How long does it take to terminate the reaction after the addition of the substrate in the ELISA test?
We know that various enzymes have specificity for the substrate, so using different kinds of enzymes requires different substrates. In the ELISA kit experiment, how long does it take to terminate the reaction after adding the substrate? According to the analysis of the ELISA technical commissioner of Shanghai Hengyuan Biological Co., the following two methods can generally be adopted:
First, arbitrarily determine a time, such as 20 minutes or 30 minutes to terminate the reaction, and determine the OD value of the tested specimen and the positive reference specimen.
Second, after preparing a batch of enzyme conjugate, the pre-test time, when the reaction temperature is fixed, when the positive reference serum OD value reaches 1.0, the reaction is terminated, and the time is recorded, for example, 30 minutes, and then the enzyme conjugate is determined by the batch. When the sample is inspected, the reaction is terminated at the same time. This method does not require calibration and is convenient.
When using an automatic analyzer abroad, the OD value of the positive reference serum on each test plate is measured at any time after adding the substrate. When it reaches 1.0, the test sample on the whole plate is immediately terminated and the result is compared. Consistent. There are also automatic enzyme labeling instruments in China, but because the solid phase carrier is not uniform enough, it is difficult to directly measure with a plate.
The concentration of the enzyme conjugate added in the ELISA, the incubation time after addition and the incubation time after the addition of the substrate. These three factors are also mutually influential. Generally speaking, if the concentration of the enzyme conjugate is low, the incubation time is required to be longer, and the concentration of the enzyme conjugate is higher, the incubation time is required to be shorter, and different factors can be changed according to the specific conditions of the experiment to obtain good results.
Due to the large number of variability factors in ELISA, a series of laboratory pre-tests, groping, or experimental studies must be performed for the diagnosis of a disease. In the case where the enzyme conjugate has been determined, the following five conditions are generally required to be predicted:
1. antigen coating concentration;
2. The serum dilution measured, that is, the measured dose response curve;
3. The time of action of the first antibody;
4. The time of action of the enzyme conjugate;
5. Substrate action time.
Once the test conditions are ascertained, the conditions must be fixed and not changed at any time during the formal measurement to maintain sufficient stability of the test system.
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