Radioimmunoassay (RIA) Rick McCosh

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

RIA Reagents Tracer: labeled antigen Antibody Introduction , Theory, Preparation of the Reagents, An actual Assay , Conclusions RIA Reagents Tracer: labeled antigen Antibody Standards: Known concentrations of unlabeled antigen Unknown samples

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

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

Separate bound from free: Introduction, Theory, Preparation of the Reagents, An actual Assay , Conclusions Introduction, Theory, Preparation of the Tracer, 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 PEG Centrifugation

Count gamma emission Counts per minute (CPM) for each tube Introduction, Theory, Preparation of the Reagents, An actual Assay , Conclusions Introduction, Theory, Preparation of the Tracer, 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 , Conclusions Introduction, Theory, Preparation of the Reagents, An actual Assay , Conclusions

Preparation of the Reagents: Antibodies and Antigens Introduction, Theory, Preparation of the Reagents, An actual Assay , Conclusions Introduction, Theory, Preparation of the Tracer, An actual Assay , Conclusions Preparation of the Reagents: Antibodies and Antigens 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.

Preparation of the Reagents: Iodination of the antigen Introduction, Theory, Preparation of the Reagents, An actual Assay , Conclusions Introduction, Theory, Preparation of the Tracer, An actual Assay , Conclusions Preparation of the Reagents: Iodination of the antigen I125 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. I125 can be added to the tube and will bind to the oxidized residues Column chromatography is used to purify the tracer

An Actual Assay: Progesterone (P4) Introduction, Theory, Preparation of the Reagents, An actual Assay, Conclusions Introduction, Theory, Preparation of the Tracer, An actual Assay , Conclusions An Actual Assay: Progesterone (P4) Total count tubes Polypropylene tube Tracer Non-specific Binding B0 Antibody labeled tube Standards ( 10, 5, 2.5, 1.25, 0.6125, 0.3125 ng/mL ) Standard High and Low pools High and low pools Samples containing unknown samples sample

An Actual Assay: Progesterone (P4) Introduction, Theory, Preparation of the Reagents, An actual Assay, Conclusions Introduction, Theory, Preparation of the Tracer, An actual Assay , Conclusions An Actual Assay: Progesterone (P4) Incubate Decant Count Calculate

An Actual Assay: Progesterone (P4) Std. Curve Introduction, Theory, Preparation of the Reagents, An actual Assay, Conclusions Introduction Theory, Preparation of the Tracer, An actual Assay , Conclusions An Actual Assay: Progesterone (P4) Std. Curve Each tube- Mean NSB = Corrected CPM Corrected CPM / B0 = % Binding Logit % binding = Ln(% binding / 1- % binding) For Standard Curve: Use SL regression to fit the model: Y = β0 + β1 X where Y = logit (%binding), X = log [sample],

Introduction Theory, Preparation of the Tracer, An actual Assay , Conclusions Std. Curve [] log[] % binding mean % dec. % logit binding 0.3 -0.52 79.9 0.80 1.38 0.6 -0.22 70.2 0.70 0.86 1.25 0.10 58.5 0.59 0.34 2.5 0.40 46.6 0.47 -0.14 5 34.6 0.35 -0.64 10 1.00 23.3 0.23 -1.19   Coefficients Standard Error t Stat P-value Intercept 0.504695 0.013009 38.79629 2.64E-06 X Variable 1 -1.67631 0.022652 -74.004 2E-07

An Actual Assay: Progesterone (P4) Samples Introduction, Theory, Preparation of the Reagents, An actual Assay, Conclusions Introduction Theory, Preparation of the Tracer, An actual Assay , Conclusions An Actual Assay: Progesterone (P4) Samples Calculate mean % binding for each sample Calculate logit % binding for each sample Solve: Y = β0 + β1 X where Y = logit (%binding), X = log [sample] Antilog of X = concentration of antigen in samples

Introduction, Theory, Preparation of the Reagents, An actual Assay, Conclusions RIA is an effective, precise and accurate method of quantifying concentrations of an antigen. Does require approval and training to work with radioactive materials Modifying an assay procedure can be difficult and time consuming

References Yalow R, Berson S. Immunoassay of endogenous plasma insulin in man. J. Clin. Invest 1960; 39: 1157-1175. Abraham G. Radioimmunoassay of steroids in biological fluids. J. Steroid Biochemistry 1975; 6: 261-270.