1.introduction immunodiagnostic
TRANSCRIPT
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INTRODUCTION TO IMMUNOASSAYS
ECi
MENU
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PLAN
Antigen
Antibodies
Structure
Function
Classes Antibodies responses
Primary
Secondary
Terms
Preparation of antibodies
polyclonal
monoclonal
the choice
Immunoassay Assay design
Assay performance
Qualitative assays
Immunoassays and problemes
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ANTIGEN
An antigen is a foreign molecule that induce the production of
antibodies
Antibody binds and destroy the invadingantigen
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ANTIBODIES
Immunoglobulins are a group of glycoproteines present in the serum /
tissue fluid of all mammals
Their production is induced by the immune system. All foreign
molecules (antigen ) can induce their formation. They are produce by the B lymphocytes and specific to their antigen
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Structures
Ig is a fourchain polypeptide structure
2 identical heavy chain, 450 amino acids long
2 identical light chain, 250 amino acids long
They are linked by disulphide bridges A amino (N) and carboxyl (C) terminal ends the peptide chain.
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Structures
N
Heavy chain
Light chain
C
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Function
Each Ig is bifunctional
one region is concerned with binding to the
antigen
a other region interacts with the hosts immune
system.
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Igclasses
They are different classes of immunoglobulines:
Ig G
is the major Ig in normal human serum.
Around 70 to 75 % of the total pool
IgG is a monomer protein
is the major antibody of the secondary immune
response
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Ig M
around 10 % of the immunoglobulin pool
pentameric structure
is the predominant early antibody
IgA
represents 15 % of the pool
polymeric structure
predominant is seromucous secretion such assaliva,colustrum
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Ig D
less than 1 % , but is know to be present in large
quantities on the membrane of B lymphocytes
function not know but may play a role in antigen
triggered lymphocyte differentiation
Ig E
through trace in serum
found of the surface of basophils
associated with immediate hypersensitivity such as
asthma
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The antibody response
Recognition of the foreign-ness of the antigen
Immune reaction induce the formation of antibody, we
distinguish a primary and secondary antibody responses
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Primary response
Following primary antigenic challenge.
There is an initial lag phase, when no antibody can be
detected
than the antibody titter rise to a plateau
there are a major proportion of IgM
finally the titter declines as the antibody are bound to the
antigen, or naturally catabolized
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Secondary response
This response appears the second time that the antigen is introduced.
The characteristics shows that the response differ in four major
respects:
the antibodies appears more quickly, the lag phase in shorter
the plateau levels of antibody are much greater, typically ten fold ormore than the primary response
consist predominately of Ig G and they persists longer
the affinity is much greater
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Commonly Used Terms
Affinity strength of binding between Ab & Ag
Avidity ability of an antiserum to bind Ag dependent on
affinity, temperature, pH, ionic strength, contaminants, etc.
Specificity ability of the Ab to recognize the antigen which
induced the immunresponse.
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Preparation of antibodies
1- Preparation of an immunogen ( which stimulates the antigenic
response- the antigen reacts immunologically with the antibody)
2- For molecules with a low MW , it is necessary to combine the
molecule with a protein. This molecules are called haptens
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Polyclonal antibodies
Immunogen are introduce into an animals that is genetically
distant from its own species origin.We commonalty use
rabbits,sheep,guinea pigs, horse, donkeys
Following a series inoculations, blood is taken and serum isseparated. The immunoglobuline fractions are separated and
tested.
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Polyclonal antibodies
The antisera will contain a heterogeneous mixture of different
antibodies varying specificity and affinity, because of the
multiplicity of antigenic sites of a single immunogen
At high dilution ( that is at low concentration ) of the antisera,
only the antibodies with the highest affinity will react.
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Polyclonal antibodies
And so the heterogeneous antiserum may behave as a
homogeneous reagent in an immunoassay.
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Monoclonal antibodies
The immunogen is injected in a mouse ( not usually used for
polyclonal Ab production- you can t get much blood out of a
mouse !)
After a good antibody response, the spleen is removed. The
cells are then fused with myeloma cells . The Hybidomas can then be grown in a cell culture
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Monoclonal antibodies
Ag ==> mouse ==> lymphocytes separation ==>fuse with myeloma cells ==> cloning
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Monoclonal antibodies: cloning
If the culture producethe desirated Ab,
it is cloned
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The choice : monoclonal or polyclonal
Advantages of monoclonal antibodies
monospecificity , even if the original immunogen was impure
affinity is defined and can be selected
clean reagents giving low non specific binding andbackgrounds
indefinite supply of Ab with constant characteristics
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The choice : monoclonal or polyclonal
Disadvantages of monoclonal antibodies
tend to have low affinities
Not useful in competition
poor curve shape
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The choice : monoclonal or polyclonal
Advantages of polyclonal antibodies
well established
simple production methods
multiplicity of antigenic site recognition confer high bindingpropriety
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The choice : monoclonal or polyclonal
Disadvantages of polyclonal antibodies
less specific , because they bind to a multiplicity of antigenic
sites
supply not indefinite ( animals may die . )
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IMMUNOASSAYS
Is a process ofmeasuring small amounts of biologicalsubstances
the measurement maybe
qualitative : you are testing for presence or absence of thesubstance
quantitative : you measure the actual amount present
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A binding agent ; one or two antibodies are used
incubation: allows the formation of the Ag-Ab
a separation step : in coated surface technology the Ab used isimmobilized . The unbound contents will be extracted by washing
IMMUNOASSAYS
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IMMUNOASSAYS
A signal generating agent , which is chemically linked to a
binding agent , e.g enzyme, HRP. This agent is called
conjugate
The substrat : we use the luminol.The enzyme usinghorseradish peroxidase oxidizes Luminol using hydrogen
peroxide. The by product is light
The name of the method is given by the signal e.g. RIA,
Chemioluminescence...
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IMMUNOASSAYS
Two Basic Immunoassay Reactions
Immunometric - two antibodies are used, each
binding to a different part of the analyte.One of the antibodies is labeled with HRP.
Competitive- Unlabeled analyte and labeledantigens compete for the binding antibody.
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Immunometric DesignAntibodies are present so that there is more than enoughto bind to the largest amount of antigen that may be
present in the patient sample
Well
Signal Reagent
Mousemonoclonalanti-wholeTSH
HRP-labeled
mousemonoclonalanti-b subunitof TSH
Thyroid StimulatingHormone
a
b
Luminescenc
e
Example Assay Design VitrosTSH assay
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Immunometric Calibration CurveThe amount of labeled antibody binding will beproportional
to the total amount ofanalyte present in the patient sample.
Immunometric Calibration Curve
1
10
100
1000
10000
100000
0 0.01 0.1 1 10 100
Concentration
LightUnits
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Competitive DesignThe two types of analytes, labeled and unlabeled, are
indistinguishable and will both compete for the limited
numberof antibodies sites.
Donkey
Anti-
sheep
Coated
Well
Signal Reagent
Luminescenc
e
Sheep anti- T4 HRP Labeled T4
T4 from Sample
Example Assay Design VitrosTT4 Assay
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Competitive Calibration CurveThe amount of labeled analyte bound to the antibody is
inverselyproportionalto the concentration of the unlabeled
analyte in a patient sample.
Competitive Calibration Curve
0
1000
2000
3000
4000
5000
6000
0 2.5 4.7 9.9 15.1 23.5 30.8
Concentration
LightUnits
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Choosing a Design
When developing new immunoassays, a format choice is often made
between immunometric or competitive assays.
The choice depends on :
* Analyte (small analytes typically do not lend themselves to sandwich
methods because their size.)
* Specificity (The use ofpaired monoclonal antibodies can enhance
the specificity ofimmunometric assays, but are less likely to measureall of the variant forms of proteins.)
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Choosing a Design
* Sensitivity (While good competitive immunoassays can
demonstrate excellent sensitivity, sandwich assays can often improve
on this
* Calibration Range (Competitive design is limited because of the
slowly diminishing signal which approaches the residual background
signal at high concentrations.)
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Assay performance characteristics
SENSITIVITY
measures the smallest concentration ofanalyte that can be reliably detected
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SENSITIVITY
Analytical sensitivity : lowest concentration at which
the kit can differentiate between analyte in a sample
and the background noise
Establish by measuring a zero standard 20 times
and calculating the 2 SD
- Functional sensitivity : lowest concentration
that can be measured in practice .
Based on the imprecision at 20 %
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SPECIFICITY
ability to measure only what you want to measure
Specificity is expressed as a % of cross reactivity
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ACCURACY
ability of the assay to report the correct value for the
concentration of analyte present
therefore reference methods and International ReferencePreparations IRP are used to define the standard, or true value,
during the calibration.
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ACCURACY
Small molecules are easily defined in terms of MW they can be
prepared and purified to a high degree and a absolute
measurement made by
gas chromatography - Mass spectrophotometry
isotopic dilution - Mass spectrophotometry
these methods are called reference methods
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measurement is not possible
International Reference Preparation serves as the definition of the true
value, and used to calibrate most of the assays
ACCURACY
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how consistently the assay gives the same result on
the same sample
Precision
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the same sample during asingle assay run
Between assay precision: variation in repeated measurements ofthe same sample from one assay run to another .
Precision
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calculation of % CV
%CV = standard deviation of the value x 100
average of the values
The precision of the immunoassay varies across the range. This gives
the concept ofprecision profile
Precision
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Recovery is usually defined as the increase in the value seen when a
know concentration ofanalyte is added to a sample
The results are repeated and reported as a mean % recovery and
ranges provided.
Recovery
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samples which lie outside the calibration range can be diluted and
measured
Serial dilution of a sample are made with a zero concentration sample ora strip serum.
Expectation is that if the sample is diluted 1/2, than the concentration
should be halved.
Dilution
A f h t i ti
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Assay performance characteristics
for QUALITATIVE ASSAYS
Qualitative assay are encountered in
infectious disease
( pregnancy such as hCG)( oncology )
Sensitivity and specificity have different meaning
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QUALITATIVE ASSAYS
Healthy Diseased
Concentration
PatientNumbers
IdealSpecificity = 100% Sensitivity = 100%
Cutoff
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QUALITATIVE ASSAYS
Healthy Diseased
Concentration
PatientNumbers
IdealSpecificity = 100% Sensitivity = 100%
Cutoff
QUALITATIVE ASSAYS
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QUALITATIVE ASSAYS
Reality
Set low (1) to identify maximum
number ofdiseased individuals
Set high (3) to identify maximumnumber ofhealthy individuals
Healthy Diseased
Concentration
Patie
ntNumbers
1 2 3
Specificity = 60%
Sensitivity = 100%
False Positives
Sensitivity = 60%
Specificity = 100%
False Negatives
What is the
optimum?
Is it position (2)?
NB100 minus
specificity
is the false
positive rate
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WELL KNOW ISSUES IN IMMUNOASSAY
High Dose Hook effect
Heterophilic antibodies
Hi h d h k
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High dose hook
Label
Antigen
Capture
Wash
Normal Conditions
Solid PhaseSolid Phase
High dose hook
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High dose hook
Label
Antigen
Capture
Wash
Hook Conditions
Solid PhaseSolid Phase
Hi h d h k
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High dose hook
Antigen
Capture
Wash
Solving the Problem...
Solid Phase Solid Phase
H ti A i l A tib d
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Human anti Animal Antibody
Human anti animal antibodies ( IgG, IgA, IgM, IgE )are the
results of an immunization by using animal derived drugs e.g.,
or after regular exposure to animals
Their concentration can reach g/l and persists for years.
The prevalence in the population is unknown, a report estimates
they are present in 3.4% of healthy individuals.
HETEROPHILIC ANTIBODIES -
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HETEROPHILIC ANTIBODIES
How can they interfere ?
Immunometric assay e.g. TSH
normal heterophilic interference
HRP
TSH
TSH-HRP is bound
giving an elevated result
heterophilic antibody
mouseanti-TSH
mouse
anti-TSH
HETEROPHILIC ANTIBODIES -
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T3
Competitive assay e.g. T3
normal heterophilic interference
sheep anti T3
ab -bound T3-HRP is washed
away giving an elevated result
HRP
donkey
anti-sheep
sheepanti-T3
O C O S
How can they interfere ?
Th l ti
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The solution
TSH
HRP
heterophilic antibody
bovine IgG
TheVitros TSH assay contains
bovine IgG which minimizes the
problem
by binding the heterophilic antibody
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antigens may be presented to the immune system
In France Rubella vaccine contain rabbit serum
The admisnitration of therapies is an other routeAnti rabbit Ab after injection of antireticulocytoxiques (
injected with human bone marrow and spleen act as a tonic
to improve senescence and reduce fatigue and debilitation
Animal derived pharmaceuticals
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Animal derived pharmaceuticals
DRUG SOURCE
Ab - targeted imaging reagents mouse
rat
Ab - targeted imaging drugs mouse
rat
Anti - thymocyte globulin horserabbit
Antin - snake venom horse
Calcitonin Salmon
factor VIII Pig
Insulin Pig
Vaccines rabbit
chicken
Human Anti Mouse Antibody: HAMA
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Human Anti Mouse Antibody: HAMA
Hama is probably the most common type and the reason is the
use of mouse monoclonal Ab for therapeutic
e.g. monoclonal anto OKT3 is widely used in transplantation as
an immunosuppressant. The prevalence is not know , but can persist up to 30 months
Assay are available on the market
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Protective Factor
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Protective Factor
Analyte K2 MABMouse
serum
Bovine
IgG
Sheep
serum
Calf
serumB2TT
Rabbit
serumSCF I SCF II
Yeast
ExtractSOD
Human
IgM interf.
Heterop.
Ab
(HAMA)
Heterop
. Ab
Heterop.
Ab
Heterop
. Ab
Heterop.
Ab
Heterop
. Ab
anti-
HRP
rogues
Azide
effects
Anti-yeast
reactive
samples
Anti-SOD
cross
reactivity
TSH ? ? ? ?
TT4 ? ?
TT3 ? ?
FT4 ? ? ?FT3 ?
T3U ? ?
E2 ? ? ?
PRL ? ? ? ?
FSH ? ? ? ?
LH ? ?
BhCG ? ? ? ?
FB-hCG ? ? ? ?AFP ? ? ? ?
PROG ?
TESTO ? ? ?
CA 15-3 ? ? ? ?
CEA ? ? ? ?
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B12PSA ? ? ? ?
CA 125 ? ? ? ? ?
CA 19-9 ? ? ? ? ? ?
HBsAg ? ? ? ? ?
Anti-HBs
Anti-HBc ? ? ? ?
HBc M
HBeAg ? ? ? ? ?
Anti-HBe ? ? ? ? ?
HAV M ? ?
Anti-HCV ? ? ?
Anti-HIV ? ? ?
Ferritin ? ? ? ? ?
B12Folate
CKMB ? ? ?
TROP I ? ? ?
CORT ? ?
NTx ?
Analyte K2 MABMouse
serum
Bovine
IgG
Sheep
serum
Calf
serumB2TT
Rabbit
serumSCF I SCF II
Yeast
ExtractSOD
Human
IgM interf.
Heterop.
Ab(HAMA)
Heterop
. Ab
Heterop.
Ab
Heterop
. Ab
Heterop.
Ab
Heterop
. Ab
anti-
HRProgues
Azide
effects
Anti-yeast
reactive
samples
Anti-SOD
cross
reactivity
ECi Assay Menu
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ECi Assay Menu
Launched (non-regulated markets)
TSH Estradiol Anti-HIV 1+2 Total PSA FerritinFree T3 Progesterone Anti-HCV CA 19-9 B12
Free T4 AFP HBsAg CA 15-3 Folate
Total T3 Free bhCG Anti-HBs CA 125 IITotal T4 Total bhCG Anti-HBc CEAT3 Uptake Testoterone Anti-HBc IgM
LH HBeAg
FSH Anti-HBe CK-MB Cortisol
Prolactin Anti-HAV IgM Troponin NTx
Anti-HAV Total Myoglobin
Roll-out Toxo,Rub,CMV