Since its establishment in the early 1970s, enzyme immunoassay has been widely used in the field of biomedical research and the diagnosis and treatment of clinical diseases due to its specific, sensitive, simple and rapid characteristics. Compared with radioimmunoassay ( In terms of RIA) technology, EIA also has the advantages of long reagent half-life, low environmental pollution, and easy handling of test waste. However, the detection sensitivity is generally lower than that of RIA. Amplifying the system makes the sensitivity of EIA to catch up with or even exceed that of RIA.
General principles for establishing positive IA measurement amplification system
EIA itself uses enzymes [horseradish peroxidase (HRP), alkaline phosphatase (AP), etc.] as markers. Therefore, the measurement amplification of EIA is always centered on the labeling enzymes. Three possible ways: â‘ increase the amount of labeled enzyme; â‘¡ the application of non-chromogenic substrate; â‘¢ attach a reaction system catalyzed by the labeled enzyme.
1. Increase the amount of labeled enzyme
Through the indirect method of biotin / avidin-enzyme or enzyme / anti-enzyme complex, the amount of labeled enzyme finally determined in the ELISA measurement can be increased. Although this method increases the sensitivity of EIA measurement due to the aggregation of labeling enzymes to a certain extent, it may also increase the non-specific background color development.
2. Application of non-color-developing substrates
Starting from the reaction substrate of the enzyme, a non-chromogenic substrate with high measurement sensitivity such as fluorescein or luminescent substance is used to improve the measurement sensitivity of EIA. The advantage of this method is that it does not require additional steps and reagent preparation. The disadvantage is the need for special measuring instruments.
3. Attach a reaction system catalyzed by the labeled enzyme
The original labeled enzyme catalyzes an additional reaction system, such as an enzyme substrate reaction cycle or an enzyme cascade reaction, so as to achieve the purpose of reaction amplification. This amplification system constructed by the coupling of several catalytic reactions is the root of enzyme amplification. The increase in sensitivity comes from true signal amplification, rather than simply converting a molecule into more easy to measure. molecule. In addition to considering biochemical factors, the most important selection of an appropriate enzyme amplification system is the stability of the enzyme and its substrate, and it must be easy to obtain because it has a greater impact on the accuracy and repeatability of the test.
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