visible standard was the first 3-4 Kongyou Ming blue gradient, then gradient 3-4 hole was not obvious, you can terminate).
7. Order of termination of the solution per well plus 50μl, terminate the reaction, then turn yellow and blue stand.Termination order of adding liquid substrate solution should be to join with the same order. In order to ensure the accuracy of results, the substrate reaction time to the solution should be terminated as soon as possible.
8. With enzyme-linked instrument in the order of 450nm wavelength measurement of optical density of each hole (OD). In Canada immediately after the termination of fluid for testing.
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