Download Principles of immunological Techniques

Principles of
immunological
Techniques
MLU 3248
by
H Weerawarna
Visiting Academic
Open University of Sri Lanka
Immunoassay
Relative concentrations of different substances in blood.
10
mmol/L
100
10
m mol/L
100
10
100
pmol/L
Glucose Calcium
Mg
Sodium
creatinine
Iron, Immunoglobulin
Oestriol
T4 Progesteron
T3
Insulin
TSH
Hormones
Drugs
proteins
Use of immunoassay
tumour markers
Vitamins
pathogens
Immunoassays
Immunological reactions
Sensitive
Antigen + antibody
+
detectors
Highly specific
RIA
FIA
EIA
Radio active
ELISA
Fluorescence
Enzymes
Immunodiagnostic techniques
Range of techniques
directly
visible
Agglutination
Precipitation
Immuno-Immuno
fluorescence
Manual
RIA
ELFA
ELISA
Automated
Antigen
As a result of the reaction of the body to
immunogen antibodies are produced
Antibody
+
antigen

antibody antigen complex
Principle of agglutination - Antigen detection
?
Specific antibodies are linked to
a solid phase
solid phase
latex
red blood cells
agglutination
Latex agglutination
Positive
Negative
latex agglutination
Principle
The Direct Pregnancy test is based upon the latex
agglutination
reaction between latex particles coated with anti-hCG
antibodies and hCG present in the test specimen.
• The presence of hCG in the urinary specimen results in
an agglutination
• which is visually differentiated from the non-agglutinating
negative control.
Characteristics of precipitation reactions
Figure 17.6
Precipitation curve
Passive Immunodiffusion
Passive diffusion method in which a concentration
gradient is established for an antigen and /or antibody.
• Diffusion reactions without electric current to speed up
reaction.
• Rate of Diffusion depends on
– Size of particles
– Temperature
– Gel viscosity
– Amount of hydration
– Interaction between matrix and reactant.
Immunodiffusion Techniques
Radial Immunodiffusion
a single diffusion technique where Ab is put into
gel and Ag is measured by the size of a
precipitin ring formed when it diffused out in all
directions from a well cut into the gel.
Ouchterlony Double Diffusion
Both Ab and Ag diffuse from wells into a gel
medium
Medium
• Agar – high molecular weight complex
polysaccharide from seaweed. Agar has
strong –ve charges.
• Agarose - purified agar , no charge,
interaction between gel and reactants are
minimized.
• Used to help stabilize the diffusion provess
and allow visualization of precipitin bands.
Radial Immunodiffusion (RID)
• Antigen from the sample is added to
antibody rich media.
• Antigen and antibody continue to react
until the zone of equivalence is reached.
• The area of ring is a measure of the
antigen concentration.
Measurement of diameter of precipitation zone
Micrometer eye piece
Ouchterlony Double Diffusion
Fusion of lines at their junction to
form an arc
serologic identity / presence of
common epitope.
Crossed lines
demonstrates 2 separate reactions
compared antigens shard no
common epitopes
Fusion of 2 lines with spur
partial identity
Rocket electrophoresis
Enzyme Linked Immunosorbent Assay
(ELISA)
Introduction
The enzyme linked immunosorbent assay (ELISA) is a versatile method
for detecting and quantifying a specific protein in a complex mixture.
Originally described by Engvall and Perlmann (1971), the method
enables analysis of protein samples.
immobilized in microplate wells using specific antibodies. The technique
has revolutionized immunology and is commonly used in medical
research laboratories.
ELISA also has commercial applications, including the detection of
disease markers and allergens in the diagnostic and food industries.
Basic elements in ELISA
All the variants of ELISA have same basic elements:
1.
Coating/Capture: direct or indirect immobilization of antigens to the
surface of polystyrene micro plate wells.
2
Plate Blocking: addition of irrelevant protein or other
cover all unsaturated surface-binding sites of the
micro plate wells.
3.
Probing/Detection: incubation with antigen-specific antibodies that
affinity-bind to the antigens.
4.
Signal Measurement: detection of the signal generated via the direct
or secondary tag on the specific antibody.
molecule to
Enzyme-linked immunosorbent assay
An antibody sandwich ELISA
Sandwich method
Ab
WASHING
+
STEP
Conjugate
Solid
Sample
phase
E
E
WASHING
E
STEP
Competitive binding Immunoassay, EIA
Colour reaction in ELISA
3,3'-5,5' tetramethylbenzidine (TMB)
TMB One-Step Substrate System is a stabilized,
ready-to-use chromogenic substrate solution for
ELISA when peroxidase is the enzyme label.
TMB can act as a hydrogen donor for the
reduction of hydrogen peroxide to water by
peroxidase enzymes such as horseradish
peroxidse.
3,3'-5,5' tetramethylbenzidine (TMB)
The TMB One-Step Substrate System contains
3,3'-5,5' tetramethylbenzidine (TMB)
and hydrogen peroxide
in an organic solvent/buffer solution.
Upon oxidation, TMB forms a blue reaction product that
can be measured at 605 nm.
Upon acidification (to stop the enzymatic reaction) the
reaction product turns yellow with an absorbance peak at
450 nm.
Sandwich ELISA protocol
1. Coat primary antibody
onto microplate.
1a. Allow antibody adsorption
and block unoccupied sites
with neutral protein (BSA).
2. Add antigen sample to be detected
into each well. Incubate 30 min at 370 C.
3. Add second primary antibody
against antigen and HRP-conjugated
secondary antibody (antibody mix)
into each well. Incubate 30 min at 370 C.
4. Develop colorimetric reaction
with appropriate substrate. Incubate
15 min at room temperature.
5. Stop reaction with 3M H2SO4. Read
absorbance in ELISA spectrophotometer
and quantitate relative antigen levels.
ELISA instrumentation
Microplate washer
multichannel
pipette.
ELISA plate, 96 wells
Microplate reader
MICROPLATE PHOTOMETER
Microplate Photometer components
MICROPLATE PHOTOMETER
Microplate Photometer components
Enzyme Multiplied Immunoassay Techniques
(EMIT)
EMIT has a wide application in therapeutic and
illicit drug monitoring.
In this type of assay, a sample of interest with
the analyte (drug) is added to a fixed quantity of
enzyme – bound drug and the anti – drug
antibody.
After the addition of substrate, absorbance
measurement s are taken at time intervals to
determine the speed of the enzyme reaction.
Assay components
drug
antibody
substrate
enzyme bound to drug
Enzyme Multiplied Immunoassay
• Techniques have a wide application in
therapeutic and illicit drug monitoring.
In this type of assay, a sample of interest with
the analyte (drug) is added to a fixed quantity of
enzyme – bound drug and the anti – drug
antibody.
After the addition of substrate, absorbance
measurement s are taken at time intervals to
determine the speed of the enzyme reaction.
Enzyme Multiplied Immunoassay
Techniques (EMIT)
The more free drug in the sample, the
faster the enzyme reaction because only
the unbound enzyme-drug complexes are
capable of binding the substrate.
The method can be used for whole blood,
serum or urine.
Enzyme Multiplied Immunoassay Techniques
(EMIT)
Ag being measured competes for the Ab binding sites
with the antigen that has been labeled with an enzyme.
The antibody reagent blocks any enzymatic activity when
it is bound with the reagent enzyme Ag complex ,
preventing the formation of the product with a substrate.
The free Ag –enzyme complexes compete with the Ag
(in the sample), forming a colour chanege which are
proportional to the concentration of the Ag present in the
tested sample
Enzyme Multiplied Immunoassay Techniques (EMIT)
Procedure
-
mix sample containing drug with antibody.
Incubate
add fixed quantity of enzyme bound drug.
Incubate.
add substrate and cofactor.
incubate.
Measure absorbance 15-45 seconds after substrate
addition.
- Absorbance -> reaction rate ->drug concentration.
- nonlinear relationship between absorbance and
concentration.
Enzyme Multiplied Immunoassay
Techniques (EMIT)
• Use for drug, hormone and metabolite
determination.
• EMIT is widely used in therapeutic and
illicit drug monitoring dg: serum Digoxin,
Cocaine, marijuana
Advantages of EMIT
homogeneous, there is no need of washing
excess of labelled enzymes.
Long shelf-life
assays are well established.
More specialty assays are available
it is a competitive assay.
Reagent separation is not required .
Faster
smaller molecules are measured
Disadvantages of EMIT
many interferences
less sensitive compared to ELISA in
hormone determination
false negatives are common
tolmetin and aspirin metabolites
Radioimmunoassay (RIA)
Radioimmunoassay (RIA) is laboratory
technique for measuring the levels of
substances which have low concentrations
in the blood. Eg hormones.
Sensitive and detection limit 10 -15 mol/l
10 3 -10 6 times more sensitive than
colorimetric assay
HISTORY OF RIA
Principle of RIA
1. plasma sample from the patient containing
the substance whose concentration is to be
measured.
2. The same substance with a radioactive
label, eg use of chloramine-T for labeling
proteins with high activity of radioiodine.
3. Receptors which bind only the substance
of interest - antibody
Production of antisera
To produce the antiserum, the antigen is injected
subcutaneously or intraperitonelly into and
experimental animal, usually the rabbit, together with
an adjuvant to stimulate the immune system.
Large molecules with MW over 10000 are naturally
antigenic and cause the production of antibodies.
Small molecules such as thyroid and steroid hormones
and drugs are not antigenic, made antigenic by
process called haptenisation, molecule is linked to a
large molecule as albumin.
After booster dose animal is bled to produce antiserum
(polyclonal)
Radioactive label
The choice of a radio active label for
the antigen will depend on its specific
activity
• Physical half life
• Biological and chemical stability
3H, 51Cr, 57Co, 59Fe, 125I
125I , ½ life 60 days
Iodine-125 Radioiodination
Iodination of peptides and proteins, as well as small
molecules, results in materials with high specific activity.
Generally, Iodine-125 is incorporated into available tyrosine
residues under oxidative conditions.
Various compatible oxidative reagents of differing strengths
exist (e.g. Chloramine-T, Iodogen, Lactoperoxidase) and
can be chosen to accommodate sensitive molecules.
Additionally, for compounds lacking a tyrosine or histidine
or are simply incompatible with other iodination techniques,
labeling of free amines can occur through the use of [125I]Bolton-Hunter Reagent.
Due to the wide variety of compounds amenable to 125I
labeling and a number of variables involved to achieve
desired specifications, please contact us to discuss your
project’s needs.
Iodination of tyrosine - chloramine-T
method
The principle of RIA
The principle of RIA (cont.)
If total Ab input is kept constant, the
value of B/F is a measure for the total Ag input
Measurement of Bound/Free Tracer
The Standard Curve
To construct a binding inhibition curve based
on known ( standard) amounts of antigen,
for use in interpolation of unknown samples.
Counting techniques
The counting of the activity of the samples
done by using
– gamma counter or g emitting labels eg; 125 I
– Liquid scintillation counter for 3 H labeled assays.
Multiwell gamma counters capable of data
processing, plotting standard curve and result
calculation are use for efficient counting.
scintillation detector configurations
Photomultiplier tube (PMT)
• Photomultiplier tube. Its basic structure is an
evacuated cylinder enclosed in glass, with a
photocathode on one end, an anode at the
• opposite end, and small curved dynodes in
between. The electrical potential to the dynodes is
what causes multiplication of the electrical signal
• created at the photocathode. The passage of an
electron through the focusing grid, its interaction
with the first dynode, and its multiplication at the
second dynode is shown, using a multiplication
factor of 3.
Photomultiplier tube.
Block diagram of a scintillation detector.
The detector/PMT assembly is usually separated
from the electronics module by a cable
e
Multi well gamma counter
Standard curve - RIA
VIDAS PRINCIPLE
Courtesy of BioMerieux,
France
VITEK
IMMUNO
DIAGNOSTIC
ASSAY
SYSTEM
VIDAS
An innovative test format
Ready-to
Readyto--use reagents
in the 1010-well strip
Specific antibodies
coated onto the SPR
SANDWICH
Detection of antigens
4-methyl umbelliferyl phosphate
Bound Ab
Alkaline phosphatase-labeled
Ab
Ag to be detected
COMPETITION
Detection of haptens
4-methyl umbelliferyl phosphate
Bound Ab
Alkaline phosphatase- labeled
hapten
Hapten to be detected
INDIRECT ELISA
Serology
4-methyl umbelliferyl phosphate
Ab to be detected
Alkaline phosphatase- labeled
Ab
Bound Ag
IMMUNOCAPTURE
Detection of specific IgM
4-methyl umbelliferyl phosphate
Ab to be
detected
alkaline phosphatase-labeled
Ab
Anti-IgM
Ag in liquid medium
DYNAMICAL REACTIONS
EXAMPLE
SPR
Strip
Washing buffer
Conjugate
Sample
Diluent
(Prewash)
Substrate
photodiode
EXAMPLE OF PROCEDURE
Detection of antigens
Cycle in and out
SPR
E
E
E
E
E
E
Target
organism
E
SAMPLE
WASHING
BUFFER
CONJUGATE
IMMUNOLOGICAL REACTION
SUBSTRATE
ENZYMATIC REACTION
- 7-1
READ FLUORESCENCE
EXCITATION
EMISSION
Filters
Lens
Xenon
Flash
Lamp
Photodiode
- 7-2
VIDAS : adapted to lab requirements
As easy as 1, 2, 3...
1- Load sample
2- Insert test
3- Press button
series :
1 to 30 tests / run
1 or more pathogens / run
Rapid
(Results in 2424-48 hours)
Software
Standardization
Controls
Traceability
Walk Away
system
VIDAS : adapted to lab requirements
High accuracy
sensitivity, specificity
> 98%
Bar-code
BarStandardization
and traceability
Ready-toReadyto-use reagents
No preparation step
Optimization of
reagents
Long shelf life
Cost reduction
Reduced lab work
Reduced time
Reduced lab materials