ELISA (enzyme linked immunosorbent assay), is a plate-based technique that can be used to detect HIV. In an ELISA, an antigen must be It is then immobilized on a solid surface, and then complexed using an antibody that's linked to an enzyme. The conjugated enzyme activity is determined by incubating the substrate with it.
The ELISA is a powerful tool to measure specific analytes in a crude preparation. A polyvinyl chloride microtiter plate (PVC), is the best choice for most applications. However, it is important to consult the manufacturer's guidelines to determine which plate is most suitable for protein binding. You can know more about competitive ELISA protocol through various online sources.
Each well should be diluted with 50 mL primary antibody (capture). Before testing the samples, it is important to determine the appropriate dilution using a checkerboard titration. The exact amount of antibody required will vary depending on the assay.
However, if maximum binding is required, you should use at least 1mg/well. This is well over the well's capacity, but binding will be faster and can be saved so that it can be used again. Allow the solution to incubate for four hours. Add approximately 50 mLs of secondary antibody solution to each well to bind it to the bottom.
Allow the plate to rest overnight at 4°C in order to ensure complete binding. Add primary capture antibodies. PBS can be used to wash the wells twice. It is easy to use a 500 mL squirt container. You can remove the antibody solution by placing the plate on a container that is suitable.