Immunoassay is a bioanalytical technique that quantifies an analyte using the interaction between an antigen and its corresponding antibody. Radioimmunoassay (RIA) is an immunoassay technique that utilizes radioactive isotopes, such as iodine-125, as labels. RIA was developed in 1959 and allowed for the first in vitro measurement of hormone levels in blood plasma. A key component of RIA is the standard curve, which is generated using known concentrations of unlabeled antigen and is used to quantify unknown samples. RIA provides high sensitivity and specificity but also involves risks associated with the use of radioactive materials.

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

2. 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

3.  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

4. 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

5. PrinciplePrinciple
 Based 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

6. 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

7. 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

8.  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.

9. 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

10.  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.

11.  Monoclonal antibodies
 Produced from hybridoma - production of very specific
antibodies that bind only to one antigen epitope

12. 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

13. 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

14. 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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15. Typical RIA Standard CurveTypical RIA Standard Curve
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16. Typical RIA Standard CurveTypical RIA Standard Curve
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17. Radioimmunoassay Procedure:Radioimmunoassay Procedure:

19. Standard Curve & Unknown SampleStandard Curve & Unknown Sample

20. Separation TechniquesSeparation Techniques

21. InstrumentationInstrumentation
Two most vital equipments :
 Centrifuge
 Radioactive Counters
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22. pellet is formed at
the bottom of the
test tube
pellet is formed at
an angle
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23. RADIOACTIVE COUNTERSRADIOACTIVE COUNTERS
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24. Gamma CounterGamma Counter

25. 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)

28. 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)

30. 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

31. 4. Centrifuge & decant supernatant containing Ab-Ag
complexes
5. Count radioactivity of liquid phase in a β- scintillation
counter.

32. 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.

33. 4.Centrifuge to separate free hormone from bound complexes.
5. Decant supernatant & radioactivity in the pellet is counted
in γ - counter.

35. 3. Solid - Phase RIA3. Solid - Phase RIA
 The radioactivity remaining in the tube is counted in a gamma
counter.

36. 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)

37. Disadvantages Of RIADisadvantages 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.

38. 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.

39.  Screening donated blood
◦ Hepatitis C
◦ Hepatitis B
 Measuring hormone levels
◦ LH
◦ TSH, T3 and T4
◦ Hormones (e.g., anabolic steroids, HGH)
APPLICATIONSAPPLICATIONS

40.  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,

41.  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

42. 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

43.  Weeks I. Chemiluminescence Immunoassay. G. Sevehla
(Eds.).Amsterdam: Elsevier. 1992; 24: 18-118.
 Pharmaceutical Drug Analysis 2nd
edition
 Slideshare.com

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