RADIOIMMUNOASSAY (RIA)RADIOIMMUNOASSAY (RIA)RADIOIMMUNOASSAY (RIA)RADIOIMMUNOASSAY (RIA)

Dr. Sigal Fleisher-BerkovichDr. Sigal Fleisher-Berkovich

Dept. of Clinical PharmacologyDept. of Clinical Pharmacology

Dr. Sigal Fleisher-BerkovichDr. Sigal Fleisher-Berkovich

Dept. of Clinical PharmacologyDept. of Clinical Pharmacology

Principles of the RadioimmunoassayPrinciples of the RadioimmunoassayPrinciples of the RadioimmunoassayPrinciples of the Radioimmunoassay

• A fixed concentration of labeled tracer antigen is incubated with a A fixed concentration of labeled tracer antigen is incubated with a constant amount of antiserumconstant amount of antiserum

• If unlabeled antigen is added to the system, there is competition If unlabeled antigen is added to the system, there is competition between labeled tracer and unlabeled antigen for the limited number of between labeled tracer and unlabeled antigen for the limited number of binding sites on the antibodybinding sites on the antibody

• The amount of tracer bound to antibody will decrease as the conc. of The amount of tracer bound to antibody will decrease as the conc. of unlabeled antigen increasesunlabeled antigen increases

• This can be measured after separating antibody bound from free This can be measured after separating antibody bound from free tracer and counting the bound fraction tracer and counting the bound fraction

RIARIA RIARIA

Increasing amount of Insulin

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Four basic necessities for RIAFour basic necessities for RIAFour basic necessities for RIAFour basic necessities for RIA

1. 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

Total Count Tubes:Total Count Tubes:

These tubes represent the total amount of radioactivity These tubes represent the total amount of radioactivity added in a RIA tube.added in a RIA tube.

They have the highest CPM’s (labeled antigen +buffer)They have the highest CPM’s (labeled antigen +buffer)

Role: Role: quality control to the counts in the rest of the quality control to the counts in the rest of the tubestubes

Total Count Tubes:Total Count Tubes:

These tubes represent the total amount of radioactivity These tubes represent the total amount of radioactivity added in a RIA tube.added in a RIA tube.

They have the highest CPM’s (labeled antigen +buffer)They have the highest CPM’s (labeled antigen +buffer)

Role: Role: quality control to the counts in the rest of the quality control to the counts in the rest of the tubestubes

TerminologyTerminologyTerminologyTerminology

TerminologyTerminologyTerminologyTerminology

Background (blank) tubes:Background (blank) tubes:

These tubes contain buffer, labeled antigen These tubes contain buffer, labeled antigen and charcoal but and charcoal but notnot antibody antibody

They have the lowest CPM’sThey have the lowest CPM’s

These counts are considered to be background countsThese counts are considered to be background counts

TerminologyTerminologyTerminologyTerminology

Standards:Standards:

Assay tubes that contain known amounts ofAssay tubes that contain known amounts ofthe compound to be measured.the compound to be measured.

Those tubes with increasing amount of Those tubes with increasing amount of standard have decreasing standard have decreasing CPMCPM’s. ’s.

RIA curve

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Unknowns:Unknowns:

Assay tubes containing the material in which the Assay tubes containing the material in which the compound you wish to measure is contained.compound you wish to measure is contained.

• Blood serum & plasmaBlood serum & plasma• UrineUrine• SalivaSaliva• CSFCSF

Unknowns:Unknowns:

Assay tubes containing the material in which the Assay tubes containing the material in which the compound you wish to measure is contained.compound you wish to measure is contained.

• Blood serum & plasmaBlood serum & plasma• UrineUrine• SalivaSaliva• CSFCSF

TerminologyTerminologyTerminologyTerminology

Precipitating agent:Precipitating agent:

It is added at the end of incubationIt is added at the end of incubation

It precipitates the “free” labeled and It precipitates the “free” labeled and unlabeled antigenunlabeled antigen

Our precipitating agent is Our precipitating agent is dextran-coated dextran-coated charcoal charcoal

Precipitating agent:Precipitating agent:

It is added at the end of incubationIt is added at the end of incubation

It precipitates the “free” labeled and It precipitates the “free” labeled and unlabeled antigenunlabeled antigen

Our precipitating agent is Our precipitating agent is dextran-coated dextran-coated charcoal charcoal

RIA TableRIA TableRIA TableRIA Table

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

RIARIARIARIA

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 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

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