RadioImmuno Assay and ELISA Importance and Applications

Radioimmunoassay
&
Enzyme Linked Immunosorbent Assay
Dr. R. SARAVANAN. Ph.D.
Professor&HOD
FACULTY OF PHARMACY, BIHER,
Chennai

Radioimmunoassay
• Radioimmunoassay (RIA) involves the identification &
separation of a protein (from a mixture) using the
specificity of antibody - antigen binding and
quantification using radioactivity
• The technique was introduced in 1960 by Berson and
Yalow as an assay for the concentration of insulin in
plasma.
• It represented the first time that hormone levels in the
blood could be detected by an in vitro assay

The virtues of a good radioimmunoassay
• PRECISION : ≈ reproducibility; characterized by
low inter-assay and intra-assay variability. (Both values
need to be < 10%)
• ACCURACY : are the figures approaching the real
concentration? (Use an independent approach to verify
e.g. a physicochemical technique)

The virtues of a good radioimmunoassay
• SENSITIVITY : how little can still be
detected? (Can be enhanced by pre-incubating
the cold hormone with the Ab, prior to tracer
addition)
• SPECIFICITY : lack of cross-reaction with
related molecules. Dilution curves of samples
and standards need to be parallel!

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]

Advantages of RIA
Highly specific: Immune reactions are
specific
High sensitivity : Immune reactions are
sensitive
Using antibodies of high affinity, it is possible
to detect a few picograms (10−12
g) of antigen
in the tube.

Disadvantages of RIA
Radiation hazards: Uses radiolabelled 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.

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

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 125
I
– Tagging should NOT affect Antigenic
specificity & Antigenic activity !

Preparation of the Specific Antibody
• Antigen injected intradermally into rabbits or
guinea pigs  antibody production
• Antibodies recovered from the serum
• Some ligands are not Antigenic
– Hormones, Steroids, Drugs  HAPTENS
– Eg: Gastrin, Morphine,
– Haptens conjugated to albumin  antigenic

Development of the Assay System
• A crucial step is separation of unbound antigens
• This achieved by binding the antibodies to the
microtitre well surface [Solid phase RIA]
• Antigens bound to the fixed antibodies remain
stuck to the inner surface
• Decanting & washing the well removes unbound
antigens
• Other techniques of separation: Centrifugation

Assay Procedure
• Add known amounts of the test sample + labelled antigen
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

RadioImmuno Assay and ELISA Importance and Applications

From the data, a standard
binding curve, like them one
shown in red, 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 antigen
concentrations can be read
directly from the standard
curve.

Radioimmunoassay laboratory of BRIT,
Navi Mumbai

A multi well automatic RIA counter

A radioimmunoassay in progress

A radioimmunoassay quantification using a
simple inexpensive counting instrument

Robotic sample processor for RIA/IRMA
that can process samples automatically

Applications of Radioimmunoassays
• Endocrinology
– Insulin, HCG, Vasopressin
– Detects Endocrine Disorders
– Physiology of Endocrine Function
• Pharmacology
– Morphine
– Detect Drug Abuse or Drug Poisoning
– Study Drug Kinetics

Applications of Radioimmunoassays
• Epidemiology
– Hepatitis B
• Clinical Immunology
– Antibodies for Inhalant Allergens
– Allergy Diagnosis
• Oncology
– Carcinoembryonic Antigen
– Early Cancer Detection and Diagnosis

• 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!]

Advantages of ELISA
• Sensitive: nanogram levels or lower
• Reproducible
• Minimal reagents
• Qualitative & Quantitative
– Qualitative  Eg HIV testing
– quantitative assays  Eg Ther. Drug Monitoring
• Greater scope : Wells can be coated with Antigens OR
Antibodies
• Suitable for automation high speed
• NO radiation hazards

Types of ELISA
1. Noncompetitive binding assay or Sandwich
method
1. Antigen measuring system [Titrewells coated with
antibodies ; Enzyme labelled antibodies]
2. Antibody measuring system [Titrewells coated with
antigens ; Enzyme labelled antiantibodies]
2. Competitive binding assay [Titrewells coated with
antibodies ; Enzyme labelled antigens]

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

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

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

Applications of 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

RadioImmuno Assay and ELISA Importance and Applications

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