The effect of coating on the ELISA kit experiment! -Huaqiang Electronic Network

The effect of the coating on the elisa kit experiment!

When using the Elisa kit for experiments, there are a lot of problems to be aware of, and the biobiologists point out problems such as incubation, color development, and coating. Among them, the coating is a problem that needs special attention, which is directly related to the effect of the experiment.

Way of coating

Fixing an antigen or antibody in a process is called coating. The protein and polystyrene solid phase carrier are bound by physical adsorption, relying on the interaction between the hydrophobic group on the structure of the protein molecule and the hydrophobic group on the surface of the solid support. This physical adsorption is non-specific and is affected by the molecular weight, isoelectric point, concentration, etc. of the protein.

Macromolecular Proteins Smaller molecular proteins usually contain more hydrophobic groups and are therefore more readily adsorbed onto the surface of the solid support. The non-protein antigen which is not easily adsorbed can be affinity-chromatized by the solid phase antibody by the indirectly coated antigen, and the purity of the antigen coated on the solid phase is greatly improved. Therefore, the antigen containing more impurities can also adopt the capture coating method.

Avidin biotin is first coated with avidin, and biotinylated DNA is added. The coating method of ELISA kit is uniform and firm, and has been expanded and applied to quantitative determination of various antigenic substances. The lipid substance cannot be combined with the solid phase carrier. It can be dissolved in an organic solvent (such as ethanol), added to the well of the ELISA plate, and opened in a refrigerator or cold air. After the alcohol is evaporated, the lipid is naturally dried. Solid surface.

Advantages: The specificity and sensitivity of the test are improved and the repeatability is also good. The amount of antigen used is small, only 1/10 or even /100 of direct coating.

Coating antigen

There are three major categories of natural antigens, recombinant antigens and synthetic peptide antigens. A synthetic polypeptide antigen is a synthetically synthesized polypeptide fragment of an amino acid sequence of an antigenic determinant. Generally, it contains only one antigenic determinant, which has high purity and high specificity. However, since the molecular weight is too small, it is often difficult to directly adsorb to the solid phase. Indirect bonding to the surface of the solid support by means of adsorption of the conjugate to the solid support.

Coating antibody

IgG has a strong adsorption force for polystyrene, and its binding occurs on the Fc segment, and the ELISA kit antibody binding site is exposed. Take antiserum or culture medium containing monoclonal antibody. It is necessary to remove the hybrid antibody before using it in ELISA to ensure the specificity of the test.

The concentration of monoclonal antibody in ascites is higher, and the specificity is also stronger. Therefore, it is not required to be subjected to absorption chromatography and directly coated. In some cases, a mixture of various monoclonal antibodies can be used to achieve better results.

Condition of coating

pH 9.6 carbonate buffer

pH 7.2 phosphate buffer

pH 7-8 Tris-HCL buffer

After the coating solution was added, it was placed in a refrigerator at 4-8 ° C and incubated at 37 ° C for 2 hours. The concentration of the ELISA kit can vary greatly depending on the nature of the carrier and the coating. Each batch of material needs to be selected by experiment and concentration of the enzyme conjugate. Typical protein coating concentrations range from 10 ng/ml to 20 ug/ml.