Question

Explain the difference between nonspecific binding and specific binding using an indirect ELISA as an example. For this assay, assume that you are determining the titer of antibody against BSA.

You bind BSA to wells of a microtiter plate, add a dilution series of the antibody in question across the wells of the plate, then detect that antibody with an enzyme-conjugated antibody/chromogenic substrate pair.

Answer 1

The significant preferred position of a roundabout ELISA is that the conjugated second neutralizer can be utilized for numerous measures, gave similar types of restricting counter acting agent is utilized for the entirety of the various tests. Another preferred position is that the subsequent immune response will give an intensification of sign, on the grounds that around 2 of these antibodies will tie to the primary counter acting agent. With an expansion in the quantity of steps, the convention ordinarily will take longer and could be increasingly inclined to blunder. Additionally there may be an increase in the background signal because of the greater number of reagents required, each one with a potential for nonspecific binding.Blocking buffers should contain the correct concentration of nonionic detergent and/or nonspecific proteins to bind to open sites on the coated plate

A protein-based blocking buffer can only be added after the coating step, because proteins in the solution become adsorbed onto all unbound sites on the plastic. Like the coated analyte or antibody, these nonspecific proteins are not removed through any washing steps. Blocking buffers need to be optimized to allow for maximal sensitivity but minimal background and should not contain components that will interfere with the detection antibody.

Blocking is regularly important to prevent the non-specific binding of detection antibodies to the multiwell plate surface itself. To achieve this assignment, blocking cradle for the most part contains a disconnected protein or a protein subsidiary that doesn't respond with any of the antibodies being utilized in the discovery step.

In case of BSA we can also use casein .i.e milk powder beacuseit is also a protein ...and we need protein based buffer so that unwanted can be blocked and only the specific binding sites will remain open