1.introduction immunodiagnostic

7/28/2019 1.Introduction Immunodiagnostic

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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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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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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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Disadvantages of monoclonal antibodies

tend to have low affinities

Not useful in competition

poor curve shape


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Advantages of polyclonal antibodies

well established

simple production methods

multiplicity of antigenic site recognition confer high bindingpropriety


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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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B12 Is the reproductibility of the measurement

how consistently the assay gives the same result on

the same sample

Precision


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B12 Two parameter are usually considered by the customer :Within -assay precision : variation in repeated measurements of

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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B12 Precision is a statistical concept and is usually quantitated through the

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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B12 tests whether an immunoassay has been calibratedcorrectly. Is a check on the accuracy and validity .

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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B12 Is a complement of the recovery study but also assess whether

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

1.introduction immunodiagnostic

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