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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
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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
Learning objectives
At the end of the presentation, participants should
• Understand direct and indirect antibody detection
• Understand the different methods for detecting antigens orantibodies
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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
Detection
• Detection of antigen-antibody complex
• Antigen-antibody complex requires specific conditions
• temperature
• pH
• Complex may be directly visible or invisible
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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
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
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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
Methods for Ag-Ab detection
• Precipitation
• Agglutination
• Hemagglutination and hemagglutination inhibition
• Viral neutralization test
• Radio-immunoassays
• ELISA
• Immunoflourescence
• Immunoblotting
• Immunochromatography
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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
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
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RPR card test
Flocculation test
(A precipitation reaction)
(1) Non Reactive (2) Weakly Reactive (3,4) Reactive
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Precipitation:
Performance, applications
• Advantages
• sensitive for antigen detection
• Limited applications
• Time taken - 10 minutes
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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
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
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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
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)
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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
Principle
• antigen binds to soluble antibody coated on carrierparticles and results in agglutination
• detects antigens
Example
• detecting cholera toxin
Reverse passive agglutination
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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
Positive
Negative
Reverse passive agglutination
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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
Agglutination:
Performance, applications
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
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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
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
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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
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
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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
Hemagglutination:
Performance, applications
Advantages
• highly specific
• can be used as gold standard
Limitations
• technically demanding
• time consuming
• cannot distinguish IgG from IgM
Time taken
• 1 day
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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
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)
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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
Neutralization:
Performance, applications
• 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
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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
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
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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
Positive
Negative
Neutralization Assay
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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
Radio-immunoassays:
Performance, applications
Adantages
• highly sensitive
• can be used for detection of small quantities
• quantification possible
Limitations
• expensive
• requires isotopes
Time taken
• 1 day
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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
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)
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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
ELISA
Micro-plate reader
96-well micro-plate Positive result
L b li t h i
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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
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
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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
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
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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
ELISA:
Performance, applications
• 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
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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
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
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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
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
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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
Immuno-fluorescence:
Performance, applications
• 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
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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
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
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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
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
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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
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
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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
Immunoblot:
Performance, applications
• Advantages
• Used for discrepant analysis
• Highly specific
• Rapid kits available
• Limitations
• Cost
• Concern validated data
• Time taken
• 1 day
I h t h
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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
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
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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
Immuno-chromatography:
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
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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
Immuno-chromatography:
Performance, applications
• 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
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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
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
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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
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
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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
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
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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
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
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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
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
Investigation strategies and methods
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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
Investigation strategies and methods