who immuno test

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E P I D E M I C A L E R T A N D R E S P O N S E

Laboratory Training for Field Epidemiologists

Antigen and antibody detection

Investigation strategies and methods

May 2007





Learning objectives

At the end of the presentation, participants should

• Understand direct and indirect antibody detection

• Understand the different methods for detecting antigens orantibodies





Detection

• Detection of antigen-antibody complex

• Antigen-antibody complex requires specific conditions

• temperature

• pH

• Complex may be directly visible or invisible





Detection

Directly visible – agglutination

Invisible

•

requires specific probes (enzyme-labelled anti-immunoglobulin, isotope-labelled anti-immunoglobulin,etc.)

• binds Ag-Ab complex and amplifys signals

• signals can be measured by naked eyes or specificequipment e.g. in ELISA, RIA, IFA





Methods for Ag-Ab detection

• Precipitation

• Agglutination

• Hemagglutination and hemagglutination inhibition

• Viral neutralization test

• Radio-immunoassays

• ELISA

• Immunoflourescence

• Immunoblotting

• Immunochromatography





Precipitation

Principle

• soluble antigen combines with its specific antibody

• antigen-antibody complex is too large to stay in solution

and precipitates

Examples

• flocculation test

• immuno-diffusion test

• counter-immuno-electrophoresis (CIEP)


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Flocculation test

(precipitation reaction)

Principle

• precipitate, a concentrate of fine particles, is usuallyvisible (macroscopically or microscopically) because

the precipitated product is forced to remain suspendedExamples

• VDRL slide flocculation test

• RPR card test• Kahn’s test for syphilis



RPR card test

Flocculation test

(A precipitation reaction)

(1) Non Reactive (2) Weakly Reactive (3,4) Reactive



Precipitation:

Performance, applications

• Advantages

• sensitive for antigen detection

• Limited applications

• Time taken - 10 minutes



E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists

Positive Negative

Ag-Ab complex

Direct agglutination

Principle

• combination of an insolubleparticulate antigen with its solubleantibody

• forms antigen-antibody complex• particles clump/agglutinate

• used for antigen detection

Examples

• bacterial agglutination tests forsero-typing and sero-groupinge.g., Vibrio cholerae,Salmonella spp




Passive (indirect) agglutination

Principle

• precipitation reaction converted into agglutination -coating antigen onto the surface of carrier particles likered blood cells, latex, gelatin, bentonite

• background clears

Examples of types

• latex agglutination

• co-agglutination

• passive hemagglutination (treated red blood cellsmade resistant)

Examples of tests - agglutination for leptospirosisWidal test (typhoid fever)




Principle

• antigen binds to soluble antibody coated on carrierparticles and results in agglutination

• detects antigens

Example

• detecting cholera toxin

Reverse passive agglutination




Positive

Negative

Reverse passive agglutination




Agglutination:


Advantages

• sensitive for antibody detection

Limitations

• Prozone phenomenon:

• requires the right combination of quantities ofantigen and antibody

• handled through dilution to improve the match

Time taken

• 10-30 minutes




Hemagglutination

Principle

• many human viruses have the ability to bind to thesurface structures on red blood cells from differentspecies thereby causing agglutination

Example

• influenza virus binds to fowl’s red blood cells




Positive Negative

Hemagglutination

inhibition for detection of Dengue antibodies

Hemagglutination inhibition

Principle

Antibodies to the virus in thepatient serum bind to the virus;blocks binding sites on the viralsurfaces

• prevents the virus fromagglutinating the red cells

Example

• detecting antibodies toinfluenza and dengueviruses




Hemagglutination:


Advantages

• highly specific

• can be used as gold standard

Limitations

• technically demanding

• time consuming

• cannot distinguish IgG from IgM

Time taken

• 1 day




Neutralization assays Principle

• antibodies in serum neutralize antigens on the surfaceof viruses(neutralizing antibodies)

• inhibited viruses cannot infect cell lines

Example

• plaque neutralization assay for dengue virus,Japanese encephalitis virus

• antibodies to bacterial toxins and other extra-cellularproducts that display measurable activities (e.g.,ASLO, diphtheria toxin, clostridium toxin)




Neutralization:


• Advantages

• Highly specific

• Often used as gold standard

•

Limitations• Technically demanding

• Time consuming

• Can only be used for viruses that can be grown

• Complexity limits the use beyond gold standard

• Time taken

• 1 week




Radio-immunoassays

• Principle

• Radioactively labelled-antibody (or antigen) competes with the patient’s

unlabelled antibody (or antigen) for binding sites on a known amount ofantigen (or antibody)

• Reduction in radioactivity of the antigen-patient antibody complexcompared with control test is used to quantify the amount of patientantibody / antibody bound

• Limited use due to the problems with handling radioisotope

• Example

• HBsAg

• Thyroid function test




Positive

Negative

Neutralization Assay




Radio-immunoassays:


Adantages

• highly sensitive

• can be used for detection of small quantities

• quantification possible

Limitations

• expensive

• requires isotopes

Time taken

• 1 day




Labeling techniqueEnzyme-linked immunosorbant

assay (ELISA)

Principle

• use of enzyme-labelled immunoglobulin todetect antigens or antibodies

• signals are developed by the action ofhydrolyzing enzyme on chromogenic substrate

• optical density measured by micro-plate reader

Examples

• Hepatitis A (Anti-HAV-IgM, anti-HAV IgG)




ELISA

Micro-plate reader

96-well micro-plate Positive result

L b li t h i




Labeling technique

Types of ELISA (Ag Ab tests)

Competitive

•Antigen or antibody are labelledwith enzyme and allowed tocompete with unlabeled ones (inpatient serum) for binding to thesame target

•Hydrolysis signal from Ag-Abcomplex (enzyme-labelled) ismeasured

•Antigen or antibody in serum is

then calculated

•No need to remove theexcess/unbound Ag or Ab fromthe reaction plate or tubes)

L b li t h i




Labeling techniqueTypes of ELISA used in the detection of

antigens and antibodies•Non-competitive

•must remove excess/unbound Agor Ab before every step of reactions

•Direct ELISA

•Indirect ELISA

•Sandwich ELISA

• Ab Capture ELISA (similar to

sandwich ELISA but in 1st step,

anti-Ig (M or G) is coated

on the plate

•Then antibodies in patient serum

are allowed to capture in next step




ELISA:


• Advantages

• Automated, inexpensive

• Objective

• Small quantities required

• Class specific antibodies measurable

• Limitations

• Expensive initial investment

• Variable sensitivity / specificity of variable tests

• Cross contamination

• Time taken - 1 day

er ormance compar son o




Performancecharacteristic

Non-competitive

ELISA

CompetitiveELISA

Capture ELISA

Purpose Antibody Antibody Best for class

specificantibody

Sensitivity ++ ++ ++

Specificity ++ ++ +++

Cost + ++ +++

Ease ofperformance

++ +++ ++

Time taken ++ + +++

er ormance compar son ovarious ELISAs for antibody

detection

L b li t h i




Labeling technique

Cell infected with Dengue virus

V. Cholerae

Immuno-fluorescence•Principle

• Use fluoresceinisothiocyanate labeled-immunoglobulin to detectantigens or antibodiesaccording to test systems

• Requires a fluorescentmicroscope

•Examples

• Herpes virus IgM• Dengue virus

• Rabies virus

• Scrub and murine typhus

I fl




Immuno-fluorescence:


• Advantages

• Sensitive and specific

• Can be used for discrepant analysis

• Limitations

• Expensive (Reagents and equipment)

• Subjective

• Cross reactivity

• Non-specific immuno-fluorescence

• Time taken

• 1 day

Labeling technique




Labeling technique

Types of immuno-fluorescence

•Direct immuno-fluorescence

• Used to detect

antigen•Indirect and sandwichimmuno-fluorescence

• Antigen detection

• Antibody detection

Direct FA Indirect FA Sandwich FA

1st

2nd

3rd

4th

Ag=

Ab=

=FITC-conjugated Ab

Steps

Legend

=FITC-conjAnti-Ig




Western-blot analysis (1)• Principle

• Antigens are separated by Poly Acrylomide GelElectrophoresis (PAGE) and trans-blotted ontonitrocellulose/nylon membranes

• Antibodies in serum react with specific antigens

• Signals are detected according to the principles of testsystems

• Antibodies against microbes with numerous cross-reacting antibodies identified more specifically

• Examples• T. pallidum, B.burgdorferi,

• Herpes simplex virus types 1 and 2 Anti HIV-1




Anti HIV-2

Western-blot analysis (2)

• Serum, saliva, urine can be tested

• Kits are commercially available

• Recombinant immuno-blotting assays (RIBA) uses

recombinant proteins

I bl t




Immunoblot:


• Advantages

• Used for discrepant analysis

• Highly specific

• Rapid kits available

• Limitations

• Cost

• Concern validated data

• Time taken

• 1 day

I h t h




Immuno-chromatography:

Principle (1)

• Dye-labelled antibody, specific for target antigen, ispresent on the lower end of nitrocellulose strip or in aplastic well provided with the strip.

• Antibody, also specific for the target antigen, is bound tothe strip in a thin (test) line

• Either antibody specific for the labelled antibody, orantigen, is bound at the control line

Lysing agend

Labled AB.

Test band

(bound AB)

Control band

(bound AB)

Nitrocellulose strip

BoundAB

Free labledAB

I h t h





Principle (2)

• If antigen is present, some labelled antibody willbe trapped on the test line

• Excess-labelled antibody is trapped on the

control line Captured Ag-labelledAb-complex

Captured labelledAb

Labelled AB-AG-complexCaptured bybound AB oftest band

Labelled AB-AG-complexCaptured bybound AB ofcontrol band

I h t h






• Advantages

• Commercially available

• Single use, rapid test

• Easy to perform

• Can detect antigen or antibody

• Can be used in the field

• Limitations

• Cost

• Concern validated data

• Time taken - 1 hour

I t t ti f ti d t ti




Interpretation of antigen detection

tests

• In general, detection of the antigen denotes a presence ofthe pathogen

• More important in some of parasitic and fungal diseases

Antigen test InterpretationPositive •Current or recent infection

Negative •No infection

•Insufficient number of

organisms

•Sensitivity of testing islow(Consider test by test)

I t t ti f i l t




Interpretation of a single, acute

IgM test

IgM test Interpretation

Negative •No current infection

Positive (Newborn) •Congenitalinfection

Positive (Adult) •Primary or current

infection

Interpretation of t o ac te and




Interpretation of two, acute and

convalescent IgG tests *

Test Interpretation

Negative •No current

infection•Past infection

•Immuno-suppression

Positive(4-fold rise or fall intiter)

•

Recent infection

* Convalescent serum collected 2-4 weeks after onset




Interpretation of a single IgG test

* Collected between onset and convalescence

Test Interpretation

Negative •No exposure or immuno-suppression

Positive(Newborn)

•Maternal antibodies crossed the placenta

Positive (Adult) •Evidence of infection at some un-determined time

•

Infection in some cases (e.g., rabies,legionella, Ehrlichia)

•May be significant if immuno-suppression(e.g., AIDS)

El t i fl i th iti it




Elements influencing the sensitivity

and specificity of a given test kit

•

Test format• Precipitation versus IFA, Rapid test versus ELISA

• Purity of the antigen used

• Crude versus purified antigen versus syntheticpeptides

• Type of the antibody used

• Polyclonal versus monoclonal antibodies

• Interfering substances in the sample

• Presence of rheumatoid factor in the serum of thepatient

• Similarity in antigenic composition of pathogens

• Cross reactivity




Developed by:

The Department of Epidemic and Pandemic Alert and

Response of the World Health Organization with the

assistance of:

European Program for Field Epidemiology

Training

Canadian Field Epidemiology Programme

Thailand Ministry of Health

Institut Pasteur


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