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A. The kit is not stored as specified and is affected by high temperature.
B. The kit and sample are not equilibrated to room temperature before use.
C. The volume and time of adding the reagent are incorrect, the pipette is not metered, and the moisture in the nozzle is too much or not clean.
D. Must be used within the validity period. Products that exceed the expiration date may produce a weak signal.
E. Incubation time and incubation temperature did not meet the requirements. When the reaction plate was placed in the incubator, pay attention to the temperature and adjust it in time.
F. During the process of washing and adding the sample, the enzyme label is deactivated by pollution and loses the ability to catalyze the color development of the color developer.
G. There is a problem with the quality of distilled water.
H, too many times of washing, or the dilution factor of the concentrated washing liquid does not meet the requirements, the washing impact force is too large, and the soaking time is too long.
I. The amount of the developer is insufficient or reversed, or added after mixing.
Problem 2: Interleukin ELISA kit color development anti-response protection from light The interleukin ELISA kit visual specimens are also colorless or near-colorless, and the color is clear. However, in the ELSIA kit, the normal human serum often shows a background of coloration. The depth of the background varies depending on the composition of the ELISA kit and the conditions of the test. Therefore, it is necessary to add a negative control in the test. . The composition of the negative control should be normal serum or the like that does not contain the test substance. When discriminating results with the naked eye, it is better to use a color deeper than the negative control as an indicator of positive specimens. At this point the enzyme catalyzes the colorless substrate to produce a colored product. The temperature and moment of reverberation are still the factors that affect color development. At a certain time, the negative holes can remain colorless, while the positive holes are colored as the time extends. Properly raising the temperature helps speed up color development. In the quantitative measurement, the reaction temperature and time after participating in the substrate should be determined according to the rules. The color of the qualitative determination of the ELISA kit can be performed at room temperature, and the time is usually not strictly controlled. Sometimes, the timing of the positive control hole and the negative control hole can be appropriately shortened or extended, and the time is determined.
The color development of the OPD substrate is usually no longer deepened after 20-30 minutes of outdoor temperature or 37 ° C reaction, and the ELISA kit is extended to reflect the reaction time, which can increase the background value. The OPD substrate liquid will change color by the light, and the stop liquid will stop responding when the color reaction is over. After the OPD product was stopped with sulfuric acid, the color developed from orange to brown.
TMB is not affected by light, and the ELISA kit can be placed on the console at room temperature, and the results of the investigation are reflected. However, in order to ensure the stability of the test results, it is advisable to read the results at the appropriate time of the rules. After TMB was subjected to HRP, the color peaked at about 40 minutes, and then gradually weakened. The ELISA kit was completely degraded to colorless after 2 hours. There are many stop solutions for TMB, and enzyme inhibitors such as sodium azide and sodium dodecyl sulfate (SDS) can stop the reaction. This kind of stopping agent can keep the blue color for a long time (12-24 hours) and it is an excellent stopping agent for visual discrimination. In addition, various types of acid stop solutions turn the blue color into yellow, at which point the absorbance can be read at a specific wavelength (450 nm).
Takes you through the two major problems of the interleukin ELISA kit: Causes of abnormalities in the results of the interleukin ELISA kit