Radioimmunoassay (RIA) &

Enzyme Linked Immunosorbent

Assay (ELISA)

By Jazira Tumusiime
BMLS, MMLS student

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Radioimmunoassay (RIA)
 When radioisotopes instead of enzymes are used as labels to
be conjugated with antigens or antibodies, the technique of
detection of the antigen–antibody complex is called as
radioimmunoassay (RIA).

 RIA was first described in 1960 for measurement of

endogenous plasma insulin by Solomon Berson and Rosalyn
Yalow of the Veterans Administration Hospital in New York.

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Radioimmunoassay (RIA)-2
 The classical RIA methods are based on the principle of competitive
binding.

 In this method, unlabeled antigen competes with radiolabeled

antigen for binding to antibody with the appropriate specificity.

 Thus, when mixtures of radiolabeled and unlabeled antigen are

incubated with the corresponding antibody, the amount of free (not
bound to antibody) radiolabeled antigen is directly proportional to
the quantity of unlabeled antigen in the mixture.

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Principle of Radioimmunoassay
 Principle: Uses an immune reaction
[Antigen – Antibody reaction] to
estimate a ligand

Ag + Ag* + Ab  AgAb + Ag*Ab + Ag + Ab*

Unbound Ag* and Ag washed out

Radioactivity of bound residue measured
Ligand conc is inversely related to
radioactivity

[Ag : ligand to be measured ; Ag*

radiolabelled ligand] 4
Advantages & Disadvantages of RIA
 Advantages
Highly specific: Immune reactions are specific
High sensitivity : Immune reactions are sensitive

 Disadvantages
Radiation hazards: Uses radio-labelled reagents
Requires specially trained persons
Labs require special license to handle radioactive material
Requires special arrangements for
• Requisition, storage of radioactive material
• radioactive waste disposal.

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Requirements for RIA
1. Preparation & characterisation of the Antigen [Ligand
to be analysed]
2. Radiolabelling of the Antigen
3. Preparation of the Specific Antibody
4. Development of Assay System

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Preparation & Radiolabelling of the Antigen
 Antigens prepared by..
Synthesis of the molecule
Isolation from natural sources

 Radiolabelling [Tagging procedure]

 3 H 14 C 125 I are used as radioactive tags
Antigens are tagged to 3 H 14 C 125I
Tagging should NOT affect Antigenic specificity & Antigenic activity

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Development of the Assay System
 A crucial step is the separation of unbound
antibodies.

 This is achieved by binding the antibodies to

the microtitre well surface [Solid phase RIA]

 Antigens(in the sample) bind to the fixed

antibodies remain stuck to the inner surface

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Assay Procedure
 Add known amounts of the test sample + labelled antibody into the
microtitre wells
 Incubate  allow the reaction to reach completion
 Decant & wash contents of the well  removes all unbound antigens
 Radioactivity remaining in the Microtitre wells measured by a Counter
[GM counter , Scintillation counter etc]
 Intensity of radioactivity is inversely correlated with the conc of antigens
in the test sample
 Sensitive to very low conc of antigens

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Enzyme Linked Immunosorbent Assay
(ELISA)
 Enzyme immunoassays (EIAs) can be used for detection of either antigens or
antibodies in serum and other body fluids of the patient.

 In EIA techniques, antigen or antibody labeled with enzymes are used.

 Alkaline phosphatase, horseradish peroxidase, and galactosidase are the

enzymes used in the EIA tests.

 The commonly used EIAs are enzyme-linked immunosorbent assays (ELISAs).

 The ELISA technique was first conceptualized and developed by Peter

Perlmann and Eva Engvall at Stockholm University, Sweden. 11
Enzyme Linked Immunosorbent Assay (ELISA)-2
 These assays involve the use of an immunosorbent specific to either the
antigen or antibody.
 Following the antigen– antibody reaction, chromogenic substrate specific to
the enzyme (o- phenyldiamine dihydrochloride for peroxidase, p-nitrophenyl phosphate for
alkaline phosphatase, etc.) is added.
 The reaction is detected by reading the optical density.
 Usually, a standard curve based on known concentrations of antigen or
antibody is prepared from which the unknown quantities are calculated.
 There are different types of ELISAs available for the detection and quantitation
of either the antigen or antibodies in serum and other body fluids.
These include:
indirect ELISA,
sandwich ELISA,
competitive ELISA,
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Enzyme Linked Immunosorbent Assay-3
 Principle:
Uses an immune reaction like RIA
Differs from RIA in detection method
Detection based on
• Enzyme catalysed reaction OR
• Fluorescent probe
• NOT radioactivity [great advantage!]

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Advantages of ELISA
 Sensitive:
 Reproducible
 Minimal reagents
 Qualitative & Quantitative
Qualitative  Eg HIV testing (Pos or neg)
quantitative assays  Eg Ther. Drug Monitoring
 Greater scope : Wells can be coated with Antigens OR Antibodies
 Suitable for automation high speed
 NO radiation hazards

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Types of ELISA
1. Non-competitive binding assay or Sandwich method
Antigen measuring system [Titrewells coated with antibodies ; Enzyme labelled
antibodies]
Antibody measuring system [Titrewells coated with antigens ; Enzyme labelled
antiantibodies]

2. Competitive binding assay [Titrewells coated with antibodies ; Enzyme

labelled antigens]

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Noncompetitive or Sandwich Assay
 Antigen measuring system
Titre wells coated with suitable antibody
Add patient sample containing the antigen
Incubate: till antigen antibody reaction is complete
Wash remove unbound antigen
Add Antibody labelled with Enzyme
Incubate till antigen binds labelled antibody
Wash  remove unbound labelled antibody
Add substrate ; incubate
Enzyme + Substrate  Product  measure colour
Colour proportional to antigen in patient sample

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Noncompetitive or Sandwich Assay
 Antibody measuring system
Titre wells coated with suitable antigen
Add patient sample containing the antibody
Incubate: till antigen antibody reaction is complete
Wash remove unbound antibody
Add Antiantibody labelled with Enzyme
Incubate till labelled antiantibodies binds antigen-antibody complex
Wash  remove unbound labelled antiantibody
Add substrate ; incubate
Enzyme + Substrate  Product  measure colour
Colour proportional to antibody in patient sample

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Competitive binding assay
 Titrewells coated with antibodies
 Known quantities of patient sample containing antigen + antigen
labelled with enzyme
 Incubate: till antigen antibody reaction is complete
 Wash remove unbound antigens
 Add substrate ; incubate
 Enzyme + Substrate  Product  measure colour
 Colour inversely related to antigen in patient sample

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Enzyme labels
 Enzyme labels should have high specific reactivity
 Should be easily coupled to ligands & the labelled complex must be
stable
 The reactivity should be retained after linking of the enzyme to the
antigen/antibody
 The chosen enzymes should not be normally present in the patient
samples
 Examples of enzyme labels
Horse radish peroxidase, Alkaline phosphatase, Glucose oxidase

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Applications of Immunoassays [RIA & ELISA]
 Analysis of hormones, vitamins, metabolites, diagnostic markers
Eg. ACTH, FSH, T3, T4, Glucagon, Insulin, Testosterone, vitamin B12,
prostaglandins, glucocorticoids,
 Therapeutic drug monitoring:
Barbiturates, morphine, digoxin,
 Diagnostic procedures for detecting infection
HIV, Hepatitis A, B etc

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Applications of Immunoassays [RIA & ELISA]
 Serum Antibody Concentrations
 Detecting potential food allergens
(milk, peanuts, walnuts, almonds and eggs)
 Disease outbreaks- tracking the spread
of disease e.g. HIV, bird flu, common, colds, cholera, STD etc
 Detections of antigens
e.g. pregnancy hormones, drug allergen, GMO, mad cow disease
 Detection of antibodies in blood sample for past exposure to disease
e.g. Lyme Disease, trichinosis, HIV, bird flu

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Assignment
Write out the principle and procedures of the
following;
Widal test
 Brucella agglutination test
 Pregnancy test
 Rheumatoid factor test

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