Problem with coating in Elisa experiment
Elisa bioanalysis is an experimental diagnostic method with high sensitivity, specificity and repeatability. Because its reagents are stable, easy to store, easy to operate, and the results are judged more objectively, it has been widely used in various fields of immunology testing.
When using the Elisa kit for experiments, there are many issues to be aware of, such as incubation, color development, and coating. Among them, the coating is a problem that needs special attention and is directly related to the effect of the experiment. Below, Wuhan New Qidi biological technology Co., Ltd. will introduce you to related questions about the coating.
The way of coating
Fixation of antigens or antibodies in the process is called coating. Proteins and polystyrene solid-phase carriers are bound by physical adsorption, relying on the interaction between hydrophobic groups on the molecular structure of the protein and the hydrophobic groups on the surface of the solid support. This physical adsorption is non-specific and is affected by the molecular weight, isoelectric point, and concentration of the protein.
Large-molecule proteins Smaller molecules usually contain more hydrophobic groups and therefore are more likely to adsorb onto the solid support surface. Non-protein antigens that are not easily adsorbed can be subjected to affinity chromatography of solid phase antibodies by indirect coating, and the purity of the antigens coated on the solid phase is greatly increased. Therefore, antigens containing more impurities can also be captured by the coating method.
Avidin Biotin Avidin is used to coat the carrier first, and biotinylated DNA is added. This coating method is uniform and firm, and has been widely used for the quantitative determination of various antigenic substances. Lipid substances can not be combined with the solid carrier, can be dissolved in an organic solvent (such as ethanol) and added to the ELISA plate wells. The refrigerator is opened overnight or the cold air is blown dry. After the alcohol volatilizes, the lipids are naturally dried. On the solid surface.
Advantages: The specificity and sensitivity of the test are improved and the repeatability is also good. The amount of antigen used is only 1/10 or even 100% of direct coating.
Coated antigen
Natural antigens, recombinant antigens and synthetic peptide antigens are three major categories. Synthetic polypeptide antigens are synthetic peptide fragments of amino acid sequences of antigenic determinants. In general, it contains only one antigenic determinant, which is of high purity and high specificity. However, due to the low molecular weight, it is often difficult to directly adsorb onto the solid phase. With the aid of adsorption of the conjugate and the solid support, indirect binding to the surface of the solid support.
Coating antibody
IgG has strong adsorption to polystyrene, and its association occurs in the Fc segment and the antibody binding site is exposed. Obtained from antiserum or culture medium containing monoclonal antibody. Must be removed before use in ELISA in order to ensure the specificity of the test.
The concentration of monoclonal antibody in ascites is higher and specificity is higher. Therefore, absorption chromatographic processing is not required and direct coating is performed. In some cases, coating with multiple monoclonal antibodies can achieve better results.
Condition of the coating
pH9.6 Carbonate Buffer
pH 7.2 phosphate buffer
pH 7-8 Tris-HCL Buffer
After the coating liquid was added, it was placed in a refrigerator at 4-8°C overnight and incubated at 37°C for 2 hours. The coating concentration can vary greatly depending on the nature of the carrier and the coating, and each batch of material must be selected by experimentation in coordination with the concentration of the enzyme conjugate. The general protein coating concentration is 10ng/ml-20ug/ml.
