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Radioimmunoasay. developed in the 1950’s, Antigen –antibody but radiation used for detection. Principle. competitive binding where a radioactive antigen (“labeled") competes with a non-radioactive antigen (unlabeled) for a fixed number of antibody or receptor binding sites.
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developed in the 1950’s, • Antigen –antibody but radiation used for detection
Principle • competitive binding • where a radioactive antigen (“labeled")competeswith a non-radioactive antigen (unlabeled) for a fixed number of antibody or receptor binding sites.
Needed substances and equipment 1. Availability of a radioactive labeled form of the antigen2. Antibodies against the antigen to be measured3. A method in which the antibody-bound tracer can be separated from the unbound tracer4. An instrument to count radioactivity
Ag* + AbAg*Ab • Ag*Ab + Ag Ag*Ab + AgAb+ Ag +Ag* Separate Ag*AbAgAb Ag Ag* Supernatant pellet Measure radioactivity, take ratio of bound/free
The target antigen is labeled radioactively and bound to its specific antibodies (a limited and known amount of the specific antibody has to be added). • A sample, for example a blood-serum, is then added in order to initiate a competitive reaction of the labeled antigens from the preparation, and the unlabeled antigens from the serum-sample, with the specific antibodies. • The competition for the antibodies will release a certain amount of labeled antigen. This amount is proportional to the ratio of labeled to unlabeled antigen. A binding curve can then be generated which allows the amount of antigen in the patient's serum to be derived. • That means that as the concentration of unlabeled antigen is increased, more of it binds to the antibody, displacing the labeled variant. The bound antigens are then separated from the unbound ones, and the radioactivity of the free antigens remaining in the supernatant is measured. A binding curve can be generated using a known standard, which allows the amount of antigens in the patient's serum to be derived. • Radioimmunoassay is an old assay technique but it is still a widely used assay and continues to offer distinct advantages in terms of simplicity and sensitivity.
Standard! • Instead of patient sample you prepare samples with Known quantity of antigens • Binding curve
Procedure • Add known amount of test sample + unlabelled antigen (patient) to microtitre plate • Incubate • Wash • Measure radioactivity in the wells – GM counter • Intensity of radioactivity inversely proportional to conc of antigen in sample
Gamma counter • measures gamma radiation in a sample. • photons are emitted as a radioactive substances decays, or attempts to become stable • these counters use crystals which emit light when photons from the gamma rays interact with them. • Samples are usually placed into test tubes, which are then placed into the machine.
Radiolabelling • Iodine-125 labels are usually applied • other isotopes such as C14 and H3 have also been used. • Preparation of radio-labeled (125-I) antigen • is prepared by iodination of the pure antigen on its tyrosine residue(s) • by chloramine-T or peroxidase methods • separating the radio-labeled antigen from free-isotope by gel-filtration or HPLC.
Why iodine-125? • Because of its relatively long half-life • emission of low-energy photons • which can be detected by gamma-counter crystal detectors
Why RIA? • Less complex than enzyme activity measurements • because of its robustness, • consistent results • and low price per test, • greatsensitivity. • possible to detect a few picograms (10−12 g) of antigen in the tube.
Why not? • Toxicity • Shelf life • Costly
