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ELISA
Enzyme Linked
Immunosorbent Assay
What is an ELISA?
• Enzyme-linked immunosorbent assay
• Name suggests three components
– Antibody
• Allows for specific detection of analyte of interest
– Solid phase (sorbent)
• Allows one to wash away all the material that is not
specifically captured
– Enzymatic amplification
• Allows you to turn a little capture into a visible color
change that can be quantified using an
absorbance plate reader
What is ELISA?
 Technique used to detect (assay) specific
molecules (e.g. proteins & carbohydrates)
in samples.
 Immunological technique: uses antibodies.
 Quantitative.
 Very sensitive.
 Commonly used in medicine and scientific
research.
Antibodies
 Proteins secreted by B-lymphocytes
(type of white blood cell), in vertebrates.
 Recognise and bind to
molecules (antigens) on foreign
particles, marking them for
destruction by T-lymphocytes.
Fab
fragments
 Each antigen may generate
several antibodies for different
sites (epitopes) on antigen.
Fc
fragments
IgG molecule
Basic steps of ELISA
Enzyme Linked Immunosorbent Assay
1. Antigen of interest is absorbed on to plastic surface
(‘sorbent’).
2. Antigen is recognised by specific antibody (‘immuno’).
3. This antibody is recognised by second antibody
(‘immuno’) which has enzyme attached (‘enzyme-linked’).
4. Substrate reacts with enzyme to produce product, usually
coloured.
Coloured product = measure (assay)
of antigen present
Substrate
Secondary
antibody
Coloured
product
Primary
antibody
Different antigens in sample
Monoclonal antibody production
(hybridoma technology)
Inject
mouse with
antigen
Obtain
Mouse spleen
B-lymphocytes
Grow mouse
myeloma (tumour)
cells in culture
Fuse
B-lymphocytes with
myeloma cells
Antibody-producing
hybridoma cells
B-lymphocyte and
myeloma mixture
Select fused
and reproducing
hybridoma cells
via growth medium
Screen
hybridomas
for antibody
production
Unlimited supply
of antibody specific
for single epitope
Keep clone
producing antibody
which best detects
antigen
Make
clones from
individual
antibodyproducing
cells
Secondary antibody production
Mouse serum
injected into a
different species,
e.g. rabbit, goat.
Polyclonal
antibodies which
can recognise any
mouse antibody
Animal makes
various antibodies
against the
different antigens
in serum
Select anti-mouse
antibodies from
plasma
Take blood
from animal
Enzymes used in ELISA
 React with a colourless substrate to produce a
coloured product.
 Must work fast at room temperature so the colour
develops quickly.
 Have minimal interference from factors in sample.
Peroxidase from horseradish
Alkaline phosphatase from E. coli
b-galactosidase from E. coli
8. Observe
colour
development
1. Add antigen
7. Add substrate
for enzyme
2. Wash with
PBST (detergent)
6. Wash with
PBST
4. Wash with
PBST
3. Add primary
antibody
5. Add secondary
antibody
Type of ELISA
Sandwich ELISA
Competitive ELISA
• Less is more. More antigen in your
sample will mean more antibody competed
away, which will lead to less signal
Uses of ELISA outside
the classroom
 Disease detection in people, animals and
plants (e.g. HIV in humans).
 Detection of allergens in food, e.g. peanuts.
 Detection of illegal drugs in humans.
 Detection of hormones, e.g. pregnancy
testing kits.
ELISA in the curriculum
 Higher
Biology, Biotechnology and Human
Biology
E.g. Biotechnology: production & use of monoclonal
antibodies
 Advanced
Higher Biology:
Biotechnology Unit
Environmental Biology Unit
Investigations
Advanced Higher Biology
Investigation ideas
 Detection of Botrytis in fruit and vegetables from
market or garden.
 Quantification of Botrytis as infection develops.
 Detection of Botrytis in tissue before symptoms
are observed.
 Investigation on effect of temperature on rate of
Botrytis development.
 Based in the Institute of Cell Biology at the
University of Edinburgh.
 Role: to enhance engagement with
biotechnology through interactions with the
scientific community, school students,
teachers and the general public.
Immunofluorescence
Introduction:
• Immunofluorescence is the labeling of antibodies or
antigens with fluorescent dyes.
• This technique is sometimes used to make viral plaques
more readily visible to the human eye.
• Immunofluorescent labeled tissue sections are studied
using a fluorescence microscope.
• Fluorescein is a dye which emits greenish fluorescence
under UV light. It can be tagged to immunoglobulin
molecules.
• There are two ways of doing IF staining
– Direct immunofluorescence
– Indirect immunofluorescence
1.
Direct immunofluorescence
• Ag is fixed on the slide
• Fluorescein labeled Ab’s are layered over it
• Slide is washed to remove unattached Ab’s
• Examined under UV light in an fluorescent microscope
• The site where the Ab attaches to its specific Ag will
show apple green fluorescence
• Use: Direct detection of Pathogens or their Ag’s in tissues
or in pathological samples
Direct immunofluorescence
2. Indirect immunofluorescence:
•
•
•
Indirect test is a double-layer technique
The unlabelled antibody is applied
directly to the tissue substrate
Treated with a fluorochrome-conjugated
anti-immunoglobulin serum
• Advantage over direct IF
– Because several fluorescent antiimmunoglobulins can bind to each antibody
present in the first layer, the fluorescence is
brighter than the direct test.
– It is also more time-efficient since it is only one
signal labelled reagent, the antiimmunoglobulin, is prepared during the lengthy
conjugation process
Indirect immunofluorescence of iron-regulated
cell wall mannoprotein FIT1 of S. cerevisiae
Confocal image to detect phosphorylated AKT (green)
in cardiomyocytes infected with adenovirus
Immunofluorescence image of Cryptosporidium parvum
oocysts
Concentrated groundwater methanotrophic bacteria on 0.2 m m
filter labeled with fluorescent antibodies.
Radioimmunoassay (RIA):
A Remarkably
Sensitive Bioassay
Biochemistry Principles
Radioimmunoassay Procedure
Standard Curve & Unknown
Sample
Characteristics of Binder and
Ligand
• Availability
– Synthetic
– Natural – Produced From Animals
– Monoclonal Antibodies
• Purity – Competing Reactions with
Impurities
• Stability – Store in Albumin Serum
• Specificity – Binding Constant
Characteristics of Tracer
• Must Have Similar Binding Properties as
Unlabeled Ligand
• Internally Labeled Ligand
– 14C and 3H
• Externally Labeled Ligand
– 131I and 125I
• By-Products or Incomplete Synthesis
– Purification by chromatography (gel filtration)
Separation of Bound and Free
Ligand
•
•
•
•
Electrophoresis
Gel Filtration
Adsorption Chromatography
Fractional Precipitation
– Centrifugation
– Filtration
• Partition Chromatography
– Dialysis
Increasing Assay Precision
Sensitivity – Decrease [Ligand]
Sensitivity – Decrease [Binder]
Comparison of Assays
• Enzymeimmunoassays (EIA)
– millimolar
• Fluoroimmunoassays (EIA)
– micromolar
• Radioimmunoassays (EIA)
– nanomolar to picomolar
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
Summary
•
•
•
•
•
•
Based on Simple Biochemistry Principles
Establish Ideal Binder and Ligand
Synthesize Tracer Ligand
Separation of Bound and Free Parts
High Precision and Sensitivity
Powerful Applications to a Wide Range of
Medical Fields