Ria.done

Presented by:Presented by: Dr. Saba Ahmed M.Phil. Pharmacology

ImmunoassayImmunoassay

Bioanalytical methods in which the quantitation of the analyte

depends on the reaction of an antigen (analyte) and an antibody

(Kellner R et. al., 1998)

Immunoassays that use radioactive isotopes -radioimmunoassay

Developed in 1959 by Rosalyn Yalow and Solomon Berson for measurement of insulin in plasma.

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

In 1977 Yalow received the Nobel Prize for her and Berson’s development of RIA

HistoryHistory

RadioimmunoassayRadioimmunoassay (RIA)An in vitro assay - measures the presence of an antigen with

very high sensitivity & utilize radioactive isotopes as a label

The amount of radioactivity measured is indicative of the amount of analyte present

Nanomolar and picomolar conc. of hormones in biological fluids can be analyzed (Patrono, C. and Peskar 1987)

Heterogenous, competitive assay

PrinciplePrincipleBased on a competitive binding reaction between

-Fixed amount of a labeled analyte ( Ag*) -Variable amount of unlabeled sample analyte (Ag)

For a limited amount of binding sites on a highly specific antibody

Essential Components Of RIAEssential Components Of RIA

• Tracer: labeled antigen• Antibody• Standards antigen: Known concentrations of

unlabeled antigen• Unknown samples• Separation method• Method for detection of the label

1.Tracer: labeled antigen1.Tracer: labeled antigen

Radioactive labels 3 H 14 C 125 I 131 I (Weeks I. 1992) (Simmonet F. et.al.,1993)

Radioisotopes of 3 H tritium (β-emitter) and 125 I 131 I iodine ( γ- emitter) - incorporated into either antigen or antibody

Tagging should NOT affect -Antigenic specificity - Antigenic activity

The labeled antigen (tracer) must be present in low conc.

Once labeled, it must maintain the same characteristics of the unlabeled antigen

14 C & 3 H - draw back of long half lives(5740 and 12.3 years respectively) - more difficult to measure, requiring cumbersome liquid scintillation counting

125 I is preferred over 131 I because of its longer half life, ease of handling, and higher counting efficiency.

2. Antibody2. Antibody

Can be either polyclonal or monoclonal

Immunizing an animal (e.g. rabbit) with the substance Ab production recovered from the serum

Some ligands are not Antigenic - need the help of an adjuvant like Freund’s complete adjuvant that helps immune response

◦ Hormones, Steroids, Drugs Haptens◦ Haptens conjugated to albumin antigenic

9

Polyclonal antibodies

Made by injecting an animal with the antigen, then purifying the antibody from serum.

Antiserum contains a mixture of antibodies - recognize and bind to the same antigen, but attach to different epitopes.

Monoclonal antibodies

Produced from hybridoma - production of very specific antibodies that bind only to one antigen epitope

3. Standard Antigen3. Standard Antigen

It is the same Ag that was injected into an animal for antibody production.

Before the antigen can be used as a standard

- specificity between this Ag & the Ag in test sample toward the antibody binding sites must be clearly established

An international organization, like WHO, issues standard preparations

Characteristics Of Standard Antigen:

Should be available in large quantity

Should not contain substances which can interfere with assays

Should be highly purified

Should be available in a form which allows convenient and accurate preparation for RIA

Development Of A Standard CurveDevelopment Of A Standard Curve In order to conduct RIA-a standard curve first to be made

Incubate fixed amount of tracer and antibody in the presence of different conc. of standard (unlabeled antigen).

Two typical RIA - Standard Curves - generated

- plotting %age of tracer bound against unlabeled compound

- plotting %age inhibition of (labeled compound)

binding A* against unlabeled compound

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Typical RIA Standard CurveTypical RIA Standard Curve


Radioimmunoassay Procedure:Radioimmunoassay Procedure:

Standard Curve & Unknown SampleStandard Curve & Unknown Sample

Separation TechniquesSeparation Techniques

InstrumentationInstrumentation

Two most vital equipments :

Centrifuge

Radioactive Counters


pellet is formed at the bottom of

the test tube

pellet is formed at an angle


RADIOACTIVE COUNTERSRADIOACTIVE COUNTERS


Gamma CounterGamma Counter

Competitive RadioimmunoassayCompetitive Radioimmunoassay

Competition between labeled and unlabeled antigen being detected for a limited number of binding sites on antibody

Depending on whether the solid phase is coated with either the antibody or the antigen (analyte)

- Antigen-capture (Darwish IA. et. al., 2001)- Antibody-capture ( Darwish IA. et. al., 2009)

Non Competitive Immunoradiometric Assay (IRMA)Non Competitive Immunoradiometric Assay (IRMA)

Analyte to be measured is 'sandwiched' between two antibodies.

1st antibody is coated onto solid support.

2nd antibody is radiolabeled for detection.

The analyte - bound by both antibodies to form a 'sandwich' complex.

Advantage Faster reaction rate & sensitivity (Lin Z et. al., 2008)

TYPES OF RADIOIMMUNOASSAYSTYPES OF RADIOIMMUNOASSAYS

1. Single Antibody RIA1. Single Antibody RIA (Charcoal-dextran Separation)

Used for measurement of steroid hormones.

Inexpensive but less sensitive than other methods

4. Centrifuge & decant supernatant containing Ab-Ag complexes

5. Count radioactivity of liquid phase in a β- scintillation counter.

2. Double Antibody RIA:2. Double Antibody RIA:

Precipitation of bound complexes with a second antibody

Used for both steroid and protein assays.

Has good sensitivity

But requires an additional incubation period that can prolong assay time.

4.Centrifuge to separate free hormone from bound complexes.

5. Decant supernatant & radioactivity in the pellet is counted in γ - counter.

3. Solid - Phase RIA3. Solid - Phase RIA

The radioactivity remaining in the tube is counted in a gamma counter.

Solid - Phase RIA:Solid - Phase RIA: Used for protein and steroid hormones.

Polystyrene test tubes, microtiter plates are used for this purpose.

Provides a simple way to separate bound and free reactants.

Advantage Centrifugation is not required to separate bound

complexes from free antigen.◦ (Janine Brown)◦ Endocrine Manual For Reproductive Assessment Of Domestic And Non-domestic Species)

Disadvantages Of RIA Disadvantages Of RIA • Radiation hazards• Requires specially trained persons• Labs require special license to handle radioactive material

• Requires special arrangements for Requisition, handling, storage of radioactive material radioactive waste disposal.

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

Short half-life time of the isotope Expensive instrumentation for the counting of radioactivity.

Advantages Of RIA:Advantages Of RIA:

◦ Highly specific: Immune reactions are specific

◦ High sensitivity : Immune reactions are sensitive

◦ Detect few picograms (10−12 g) of antigen in the tube.

Screening donated blood◦ Hepatitis C ◦ Hepatitis B

Measuring hormone levels ◦ LH◦ TSH, T3 and T4◦ Hormones (e.g., anabolic steroids, HGH)

APPLICATIONSAPPLICATIONS

Analysis of hormones, vitamins, metabolites, diagnostic markers .

o ACTH, FSH, Glucagon, Insulin, Testosterone, o Vitamin B12, Prostaglandins, Glucocorticoids

Early Cancer Detection and Diagnosis

Measuring toxins in contaminated food

Therapeutic drug monitoring: ◦ Barbiturates, morphine, digoxin,

Detecting infections ◦ sexually-transmitted agents like◦ HIV, syphilis, and chlamydia ◦ Hepatitis B and C

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

Detecting illicit drugs, e.g., ◦ cocaine ◦ opiates ◦ Δ-9-tetrahydrocannabinol,

0r drug poisoning

ReferencesReferences Blake DA. One-step competitive immunoassay for cadmium ions: development

and validation for environmental water samples. Anal. Chem. 73 (2001) 1889–1895.

Darwish IA, Blake DA. One-step competitive immunoassay for cadmium ions: development and validation for environmental water samples. Anal. Chem. 73 (2001) 1889–1895

Janine Brown, Ph.D.Sue Walker, M.S.Karen Steinman, B.S.Conservation & Research CenterSmithsonian's National Zoological Park1500 Remount RoadFront Royal, Virginia, 22630

Kellner R, Mermet JM, Otto M, Widmer HM. Analytical Chemistry.New York: Wiley-VCH. 1998; pp405-429. :

Patrono, C. and Peskar, B.A. (eds) Radioimmunoassay in basic and clinical pharmacology. Heidelberg, Springer-Verlag, 1987

Lin Z, Wang X, Li ZJ, Ren SQ, Chen GN, Ying XT, Lin JM. Development of a sensitive, rapid, biotin–streptavidin based chemiluminescent enzyme immunoassay for human thyroid stimulating hormone. Talanta 75 (2008) 965-972.

Simmonet F, Guilloteau D. in: R. F. Masseyeff, W. H. Albert, N. A.Staines (Eds.). Method of Immunological Analysis. New York: VCH. 1993; 1: 270-282

Weeks I. Chemiluminescence Immunoassay. G. Sevehla (Eds.).Amsterdam: Elsevier. 1992; 24: 18-118.

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