Radioimmunoassay (RIA)

RIA Purpose is to determine the concentration of an antigen in solution Competitive binding assay Originally developed by Yalow and Berson in 1960 for insulin Introduction, Theory, Preparation of the Reagents, An actual Assay, Conclusions

Principle of Radioimmunoassay Principle: Uses an immune reaction [Antigen – Antibody reaction] to estimate a ligand Ag + Ag* + Ab  AgAb + Ag*Ab + Ag + Ab* – Unbound Ag* and Ag washed out – Radioactivity of bound residue measured – Ligand conc is inversely related to radioactivity [Ag : ligand to be measured ; Ag* radiolabelled ligand]

Antibody Introduction, Theory, Preparation of the Reagents, An actual Assay, Conclusions

Labeled Antigen + Sample Introduction, Theory, Preparation of the Tracer, An actual Assay, ConclusionsIntroduction, Theory, Preparation of the Reagents, An actual Assay, Conclusions

Separate bound from free: Antibody labeled tubes can be simply decanted Liquid-phase antibodies need to be precipitated Use a second antibody Centrifugation Introduction, Theory, Preparation of the Tracer, An actual Assay, ConclusionsIntroduction, Theory, Preparation of the Reagents, An actual Assay, Conclusions

Count gamma emission Counts per minute (CPM) for each tube A sample containing a higher concentration of the unknown antigen will have a lower CPM Introduction, Theory, Preparation of the Tracer, An actual Assay, ConclusionsIntroduction, Theory, Preparation of the Reagents, An actual Assay, Conclusions

Preparation of the Reagents: Antibodies and Antigens Introduction, Theory, Preparation of the Tracer, An actual Assay, Conclusions Polyclonal antibodies are made by injecting an animal with the antigen, then purifying the antibody from serum. Molecules smaller than ~1000 d are not generally immunogenic Steroids are covalently bond to protein carriers which are immunogenic, antibodies can then be purified and their specificity verified. Introduction, Theory, Preparation of the Reagents, An actual Assay, Conclusions

Preparation of the Reagents: Iodination of the antigen Introduction, Theory, Preparation of the Tracer, An actual Assay, ConclusionsIntroduction, Theory, Preparation of the Reagents, An actual Assay, Conclusions I 125 is the radioactive label most often used. Gamma emission at 35keV Available commercially as NaI Proteins with surface tyrosine groups can be oxidized with commercially available products. I 125 can be added to the tube and will bind to the oxidized residues Column chromatography is used to purify the tracer

Advantages & Disadvantages of RIA Advantages – Highly specific: Immune reactions are specific – High sensitivity : Immune reactions are sensitive Disadvantages – Radiation hazards: Uses radiolabelled reagents – Requires specially trained persons – Labs require special license to handle radioactive material – Requires special arrangements for Requisition, storage of radioactive material radioactive waste disposal.

Applications of Immunoassays [RIA] Analysis of hormones, vitamins, metabolites, diagnostic markers – Eg. ACTH, FSH, T3, T4, Glucagon, Insulin, Testosterone, vitamin B12, prostaglandins, glucocorticoids, Therapeutic drug monitoring: – Barbiturates, morphine, digoxin, Diagnostic procedures for detecting infection – HIV, Hepatitis A, B etc

Conclusions: RIA is an effective, precise and accurate method of quantifying concentrations of an antigen. Introduction, Theory, Preparation of the Reagents, An actual Assay, Conclusions