RADIOIMMUNOASSAY (RIA) Dr. Sigal Fleisher-Berkovich Dept. of Clinical Pharmacology

Principles of the Radioimmunoassay A fixed concentration of labeled tracer antigen is incubated with a constant amount of antiserum If unlabeled antigen is added to the system, there is competition between labeled tracer and unlabeled antigen for the limited number of binding sites on the antibody The amount of tracer bound to antibody will decrease as the conc. of unlabeled antigen increases This can be measured after separating antibody bound from free tracer and counting the bound fraction

RIA radioactivity Increasing amount of Insulin

Four basic necessities for RIA Antiserum to the compound to be measured 2. A radioactively labeled form of the compound 3. A method whereby antibody-bound tracer can be separated from “free” tracer 4. A standard unlabeled material

Terminology Total Count Tubes: These tubes represent the total amount of radioactivity added in a RIA tube. They have the highest CPM’s (labeled antigen +buffer) Role: quality control to the counts in the rest of the tubes

Terminology Background (blank) tubes: These tubes contain buffer, labeled antigen and charcoal but not antibody They have the lowest CPM’s These counts are considered to be background counts

Terminology Standards: Assay tubes that contain known amounts of the compound to be measured. Those tubes with increasing amount of standard have decreasing CPM’s. RIA curve

Terminology Unknowns: Assay tubes containing the material in which the compound you wish to measure is contained. Blood serum & plasma Urine Saliva CSF

Terminology Precipitating agent: It is added at the end of incubation It precipitates the “free” labeled and unlabeled antigen Our precipitating agent is dextran-coated charcoal

RIA Table No of tubes: 2 2 4 2 2 buffer (ml) 0.8 0.6 0.1 - - Total Blank Zero standard assay No of tubes: 2 2 4 2 2 buffer (ml) 0.8 0.6 0.1 - - Standard (ml) - - - 0.1 - Sample (ml) - - - - 0.1 Antibody (ml) - - 0.5 0.5 0.5 Incubation for 30 min Labeled PG (ml) 0.1 0.1 0.1 0.1 0.1 Incubation for 1-24 hr Charcoal 0.2 0.2 0.2 0.2

RIA Advantages: Extremely sensitive method Large number of samples can be processed Small changes in hormone concentrations can be reproducibly measured Disadvantages: Can’t determine if hormone measured has biological Activity Peptide hormones can be denatured and not active but still retain their antigenic character

Prostaglandin E2 Prostaglandins: a 20-carbon unsaturated fatty acids derived from arachidonic acid The inflammatory response is always accompanied by the release of prostaglandins, the predominant product being PGE2 PGE2 mediates: fever, pain vasodilation