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RadioImmuneAssay

(RIA)

•By:Sondos HassounehAreej Abu haniehShireen Rawajbeh

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IMMUNOASSAYS:

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Immuno” refers to an immune response that causes the body to generate

antibodies Assay” refers to a test..• Immunoassays are chemical tests used to detect or quantify a

specific substance, the analyte, in a blood or body fluid sample, using an immunological reaction. Immunoassays are highly sensitive and specific. Their high specificity results from the use of antibodies and purified antigens as reagents.

Immunoassays measure the formation of antibody-antigen complexes and detect them via an indicator reaction. High sensitivity is achieved by using an indicator system (e.g., enzyme label) that results in amplification of the measured product.

Immunoassays may be qualitative (positive or negative) or quantitative (amount measured). 

Definition :

The purpose of an immunoassay is to measure (or, in a

qualitative assay, to detect) an analyte. Immunoassay is the method of choice for measuring analytes normally present at very low concentrations that cannot be determined accurately by other less expensive tests Common uses include measurement of drugs, hormones, specific proteins, tumor markers, and markers of cardiac injury

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ANTIBODIES and ANTIGENS:

• An antibody is a protein (immunoglobulin) produced by B-lymphocytes (immune cells) in response to stimulation by an antigen.

An antigen is a substance with the ability to induce an immunological response.

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LABELS IN IMMUNOASSAYSImmunoassays require the use of labeled

materials in order to measure the amount of antigen or antibody present. A label is a molecule that will react as part of the assay, and in doing so produce a signal that can be measured in the solution. Examples of a label include a radioactive compound, or an enzyme that causes a change of color in a solution or substance that produce light .

Labels may be applied to

either the antibody

..or the antigen.

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Categories of Immunoassay TestsCompetitiveNoncompetitiveHomogeneousHeterogeneous

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Competitive Assays:• Unlabeled analyte (antigen) in the test sample is measured

by its ability to compete with the labeled antigen in the immunoassay.

• In a competitive immunoassay, less label measured in the assay means more of the unlabeled (test sample) antigen is present.

•Noncompetitive (sandwhich) immunoassays generally provide the highest level of assay sensitivity and specificity. This format is referred to as a “sandwich” assay because the analyte is bound (sandwiched) between two highly specific antibody reagents. •The reaction mixture typically includes an excess of labeled antibody, so that all drug/metabolite is bound. The amount of antibody-antigen complex is then measured to determine the amount of drug present in the sample. The measurement of labeled analyte, usually antibody, is directly proportional to the amount of antigen present in the sample.

Noncompetitive Assays

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Heterogeneous and

Homogeneous immunoassays Methods

HETEROGENEOUS IMMUNOASSAYS : Called separation assay Require multiple steps Unbound analyte is separated or washed

away, and the remaining labelled, bound analyte is measured.

• Those that do not require separation are referred to as HOMOGENEOUS IMMUNOASSAYS.

• Homogeneous methods have generally been applied to the measurement of small analytes such as Ab used and therapeutic drugs

There are several different methods

used in immunoassay tests:

Radioimmunoassay (RIA) Enzyme Immunoassay (ELISA) Fluorescence Polarization Immunoassay (FPIA)

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it begin with the separation of a protein (from a

mixture) using the specificity of antibody - antigen binding and quantify it using radioactivity .

The technique begin in 1960 by berson and yalow to test the blood Insulin

it begin with the separation of a protein (from a mixture) using the specificity of antibody - antigen binding and quantify it using radioactivity .

The technique begin in 1960 by berson and yalow to test the blood Insulin .

Radioimmunoassay (RIAs) utilize a radioactive label (usually 125I, 3H or 14C), which emits radiation that can be measured with a beta or gamma counter.BUT radioactive materials are not administered to the individual but are used as reagents.

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The technique of radioimmunoassay used in

many research and clinical practice in many areas:

Diagnosis of allergy

Blood Banking

Endocrinology

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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]

Principle of Radioimmunoassay

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A mixture is prepared of

radioactive antigen

antibodies against that antigen.

Known amounts of unlabeled ("cold") antigen are added to samples of the mixture. These compete for the binding sites of the antibodies.

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+

+P

P*

P*Q

Radioactive tag

Analyte

Binding agentFree Bound

QPQ

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At increasing concentrations of unlabeled antigen,

an increasing amount of radioactive antigen is displaced from the antibody molecules.

The antibody-bound antigen is separated from the free antigen in the supernatant fluid, and

The radioactivity of each is measured.

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1. Preparation & characterisation of the Antigen

[Ligand to be analysed] 2. Radiolabelling of the Antigen3. Preparation of the Specific Antibody4. Development of Assay System

Requirements of RIA

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Antigens prepared by..

Synthesis of the molecule Isolation from natural sources

Radiolabelling [Tagging procedure] 3 H , 14 C, 125 I are used as radioactive tags Antigens are tagged to 3 H , 14 C, 125 I Tagging should NOT affect Antigenic

specificity & Antigenic activity !

Preparation and radiolabeling of the

antigen

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Antigen injected intradermally into rabbits or

guinea pigs antibody production

Antibodies recovered from the serum.

Preparation of the specific antibody

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Crucial step is separation of unbound antigens

Antibodies bind to microtitre well surface [Solid phase RIA]

Antigens bound to the fixed antibodies remain stuck to the inner surface

Decanting & washing the well removes unbound antigens

Other techniques of separation: Centrifugation, Precipitation and Electrophoresis

Development of the assay system

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Video for RIA

https://www.youtube.com/watch?v=HOHczdglqHI

https://www.youtube.com/watch?v=q4dY52v-IsY

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Radioimmune procedure

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From these data, a

standard binding curve, like thee one shown in red, can be drawn.

The samples to be assayed (the unknowns) are run in parallel.

Calibration Curve

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General uses

In Endocrinology Insulin, HCG, Vasopressin Detects Endocrine Disorders Physiology of Endocrine Function

In Pharmacology Morphine Detect Drug Abuse or Drug Poisoning Study Drug Kinetics

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Clinical Immunology

Antibodies for Inhalant Allergens Allergy Diagnosis

Oncology Carcinoembryonic Antigen Early Cancer Detection and Diagnosis

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tracking of leukemia virus

diagnosis and treatment of peptic ulcers

research with Neurotransmitters

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Screening donated blood for evidence of viral

contamination by HIV-1 and HIV-2 (presence of anti-HIV

antibodies) Hepatitis C (presence of antibodies) Hepatitis B (testing for both antibodies and a

viral antigen) Measuring hormone levels

HCG (as a test for pregnancy)

APPLICATIONS

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Negative :

Positive:

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LH (Luteinizing hormone , determining the time of ovulation)

TSH, T3 and T4 (for thyroid function) Hormones (e.g., anabolic steroids, HGH) that may

have been used illicitly by athletes.

Detecting infections sexually-transmitted agents like HIV, syphilis, and chlamydia hepatitis B and C Toxoplasma gondii

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Detecting allergens in food and house dust .

RAST: The radioallergosorbent test to detect

specific IgE antibodies to suspected or known allergens . IgE is the antibody associated with type I allergic response : Pollen (is a fine to coarse powder containing the microgamatophytes of seeds)

The amount of radioactivity is proportional to the serum IgE for the allergen.

RAST rating

IgE level (KU/L) COMMENT

0 < 0.35 Absent or undetectable allergen specific IgE

1 0.35 - 0.69 Low level of allergen specific IgE

2 0.70 - 3.49 Moderate level of allergen specific IgE

3 3.50 - 17.49 High level of allergen specific IgE

4 17.50 - 49.99 Very high level of allergen specific IgE

5 50.0 - 100.00 Very high level of allergen specific IgE

6 > 100.00 Extremely high level of allergen specific IgE 36

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Measuring "rheumatoid factors" and other auto antibodies in autoimmune diseases like lupus erythematosus.

Measuring toxins in contaminated food Detecting illicit drugs, e.g.,

cocaine opiates Δ-9-tetrahydrocannabinol, the active

ingredient in marijuana

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Detection of drug:

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ADVANTAGES Radioimmunoassay is widely-used

because of its great sensitivity.

Using antibodies of high affinity, it is possible to detect a few pictograms (10−12 g) of antigen in the tube.

The greater the specificity of the antiserum, the greater the specificity of the assay

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RIA has become a major tool in the clinical

laboratory where it is used to assay . plasma levels of:

most of our hormones; digitoxin or digoxin in patients receiving

these drugs; certain abused drugs.

Presence of hepatitis B surface antigen (HBsAg) in donated blood.

Anti-DNA antibodies in systemic lupus erythematosus (SLE).

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DRAWBACKS

The main drawbacks to radioimmunoassay are the expense and hazards if preparing and handling the radioactive antigen.

Both 125I or 131I emit gamma radiation that requires special counting equipment;

The body concentrates iodine atoms — radioactive or not — in the thyroid gland where they are incorporated in thyroxine (T4).

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Labs require special license to

handle radioactive material

Requires special arrangements for Requisition storage of radioactive

material radioactive waste disposal.

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http://www.discoveriesinmedicine.com/Ni-Ra/R

adioimmunoassay-RIA.html http://www.encyclopedia.com/medicine/divisio

ns-diagnostics-and-procedures/medicine/radioimmunoassay

Textbook of microbiology by Prescott, Harley. Practical analytical chemistry by BECKEET. http://www.millipore.com/immunodetection/

id3/radioimmunoassay.

References

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