The lab needs to do the elisa experiment. What problems do you need to pay special attention to when doing it? For example, sample dilution, sample addition, incubation and so on. Problems that should be noted in the elisa experiment, including sample dilution, kit balance, sample and reagent mixing, sample loading, incubation, etc., I hope to be useful to you. Elisa kit has many advantages such as high accuracy, high sensitivity and strong specificity, which is very popular among users. However, there are many issues to be aware of during the experiment. In this regard, Wuhan Fulai Bioengineering reminds you that you need to follow some rules in order to conduct experiments better and achieve better experimental results. The Elisa method is widely used in various antigen and antibody assays. There are many influencing factors in the Elisa assay. In addition to the normal reaction, some wrong results are sometimes encountered. The main causes of ELISA error results are: specimen factors; reagent factors; operational factors. 1. Sample dilution 2. Kit balance 3. Mixing samples and reagents 4. Loading 5. Incubation 6. Washing board 7. Color development 8. Colorimetric
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The dilution factor of the sample of the general product is determined by a large number of tests to ensure the sensitivity and specificity of the test. The enzyme-linked immunoreactivity is a highly sensitive reaction. If the serum is not diluted, a strong non-specific reaction will inevitably occur and a false positive will occur.
All reagents and slats in Elisa should be equilibrated to room temperature (about 25 ° C) before the test, generally at room temperature for 20 to 30 minutes. Too short a balance time will result in insufficient reagent mixing, a relatively short incubation time, and insufficient ELISA. The room temperature is low in winter, and the kit can be placed in a 37 ° C incubator for 20 minutes.
Samples must be thoroughly mixed before and after dilution, and all reagents should be shaken before loading to ensure uniformity of the test.
Loading is a very important step. Do not add too fast, avoid adding it to the upper part of the hole wall, and do not spill and create bubbles. The loading is too fast and the accuracy and uniformity of the micro-dosing cannot be guaranteed. The non-coated area applied to the upper part of the pore wall is liable to cause non-specific adsorption.
Incubation is one of the most critical factors influencing the success or failure of an assay in an ELISA assay. ELISA as a solid phase immunoassay, the antigen-antibody binding reaction is carried out on a solid phase. In order to completely bind the antigen or antibody in the liquid phase to a specific antibody or antigen on the solid phase, it must react under certain temperature conditions. time.
Solid phase immunoassay is a heterogeneous immunoassay technique that separates the antigen or antibody that specifically binds to the solid phase from the non-specific components adsorbed during the incubation to ensure the specificity of the ELISA assay. Sex. Do not spill the washing liquid as much as possible; after standing for the washing liquid, let it stand for 1 minute. After washing the liquid in the plate hole, be sure to shoot it vigorously; replace the absorbent paper in time, especially the absorbent paper that has taken the enzyme label. Discard, otherwise it may affect the test results.
Color control must be controlled, according to the kit instructions. In general, the color development time is too short, and the result is low; the color development time is too long, the blank is increased, or the non-specific color development is increased.
Colorimetry should pay attention to the choice of wavelength. The TMB is used as a substrate and the OPD is used as a substrate. The former has a colorimetric wavelength of 450 nm and the latter has a wavelength of 492 nm. The filter needs to be replaced at any time according to requirements. Therefore, the problem of misuse of the filter is apt to occur.