Merck Free Digital Learning Webinar Series 1 5/19/2020

Basic Immunobiology techniques

Radioimmunoassay

Ankur Gautam, PhD.,

Merck High End Skill Development Centre
Chandigarh, India
18th May, 2020

Merck Free Digital Learning Webinar Series 1

Basic Immunobiology techniques

Diagnosis of fungal diseases Detection and localization Detection and

Presence of Ag or Ab in serum of Ag on tissue quantitation of Ag
Immunoelectrophoresis Immunohistochemistry Radioimmunoassay
20.04.2020 04.05.2020 18.05.2020

27.04.2020 11.05.2020
Immunoblotting ELISA
Detection of specific protein in Detection and quantitation of Ag
mixture (Gene Expression)

Immunoassay
Immuno: Related to antigen and antibody

Assay : Test Or Technique

A Test to detect or quantify biological substances using specific

antibody-antigen reaction.

https://knowyourphrase.com/needle-in-a-haystack https://www.shutterstock.com/image-photo/hand-lens-that-magnifies-needle-haystack-173361524

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Merck Free Digital Learning Webinar Series 1 5/19/2020
Basic Immunobiology techniques

Labels in Immunoassay

❑ Either antigen or antibody can be labeled or tagged.

Ab

❑ Labels could be fluorophore, Enzyme, Radioisotopes,

Chemiluminescent compound. Label Label

Ag Ag

❑ Labeled components also called tracer. Ab

Types of Immunoassay Enzyme

❑ Enzyme-linked Immunosorbent assay (ELISA)

❑ Fluro immunoassay (FIA)

❑ Chemiluminescence immunoassay (CLIA)

❑ Radioimmunoassay (RIA)

Label
Immunoassays
Antibody
Unlabeled labeled

Ag
Antigen

Immunoelectrophoresis

Immunoblotting

Immunohistochemistry ELISA

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RIA

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Merck Free Digital Learning Webinar Series 1 5/19/2020
Basic Immunobiology techniques

Outline - RIA

❑ Introduction

❑ Principle

❑ Procedure

❑ Types

❑ Advantages and disadvantages

❑ Applications

https://www.alamy.com/stock-image-rosalyn-yalow-1977-167023231.html

Radioimmunoassay

Method of quantifying minute amounts of biological substances in the body

using Ag-Ab interaction and with the use of radioactive label.

History
❑ Rosalyn Yalow and Solomon A. Berson

❑ Veterans Administration (VA) hospital in the Bronx, New York

❑ Dicovered RIA in 1959. Rosalyn Yalow

(1921–2011)
❑ Substances tracked: Insulin

Nobel Prize in Physiology or Medicine 1977

Solomon A. Berson
(1918–1972)

https://www.nobelprize.org/womenwhochangedscience/stories/rosalyn-yalow

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Basic Immunobiology techniques

RIA Principle

RIA determines the concentration of antigen (Ag) in a given sample based on………..

❑ Competitive binding 125I

❑ Radiolabeled antigen (Ag*) and unlabeled

antigen (Ag) competes for its binding site on

fixed amount of specific high affinity Ab.

Insulin

labeled Unlabeled
Unlabeled Insulin Labeled Insulin

Unlabeled Insulin Labeled Insulin

Ab for insulin

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Types of RIA

Qualitative Quantitative

Ag
Serum

Amount = ? ng/ml

No antigen

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Types of RIA

Ab
Solid Phase

Solid surface

Ab

Liquid Phase
Buffer

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Basic Immunobiology techniques

QUIZ

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Q1. Which of the following statement is not correct for RIA:

(A). It is an immunoassay
(B). It is used radioisotope label
(C). It was discovered in 1977
(D). Rosalyn Yalow discovered the RIA

Correct Answer: C

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Q2. Which of the following is not come under labeled immunoassay:

(A). ELISA
(B). Immunoelectrophoresis
(C). Immunohistochemistry
(D). Immunoblotting

Correct Answer: B

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Basic Immunobiology techniques

Requirements

Sample: Concentration of Ag needs to be determined in a sample Antigen X

Antigen X*: Labeled with radioisotope (fixed amount)

Antigen X: Unlabeled (0.1, 1.0, 10 and 100 ng)

Gamma counter

Anti-X antibody: Fixed amount (limiting)

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Radioisotopes
-
Neutron
Atoms of the same element having
+ Proton
the same number of protons
- Electron but different number of neutrons

- - - Unstable

+ + +
Proton same
Neutron Different Emit particles
or radiations
Protium Deuterium Tritium
1H 2H 3H

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Solid Phase RIA (Procedure) Coat Ab (equal amt)

Add labeled Ag* (equal amt)

in all wells

Washings
C 1 2 3 4 S

All wells - fixed amt. of antibody (anti X antibody) Add increasing conc. Of
unlabeled Ag in wells 1-4
C: Only Labeled Ag X* (no unlabeled Ag X)

1: Labeled Ag X* (fixed) & 0.1 ng of unlabeled Ag X Washings

2: Labeled Ag X* (fixed) & 1.0 ng of unlabeled Ag X
3: Labeled Ag X* (fixed) & 10 ng of unlabeled Ag X
4. Labeled Ag X* (fixed) & 100 ng of unlabeled Ag X Add sample (x ng) in well S

S: Labeled Ag X* (fixed) & sample (x ng Ag X)

Detection

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Merck Free Digital Learning Webinar Series 1 5/19/2020
Basic Immunobiology techniques

Procedure
0.1 ng
1.0 ng

Washing Washing

Radioactivity 100% Radioactivity 90% Radioactivity 70%

Sample
10 ng 100 ng X ng

Washing Washing

Radioactivity 50% Radioactivity 30% Radioactivity 60%

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Concentration of
unlabeled Antigen X
Radioactivity (%)
Procedure
(ng)
0.0 100
100
0.1 90
1.0 70 80
Radioactivity (%)

10 50
60 Concentration of
100 30 antigen 5 ng in sample
1000 10 40

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0.0
0 0.1 1 10 100 1000
Concentration of unlabeled Antigen X (ng)

Concentration of Radioactivity (%)

Antigen X in sample
(ng)
5 60

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Liquid Phase RIA

Ag * Ag

2° Ab Precipitation

Bead

Gamma counter

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Merck Free Digital Learning Webinar Series 1 5/19/2020
Basic Immunobiology techniques

RIA Principle

Ab + Ag* Ab-Ag* 100% Radioactivity

+
Ag*: Labelled
Ag Ab-Ag Decrease in Radioactivity Ag : Unlabeled

The proportion of Ab-Ag* and Ab-Ag at equilibrium will be equal to

the original proportion to Ag* and Ag

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QUIZ

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Q3. Which of the following component is limiting in Radioimmunoassay:

(A). Antibody
(B). Labeled antigen
(C). Unlabeled antigen
(D). Non of the above

Correct Answer: A

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Basic Immunobiology techniques

Q4. Radioimmunoassay is a type of

(A). Uncompetitive immunoassay
(B). Noncompetitive immunoassay
(C). Competitive immunoassay
(D). Both A & B

Correct Answer: C

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Q5. Which is the correct order of steps for performing RIA

(A). Ab coating, addition of labeled Ag, detection, and addition of unlabeled Ag

(B). Ab coating, addition of unlabeled Ag, addition of labeled Ag, and detection
(C). Ab coating, addition of labeled Ag, addition of unlabeled Ag and detection
(D). Addition of labeled Ag, Ab coating, addition of unlabeled Ag and detection

Correct Answer: C

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Advantages

❑ Highly specific (Ab-Ag reaction)

❑ Highly sensitive (radioactivity)

❑ Can detect antigens in nanogram or picogram

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Basic Immunobiology techniques

Disadvantages

❑ Not all labs can used (radioactivity facility not available)

❑ Isotope half lives is short (e.g. 125I is 60 days).

❑ Exposure to radioactivity- Hazardous.

❑ Instrument is expensive (gamma counter, beta counter).

❑ Time consuming.

ELISA was invented to overcome the above limitations

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RIA Vs ELISA
Characteristics RIA ELISA
Sensitivity High Moderate to high
Cost High Low
Time duration More Less
Ease of handling Tedious easy
Disposal Care has to be taken No special care required

Radiation hazard Hazardous No hazard

Equipment Complex Easy
Mechanism Based on measurement Based on measurement
of radioactivity of enzymatic activity

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Applications
Hormone

Infectiou
Vitamins
s agents

RIA

Early Serum
detection proteins

Drugs

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Merck Free Digital Learning Webinar Series 1 5/19/2020
Basic Immunobiology techniques

QUIZ

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Q6. Which of the assay is used for localization of antigen on tissue

(A). Immunoblotting
(B). Immunohistochemistry
(C). Radioimmunoassay
(D). Immunoelectrophoresis

Correct Answer: B

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Q7. In which techniques multiple antigens can be detected

(A). ELISA and Immunoelectrophoresis
(B). Immunoelectrophoresis and Radioimmunoassay
(C). Immunohistochemistry and Radioimmunoassay
(D). Immunohistochemistry and immunoblotting

Correct Answer: D

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Merck Free Digital Learning Webinar Series 1 5/19/2020
Basic Immunobiology techniques

Our Team
anil-kumar.badana@external.mergroup.com

ankur.gautam@external.merckgroup.com

senthil.karunakaran@merckgroup.com

parul.tomar@merckgroup.com

pankajkumar.joshi@merckgroup.com

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THANK YOU

New students please send their details

ankur.gautam@external.merckgroup.com
parultomar@merckgroup.com

https://www.linkedin.com/groups/10544590/

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