Some notes on ELISA applications: overcoming non-specific adsorption

Some notes on ELISA applications: overcoming non-specific adsorption

In ELISA, non-specific adsorption is a problem that deserves attention. If the antigen or antibody is not purified, non-specific adsorption is unavoidable in the experimental operation.

However, even if the purification of antigen and antibody reaches a certain level, there is still the possibility of non-specific adsorption. For example, the adsorption on the surface of the plastic carrier, in order to avoid non-specific color development, you need to use PBS or physiological saline containing Tween20 as the eluent. Tween (Tween) refers to polyoxyethylene sorbitan fatty acid ester, which is non-ionic The surface tension substance is often used as a cosolvent. Tween numbering is based on the type of fatty acid it binds to the polymerized sorbitol, Tween 20 for lauric acid, Tween 40 for palmitic acid, Tween 60 for stearic acid, and Tween 60 for oleic acid. Tween 80. In immunoenzyme technology, PBS containing Tween 20 is commonly used as a detergent or as a diluted sample and enzyme-labeled antibody (antigen). Its washing effect is better than that of bovine serum protein solution (0.5%), with reduced non-specific adsorption And enhance the role of antigen and antibody binding. The dosage of Tween 20 is generally 0.06% -0.1%. Some scholars believe that Tween20 can also enhance the specific response. In addition, the detection of serum and enzyme-labeled antibodies diluted with 0.5% -1% bovine serum albumin (or 0.1% gelatin) can also enhance the specific adsorption. The sample is first incubated with diluted healthy serum, which also helps to remove non-specific color development. In addition, the most suitable time for the reaction and the optimal concentration of the reactants also help to overcome non-specific adsorption. The reaction generally takes 4 ~ 5h (25 ℃). If you want to reduce the reaction time, you can increase it to 37 ℃. Although the reaction temperature is 37 ℃, it has a slight effect on the sensitivity and accuracy, but it reduces the holding time (takes 2 ~ 3h ), Has little effect on the results, therefore, 37 ℃ is often used. The concentration of enzyme-labeled antibody is too high, prone to false positives; too low, it is not sensitive. The optimal concentration of the test sample, enzyme-labeled antibody and anti-antibody used can only be obtained through experiments. Within a certain range, the reaction intensity of ELISA has a linear relationship with the logarithm of the concentration of the sample to be tested. Only by measuring a series of samples of different dilutions can the optimal dilution range of the samples to be tested be obtained. In addition, due to the excessive amount of antigen added to the solid phase carrier for sensitization, the prozone effect caused by it can also be eliminated by testing the dilution of the antiserum, because in the case of the optimal amount of antibody, the excess antigen Does not interfere. Regarding the optimal concentration of enzyme-labeled antibody, a dose response curve (a series of diluted positive and negative sera that react with the same concentration of enzyme-labeled antibody) can be used to select the same dilution of positive and negative sera for different dilutions of enzyme Labeled antibody reaction is determined. When the ratio of the absorption value of the positive serum to the absorption value of the negative serum is the maximum, the concentration of the enzyme-labeled antibody is the optimal concentration.