Immunoassay - RIA & ELISA

Objectives
Principle of immunoassay
Types of immunoassays
Principle of radioimmunoassay & ELISA
Various types of ELISA
Steps of ELISA
Urine pregnancy test by lateral flow immunoassay
2

Immunoassay
Immunoassay is an analytical technique used for the
quantification of an analyte based on the antigen-antibody
reaction.
3

Labels in immunoassay
The advantage of immunoassay is that either
the antigen or antibody can be labelled.
This label can be an enzyme, radioactive
compound, chemiluminescent or fluorescent.
The labelled component of an immunoassay is
sometimes called the tracer.
4

Types of immunoassay
Radioimmunoassay (RIA)
Enzyme-linked immunosorbent assays (ELISA)
Fluoroimmunoassay (FIA)
Chemiluminescence immunoassay (CLIA)
5

Constituents of labelled immunoassay
Specific antibody/antibodies (labelled)
Antigen (known/unknown)
A method to separate the unbound
components from bound immune complex
A method for detection of the label
Standards
6

Radioimmunoassay
Dr. Rosalyn Yalow and Dr. Solomon
Berson
Physiology or Medicine
1977
7

RIA-Principle
Competitive binding assay
“Radio labelled antigen (Ag*) competes with
the unlabelled antigens for the binding site of the
fixed amount of antibody (Ab).”
8

RIA- Principle:
Ab + Ag* ⇌ Ab-Ag*
+
Ag ⇌ Ab-Ag
The proportion of Ab-Ag* and Ab-Ag at equilibrium will be
equal to the original proportion of Ag* to Ag
31

Disadvantages of RIA
Very high cost of equipments and reagents.
Half life of I125 is 60 days only.
Hazards of radioactivity.
Lengthy procedure.
35

Enzyme-Linked
Immunosorbent Assay
ELISA is the simplified and modified version of RIA.
Instead of the radiolabelled antibodies, enzyme-
linked antibodies are used in ELISA.
It eliminates all the hazards associated with
radioactivity and the equipment needed for
measuring radioactivity.
36

Types of ELISA
1. Direct ELISA
2. Indirect ELISA
3. Sandwich ELISA
4. Competitive ELISA
37

Direct ELISA
Advantages:
Quick because only one antibody and fewer steps are
used.
39

Direct ELISA
Disadvantages:
Immunoreactivity of the antibody might be affected by
labeling.
Labelling primary antibodies for each specific ELISA system
is time-consuming and expensive.
Less signal amplification.
40

Indirect ELISA - Advantages
Versatile - any primary antibodies can be made in
one species and the same labelled secondary
antibody can be used for detection.
Maximum immunoreactivity of the primary antibody
is retained.
Sensitivity is increased because of signal
amplification.
42

Signal-Amplification in ELISA
Enzyme labelled secondary antibodies are responsible for
the signal amplification.
Each primary antibody can be bound by more than one
secondary antibody.
Similarly, each enzyme molecule will process multiple
substrates into the product.
This leads amplification at each step, responsible for high
sensitivity of the ELISA.
43

Sandwich ELISA
The antigen is sandwiched between two primary
antibodies recognizing two different epitopes of the
antigen.
The first antibody is coated on the plates.
The second antibody, which is added after the antigen
addition, is linked to the enzyme. It is an Ag detection
test.
The first antibody is known as ‘capture’ antibody and
the second antibody is known as ‘detection’ antibody.
46

Sandwich ELISA
It is important to note that the antibodies used should
be able to recognise the different epitopes of the same
antigen.
So, haptens can’t be detected using sandwich ELISA.
47

Competitive ELISA
Competitive ELISA is similar to RIA.
Both unknown antigen (sample) and the known
antigen (standard) compete with each other for a fixed
amount of antibody.
Ag is detected.
Normally used for hapten detection.
48

Comparison of the 4 types of
ELISA
Type of ELISA Well
coated
with
Detects in
serum
Example
Direct Antigen Antigen Beta hCG
Indirect Antigen Antibody Anti HBV
antibody
Sandwich Antibody Antigen HIV: p24 antigen
Competitive Antigen Antigen Hapten detection
50

Identify the type of ELISA:
51

Which ELISA will you use?
Histamine
Complement C3
Insulin
Interleukins
Leukotrienes
Hepatitis B surface antigen
Dengue IgM Antibodies
52
- Competitive
- Competitive
- Sandwich
- Sandwich
- Competitive
- Indirect
- Sandwich

Steps of ELISA
1. Coating
2. Blocking
3. Addition of Antibodies
4. Washing
5. Detection
53

Blocking
Blocking is done after coating and it ensures no empty
spaces are left on the plate surface.
Blocking buffers are used to prevent non-specific
binding of proteins to the plate.
Optimal blocking buffer maximizes the signal to noise
ratio during estimation.
55

Blocking agents
1% Bovine serum albumin (BSA)
Serum
Non-fat dry milk (NFM)
Casein or Gelatin in PBS
56

Detection
The final stage in all ELISA systems is a detection
step.
This involves the introduction of an enzyme-
substrate system.
The enzyme converts the substrate to a
detectable product.
62

Commonly used enzymes
Horseradish peroxidase
Alkaline phosphatase
63

ALP chromogenic substrate
1. Nitrophenyl phosphate (NPP)
2. p-nitrophenyl pyrophosphate (PNPP)
3. Bromo chloro indolyl phosphate (BCIP) & Nitro
blue tetrazolium (NBT)
4. Fluorescein diphosphate
67

Human chorionic gonadotrophin
hCG is synthesized in the syncytiotrophoblast cells of
the placenta.
Minute amounts are also made in the pituitary.
A single gene located on chromosome 6 encodes the
α subunit.
Chromosome 19 contains genes for beta subunit.
71

Alpha subunit is shared
72
Alpha subunit is shared by glycoprotein
hormones (TSH, LH [luteinizing hormone],
FSH, and hCG).

hCG and pregnancy
Synthesis of hCGβ peaks at about 8 to 10 weeks of
gestation.
74
First-morning specimens are preferred for
qualitative urine pregnancy tests because they are
concentrated and contain abundant hCG.

Immunoassay - RIA & ELISA

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