The most common method used in antigen ELISA test kits involves the detection of excess antigen using solid-phase antibody and enzyme-labeled antibody. In this approach, the antigen is not typically involved in forming a sandwich complex, which can lead to results that are lower than the actual antigen concentration. In severe cases, this may even result in false negative outcomes due to the hook effect.
In this method, a mixture of the sample being tested and a known amount of enzyme-labeled antigen is added to a reaction tube containing the solid-phase antibody. If the sample does not contain any antigen, the enzyme-labeled antigen will bind freely to the solid-phase antibody. However, if the sample contains the target 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 solid phase.
A reference tube is prepared by adding only the enzyme-labeled antigen, allowing it to bind fully to the solid-phase antibody. After incubation, the reference tube achieves maximum binding. Following washing steps, the bound enzyme-labeled antigen is retained while unbound material is removed.
During the substrate addition step, the enzyme catalyzes the substrate into a colored product. The reference tube shows the highest color intensity due to the maximum binding of the enzyme-labeled antigen. The difference in color between the reference tube and the test tube reflects 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 enzymatic activity. Throughout the assay, 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 is directly proportional to the amount of the target substance in the sample.
This technique allows for precise quantification of antigens or antibodies in a sample. By measuring the color change resulting from the enzyme-substrate reaction, the ELISA test kit provides a reliable and widely used method for detecting and quantifying biological molecules. Its sensitivity and specificity make it a valuable tool in diagnostic and research settings.
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