In RIA technique, Radio labelled antigen are allow to react with known quantity of antibody. Unlabeled antigens are added later on. There is a competition between radio labeled antigen and unlabeled antigen and radio labelled antigens are replaced by unlabeled antigen from antigen antibody complex. What is the reason of replacing radio labeled antigen replaced by unlabeled antigen ?
Normally, you first add mix labelled antigen and your sample containing an unknown amount of antigen, then add a limiting amount of antibody. As you dilute your labelled antigen with the unlabelled antigen from your sample, the (roughly) constant amount of total antigen pulled down by the antibody contains only a fraction of labelled antigen. The more unlabelled antigen where is in your sample, the less radioactivity is pulled down by your antibody. By comparing to a standard curve where you add known amounts of unlabelled antigen, you can determine the amount of antigen in your sample.
Normally, you work from the assumption that cold and hot antigen bind to the antibody with the same affinity, although some labelling techniques such as iodination can interfere with the binding of your hot antigen if tyrosine residues within the epitope get labelled.
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