As an auxiliary material in ELISA detection experiments, an Enzyme plate plays a decisive role and directly affects the final experimental results. The quality of the Enzyme plate mainly depends on its sensitivity to protein adsorption, the difference in protein adsorption capacity between plates and holes, and the difference between batches of the purchased enzyme plate. Therefore, selecting an enzyme plate product with high protein adsorption sensitivity, the small difference between holes of protein adsorption capacity, and a small difference between batches is the guarantee for the experimenter to obtain reliable and stable experimental results.

Enzyme plate classification: according to different classification standards, the plate has different classifications.

1: According to the number of holes, it can be divided into 96 holes, 48 holes, etc. Because the enzyme plate is mainly used with the microplate reader, the plate meter on the market is at most 96 holes, so the microplate is 96 holes.

2: According to the different bottoms, it is divided into flat bottom, U bottom, V bottom, etc.

The refractive index of the flat base is low, which is suitable for detection in the microplate;

The U microplate refractive index is high, convenient for sampling, sampling, and mixing, and can directly observe the color change without placing it on the microplate, so as to determine whether there is a corresponding immune reaction.

The microplate of the V base can accurately absorb the sample.

3: According to the different binding abilities of the microplate and protein and other molecules, it is divided into high binding force, medium binding force, and amination.

(1) High binding force

After the surface of this microplate, its protein binding ability is greatly enhanced, up to 300~400ng IgG / cm2, and the molecular weight of the main bound protein is> 10 kD. The use of this class of microplate can improve the sensitivity, and can reduce the concentration and amount of coated protein relatively, which is easier to produce non-specific reactions. After coating with antigen or antibody, the non-ionic detergent cannot effectively seal the site of the unbound protein, and the protein should be used as the sealant agent.

(2) Medium binding force

Such microplate plates passively bind to the protein through hydrophobic bonds on the surface, and are suitable as solid-phase carriers for macromolecular proteins with molecular weight> 20 kD, with a protein binding capacity of 200 to 300 ng IgG / cm2. Due to the characteristics of its only binding to macromolecules, it is suitable for solid-phase carriers as unpurified antibodies or antigens to reduce the potential for nonspecific cross-reactivity. The plate can be an inert protein or a nonionic detergent as a sealing solution.

(3) Amination

This microplate after a surface modification has a positively charged amino group, whose hydrophobic bond is replaced by a hydrophilic bond. This class of microplate is suitable as a solid-phase carrier for small-molecule proteins. Using a suitable buffer and pH, the surface binds to negatively charged small molecules via ionic bonds. Due to the hydrophilic properties of its surface and its ability to be covalently bound by other cross-linkers, it can be used to fix protein molecules soluble in decontamination agents such as Triton-100, Tween 20, etc. The defect of this plate is because of reduced hydrophobicity; moreover, the surface needs to be effectively closed. Because of the hydrophilic and covalent surface properties, the sealing solution used must be able to interact with any functional group in the nonreactive amino group and the selected crosslinker.

4. According to the color can be divided into transparent, black, and white.

Transparent is the most commonly used for the most general enzyme-linked immunization experiments. In Relcontrast to the transparent microplate, there are also opaque microplates for luminous detection, generally black and white. The black microplate itself has light absorption, so its signal is much lower than the white microplate, so it is generally used to detect strong light, such as fluorescence detection. The white microplate is used for weak light detection, often used for general chemiluminescence. In addition, the black microplate can also weaken the problem of non-specific reactions. At the same time, it is important to note that with the general microplate can not be luminous detection, because the light emitted from the chemiluminescence reaction is isotropic, if, with a transparent microplate, the light will not only spread from the vertical direction, but also from the horizontal direction, make light easily through the gap between the hole and hole wall, resulting in the light of the light absorption value of the hole is affected by the adjacent hole emitted light.

A good microplate should be of good adsorption performance, low blank value, high transparency of the hole bottom, and similar performance between the plates and between the holes of the same plate. Due to the difference in raw materials and the difference in the production process, the quality of various products is very different, therefore, the performance of each batch of microplate must be checked in advance before use. The commonly used inspection methods are a certain concentration of human IgG (generally 10 ng/ml) coated with ELISA plate wells, after washing, adding an appropriate dilution of enzyme-labeled anti-human IgG antibody to each well, washing after heat preservation, adding substrate color, stop the enzyme reaction, and measure the absorbance of the solution in each well respectively. The reaction conditions were controlled so that the reading of each well was kept at an absorbance of around 0.8. The average of the total readings was calculated. The difference between the mean of all individual readings and all readings shall be less than 10%. The following three microplates are A, B, and C as an example.

Direct method: to detect the adsorption of Human IgG on the surface of the microplate

Double antibody sandwich method: antigen in serum positive for anti-human antibody

As can be seen from the above figure, in these three types of bio enzyme plates, class A microplate has a better protein adsorption effect, but also improves the sensitivity of protein adsorption, which can provide more reliable experimental data. In addition, you can ask about the plate of the batch difference. The following is the batch difference of a certain plate. As can be seen from the within-batch difference data chart, the microplate has good inter-batch stability, and the within-batch coefficient of variation (CV) difference is around 5.0%, which is significantly lower than the within-batch variation coefficient below 10.0% in the quality control standard for clinical immune reaction. Therefore, so it is also suitable for ELISA experiments.