After being blocked, the ELISA kit plate can only be stored at low temperature for about 2 weeks. In order to extend the stability of the antigen on the solid phase plate, we added a step to protect the plate. Add appropriate amount of sugar, irrelevant protein, gentamicin and divalent metal ions to 10mM phosphate as the protective agent of the enzyme plate. After blocking, add the protective solution and incubate in a 4 ° C refrigerator overnight. Protected microplates were compared, and then protected and unprotected microplates were placed in a 37-oven for 15 days to investigate the effect of the protective solution on the stability of microplates.
1. Optimization of dilution of enzyme-labeled antibody
Antigen coating concentration is 4ug / ml, serial dilution of enzyme-labeled antibody: 1: 100, 1: 200; 1: 300; 1: 400; 1: 500; 1: 600; 1: 1000; after incubation and washing 1. After the color development is terminated, analyze it with an automatic microplate reader, and select the dilution of the enzyme-labeled antibody with an A value of about 1.5 as the optimal working concentration.
2. Optimization of enzyme-labeled antibody protection solution
Enzyme-labeled antibodies at low concentrations are extremely susceptible to inactivation by the effects of high temperature, microorganisms, and proteases, which seriously affects the shelf life of products. In the past, enzyme-labeled antibodies were usually lyophilized, but such storage is not only due to the process of lyophilization Can affect the activity of enzyme-labeled antibodies, and is quite inconvenient in clinical applications. Appropriate buffers and the addition of irrelevant proteins, sugars, preservatives, and some amino-containing compounds will have a certain protective effect on the activity of the enzyme-labeled antibody, so we designed a set of protective agents to protect the enzyme-labeled antibody, and The enzyme-labeled antibody without any glycoprotein added was used as a control. The protected enzyme-labeled antibody and the unprotected enzyme-labeled antibody were placed in a 37 ° C oven for 6 days to investigate the effect of the protective solution on the enzyme-labeled antibody.
When coating antigen, in order to get the best coating effect, we used carbonate buffer (50mM ph9.6), phosphate buffer (50mM ph7.6), and TRIS hydrochloric acid buffer (50 mM ph7. 6) The three buffers are used as coating solutions. The antigen coating concentration is 5ug / ml and 2.5ug / ml. The working concentration of the enzyme-labeled antibody is 1: 400 and 1: 300. Analyze with an automatic microplate reader to choose the best Coating buffer system. At the same time, in order to ensure the long-term storage of the buffer solution, we added 0.01% thimerosal as a preservative in the coating solution.
3. Optimization of the optimal antigen coating concentration
Dilute the coating antigen (ALB) to 0.1ug / ml, 1.0ug / ml, 2.0ug / ml, 4.0 ug / ml, 6.0 ug / ml, 8.0 ug / ml, 10.0 ug with optimized coating buffer / ml and other six concentrations, coated microplate, 100ul per well; competition antigen concentration is 4.0 ug / ml, 2.0 ug / ml; enzyme-labeled antibody dilution is 1: 300, used after incubation, color development, and termination Automatic microplate reader analysis. The intensity of color development is proportional to the amount of coated antigen within a certain range, but with the increase of the concentration of the coated antigen, the intensity of color development decreases instead. We chose the critical coating value as the amount of antigen coated by the ELISA kit.
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