Question

Explain how antibody-antigen interaction is applied in RIA or ELISA.

Answer 1

• Enzyme linked immuno sorbent assay ( ELISA) is a commonly used analytical biochemistry assay, which uses the solid phase of enzyme immuno assay to detect the presece of a ligand in a liquid sample using antibodies directed against the protein to be measured.
• In simple form of ELISA, antigen from the sample is attatched to a surface.
• Then a matching antibody is applied over the surface so it can bind to antigen.This antibody is linked to an enzyme , and in the final step the substance containing enzyme's substrate is added .
• The subsequent reaction produce detectable signal, most commonly a colour change.
• There are 4 types ELISA, they are Direct ELISA, Sandwich ELISA, Competitive ELISA, Reverse ELISA.
• Radio immuno assay (RIA) is an invitro assay which measures the presence of antigen with high sensitivity.
• The target antigen is labeled radioactively and bound to specific antibodies ,with limited and known amount of specific antibody is added.
• A blood sample is added to initiate a competitive reaction of labeled antigen from the preparation, with unlabeled antigen from serum sample,with specific antibodies.
• Th competition for antibodies will provide certain amount of labelled antigen, this amount is proportional to ratio of labeled to unlabeled antigen.
• A binding curve is obtained which allows the amount of antigen in the patients serum is derived,which means concentration of unlabeled antigen is increased , more of its bind to antibody.Bound antigen are separated from unbound one and radioactivity of free antigen in the remaining supernatant is measured.
• A binding curve can be generated using a known standard , which allows the amount of antigen in the patient's serum to be derived.