The most common method used in antigen ELISA test kits involves the use of excess antigen, solid-phase antibody, and enzyme-labeled antibody. In this approach, the antigen is detected without forming a sandwich complex, which can lead to results that are lower than the actual concentration. In severe cases, this may even result in a false negative outcome.
The process begins by mixing the sample to be tested with a known amount of enzyme-labeled antigen, which then reacts with the solid-phase antibody. If the sample does not contain the target antigen, the enzyme-labeled antigen will bind freely to the solid-phase antibody. However, if the sample contains the antigen, it will compete with the enzyme-labeled antigen for binding sites on the solid-phase antibody. This competition reduces the amount of enzyme-labeled antigen that can bind to the carrier.
In the reference tube, only the enzyme-labeled antigen is added. After incubation, the binding between the enzyme-labeled antigen and the solid-phase antibody reaches its maximum level. Following this, all tubes are washed to remove unbound components.
Next, the substrate is added, and a color development reaction occurs. The reference tube shows the highest color intensity due to the maximum binding of the enzyme-labeled antigen. The difference in color intensity between the reference tube and the test tube indicates the amount of antigen present in the sample. The lighter the color in the test tube, the higher the antigen concentration in the sample.
The enzyme-labeled antigen or antibody used in the ELISA test kit retains both its immunological activity and the enzymatic activity necessary for the reaction. Throughout the procedure, the antigen-antibody complexes formed on the solid-phase carrier are separated from other substances through washing. Finally, the amount of enzyme bound to the solid-phase carrier is directly proportional to the amount of the target substance in the sample.
After adding the enzyme's substrate, the enzyme catalyzes the conversion of the substrate into a colored product. The intensity of the color produced is directly related to the concentration of the analyte in the sample. This principle allows the ELISA test kit to accurately detect and quantify antigens or antibodies in various biological samples.
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