RADIOIMMUNOASSAYRADIOIMMUNOASSAY

Presented by:Chakravarthy.RY10MPH1203

Guided by:Dr. Abdul Rehaman Asst prof M.ph.,PhD.,

PHARMACEUTICS

NIRMALA COLLEGE OF PHARMACY

The technique was introduced in 1960 by Berson and Yalow as an assay for the concentration of insulin in plasma.

It represented the first time that hormone levels in the blood could be detected by an invitro assay.

Dr. Rosalyn Yalow became the firstfemale to win a Nobel Prize withher work on the radioimmunoassay

HISTORY

ANTIGENSANTIGENSAn antigen is a substance with the ability to

induce an immunological response. They

typically enter the body from an infection.

They are recognized at their epitopes by B

cells or by the T cell receptor on T cells.

Proteins or glycoproteins make the best

antigens because they are the best at

stimulating antigen recognition molecules.

Some immunoassays test for antigens, rather

than antibodies.

ANTIBODIESANTIBODIES

• Antibodies are produced by the

B-lymphocytes. They are glycoproteins

belonging to the “immunoglobulin supergene

family” that are produced in response to a

foreign substance in the body.

• Antibodies have a generally common structure,

but have regions that vary among them to

accommodate the specific antigens.

PRODUCTION OF PRODUCTION OF ANTIBODIESANTIBODIES

Polyclonal antibodies are more likely to

produce a false positive because they are less

specific to antigen epitopes and have varying

binding affinities.

◦ A mouse (or rabbit) is immunized by being

injected with an antigen; the antigen

generates an antibody response in the animal.

◦ The animal is bled and the antibodies are

collected.

LABELS IN LABELS IN IMMUNOASSAYSIMMUNOASSAYS

Immunoassays 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 its fluorescence .

PRINCIPLEPRINCIPLE

Radioimmunoassay (RIA) involves the separation of a protein (from a mixture) using the specificity of antibody - antigen binding and quantitation using radioactivity.

Radioimmunoassay (RIAs) utilize a radioactive label (usually 125I, 3H or 14C), which emits radiation that can be measured with a beta or gamma counter.

The technique of radioimmunoassay has revolutionized research and clinical practice in many areas, e.g.,

◦blood banking

◦diagnosis of allergies

◦endocrinology

THE TECHNIQUETHE TECHNIQUE

A mixture is prepared of ◦radioactive antigen Because of the ease with which iodine

atoms can be introduced into tyrosine residues in a protein, the radioactive isotopes 125I or 131I are often used.

◦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.

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.

Gamma Counter

CALIBRATION CURVECALIBRATION CURVE

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.

After determining the ratio of bound to free antigen in each unknown, the antigen concentrations can be read directly from the standard curve.

SEPARATING BOUND FROM SEPARATING BOUND FROM FREE ANTIGENFREE ANTIGEN

Precipitate the antigen-antibody complexes by adding a "second" antibody directed against the first.

For example, if a rabbit IgG is used to bind the antigen, the complex can be precipitated by adding an antirabbit-IgG antiserum (e.g., raised by immunizing a goat with rabbit IgG).

ADVANTAGESADVANTAGES

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

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).

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)

APPLICATIONSAPPLICATIONS

Negative :

Positive:

◦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

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 < 0.35Absent 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 > 100.00Extremely high level of allergen

specific IgE

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

Detection of drug: Detection of drug:

DRAWBACKSDRAWBACKSThe 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).

ISOTOPESISOTOPESISOTOPE HALF-LIFE USES

Carbon-11 20.3m Brain scans

Chromium-51

27.8d Blood Volume determination

Cobalt-57 270d Measuring vitamin B12 uptake

Cobalt-60 5.26y Radiation cancer therapy

Gadolinium-153

242d Determining bone density

Gallium-67 78.1 Scan for lung tumors

Iodine-131 8.07d Thyroid therapy

Iridium-192 74d Breast cancer therapy

Iron-59 45d Detection of anemia

Phosphorous-32

14.3d Detection of skin cancer or eye tumors

Plutonium-238

86y Power for pacemakers

Slenium-75 120d Pancreas scans

Sodium-24 15.0h Locating obstructions in blood flow

Technetium-99

6.0h Imaging of brain, liver, none marrow, kidney, lung or heart

Thallium-201

73h Detecting heart problems with treadmill stress test

Tritium 12.3y Determining total body water

Xenon-133 5.27d Lung imaging

REFARENCES

Textbook of microbiology by Prescott, Harley.

Practical analytical chemistry by BECKEET.

http://en.wikipedia.org/wiki/Radioimmunoassay

http://en.wikipedia.org/wiki/RIAhttp://en.wikipedia.org/wiki/RAST_testhttp://www.millipore.com/

immunodetection/id3/radioimmunoassay.

Thank“u”