RADIOIMMUNOASSAY (RIA) 3

8/2/2019 RADIOIMMUNOASSAY (RIA) 3

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RADIOIMMUNOASSAY (RIA)

Enzyme-Linked ImmunosorbantAssay (ELISA)


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Introduced in 1960 by Berson and Yalow as an assayfor the concentration of insulin in plasma. Itrepresented the first time that hormone levels in thebloodcould be detected by an in vitro assay.

Radioimmunoassay (RIA) The technique used inmany areas, e.g.,

Blood banking

Diagnosis of allergies

Endocrinology most of our hormones ;

digitoxin or digoxin in patients receiving these drugs ;

certain abused drugs

for the presence of hepatitis B antigen (HBsAg) indonated blood ;

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


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The Technique

A mixture is prepared of

radioactive antigen Iodine atoms can be introduced into tyrosine residues in a protein,

the radioactive isotopes125I or131I are often used.

antibodies against that antigen.

Known amounts of unlabeled ("cold") antigen areadded 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 displacedfrom the antibody molecules.

The antibody-bound antigen is separated from the free

antigen in the supernatant fluid, and


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Separating Bound from Free 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).


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Gamma Counter


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the radioactivity of each is measured.

From these data, a standard binding curve,

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 antigenconcentrations can be read directly from thestandard curve


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4Ag* + 4 Ab 4Ag*Ab

4Ag + 4Ag* + 4 Ab 2Ag*Ab + 2AgAb + 2Ag* + 2 Ab

12Ag + 4Ag* + 4 Ab Ag*Ab + 3AgAb + 3Ag* + 9Ag


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Radioimmunoassay is widely-used because

of its great sensetivity.

it is possible to detect a few picograms (1012g) of antigen in the tube.

The main drawbacks to radioimmunoassay

are the expense

hazards if preparing and handling the

radioactive antigen. Both 125I or131I emit gamma radiation that

requires special counting equipment;


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Despite these drawbacks, RIA has become amajor 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

for the presence of hepatitis B surface antigen(HBsAg) in donated blood;

anti-DNA antibodies in systemic lupuserythematosus (SLE).


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Enzyme-Linked Immunosorbent Assay

(ELISA)

ELISA is a widely-used method for measuringthe concentration of a particular molecule

(e.g., a hormone or drug) in a fluid such as

serum or urine. It is also known as enzyme

immunoassay orEIA .

The molecule is detected by antibodies that

have been made against it; that is, for which it

is the antigen. Monoclonal antibodies areoften used.


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The test requires :

the antibodies fixed to a solid surface,

such as the inner surface of a test tube ;

a preparation of the same antibodies

coupled to an enzyme. This is one (e.g. B-

galactosidase that produces a coloredproduct from a colorless substrate .


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The tubes are filled with the antigen solution (e.g., urine) tobe assayed. Any antigen molecules present bind to theimmobilized antibody molecules .

The antibody-enzyme conjugate is added to the reactionmixture. The antibody part of the conjugate binds to anyantigen molecules that were bound previously, creating anantibody-antigen-antibody "sandwich ."

After washing away any unbound conjugate, the substratesolution is added .

After a set interval, the reaction is stopped (e.g., by adding1 N NaOH) and the concentration of colored productformed is measured in a spectrophotometer. The intensity

of color is proportional to the concentration of boundantigen .


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ELISA can also be adapted to measure the concentrationofantibodies .In this case ,

The wells are coated with the appropriate antigen .

The solution (e.g., serum) containing antibodies is added .

After they have had time to bind to the immobilizedantigen ,

an enzyme-conjugated anti-immunoglobulin is added,

consisting of an antibody against the antibodies being tested for. For example, if

human anti-HIV antibodies are being assayed, then antibodies(raised in a goat or rabbit against human immunoglobulins) areconjugated to

the enzyme.

After washing away unreacted reagent, the substrate isadded .

The intensity of the color produced is proportional to theamount of enzyme-labeled antibodies bound and thus to

the concentration of the antibodies being assayed.


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Some examples :

screening donated blood for evidence of viral contamination by HIV-1 and 2 presence of anti-HIV antibodies

Hepatitis B and C

measuring hormone levels

HCG as a test for pregnancy (

LH determining the time of ovulation (

TSH T3 and T 4

detecting infections sexually-transmitted agents like HIV, syphilis and chamydia

Toxoplasma gondii

detecting alleregens in food and house dust

measuring "rheumatoid factors" and other autoantibodies in autoimmunediseases like lupus erythematosus

measuring toxins in contaminated food

detecting illicit drugs, e.g ., cocaine


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Sandwich Method


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Direct and Indirect Methods


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Comparison of ELISA and RIA

ELISA RIA

Specificity + +

Sensetivity + +

Cost + -

Reagent shelf life + -

Fully automated - +

Safety + -

Small diagnostic laboratories + -


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Thank you

RADIOIMMUNOASSAY (RIA) 3

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