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
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 objectivesLearning objectives
At the end of the presentation, participants should
• Understand direct and indirect antibody detection
• Understand the different methods for detecting antigens or antibodies
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
• Detection of antigen-antibody complex
• Antigen-antibody complex requires specific conditions• temperature
• pH
• Complex may be directly visible or invisible
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
DetectionDetection
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 specific equipment e.g. in ELISA, RIA, IFA
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 detectionMethods for Ag-Ab detection• Precipitation
• Agglutination
• Hemagglutination and hemagglutination inhibition
• Viral neutralization test
• Radio-immunoassays
• ELISA
• Immunoflourescence
• Immunoblotting
• Immunochromatography
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
PrecipitationPrecipitation
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)
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
Flocculation test Flocculation test (precipitation reaction)(precipitation reaction)
Principle
• precipitate, a concentrate of fine particles, is usually visible (macroscopically or microscopically) because the precipitated product is forced to remain suspended
Examples
• VDRL slide flocculation test
• RPR card test
• Kahn’s test for syphilis
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
RPR card test
Flocculation test Flocculation test (A precipitation reaction)(A precipitation reaction)
(1) Non Reactive (2) Weakly Reactive (3,4) Reactive
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:Precipitation:Performance, applicationsPerformance, 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 Direct agglutination
Principle
• combination of an insoluble particulate antigen with its soluble antibody
• forms antigen-antibody complex
• particles clump/agglutinate • used for antigen detection
Examples
• bacterial agglutination tests for sero-typing and sero-grouping e.g., Vibrio cholerae, Salmonella spp
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 Passive (indirect) agglutination
Principle
• precipitation reaction converted into agglutination - coating antigen onto the surface of carrier particles like red blood cells, latex, gelatin, bentonite
• background clears
Examples of types
• latex agglutination
• co-agglutination
• passive hemagglutination (treated red blood cells made resistant)
Examples of tests - agglutination for leptospirosis Widal test (typhoid fever)
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 carrier particles and results in agglutination
• detects antigens
Example
• detecting cholera toxin
Reverse passive agglutinationReverse passive agglutination
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 agglutinationReverse passive agglutination
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:Agglutination:Performance, applicationsPerformance, applications
Advantages
• sensitive for antibody detection
Limitations
• Prozone phenomenon:
• requires the right combination of quantities of antigen and antibody
• handled through dilution to improve the match
Time taken
• 10-30 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
HemagglutinationHemagglutination
Principle
• many human viruses have the ability to bind to the surface structures on red blood cells from different species thereby causing agglutination
Example
• influenza virus binds to fowl’s red blood cells
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 inhibitionHemagglutination inhibition
Principle
Antibodies to the virus in the patient serum bind to the virus; blocks binding sites on the viral surfaces
• prevents the virus from agglutinating the red cells
Example
• detecting antibodies to influenza and dengue viruses
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:Hemagglutination:Performance, applicationsPerformance, applications
Advantages
• highly specific
• can be used as gold standard
Limitations
• technically demanding
• time consuming
• cannot distinguish IgG from IgM
Time taken
• 1 day
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 assaysNeutralization assaysPrinciple
• antibodies in serum neutralize antigens on the surface of 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-cellular products that display measurable activities (e.g., ASLO, diphtheria toxin, clostridium toxin)
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:Neutralization:Performance, applicationsPerformance, 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
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-immunoassaysRadio-immunoassays
• Principle• Radioactively labelled-antibody (or antigen) competes with the patient’s
unlabelled antibody (or antigen) for binding sites on a known amount of antigen (or antibody)
• Reduction in radioactivity of the antigen-patient antibody complex compared with control test is used to quantify the amount of patient antibody / antibody bound
• Limited use due to the problems with handling radioisotope
• Example• HBsAg
• Thyroid function testR
espo
nse
Antibody
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
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:Radio-immunoassays:Performance, applicationsPerformance, applications
Adantages
• highly sensitive
• can be used for detection of small quantities
• quantification possible
Limitations
• expensive
• requires isotopes
Time taken
• 1 day
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
Enzyme-linked immunosorbant assay Enzyme-linked immunosorbant assay (ELISA)(ELISA)
Principle
• use of enzyme-labelled immunoglobulin to detect antigens or antibodies
• signals are developed by the action of hydrolyzing enzyme on chromogenic substrate
• optical density measured by micro-plate reader
Examples
• Hepatitis A (Anti-HAV-IgM, anti-HAV IgG)
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 ELISA
Antibody
Res
pons
e
Micro-plate reader
96-well micro-plate
Positive result
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 (Ag Ab tests)Types of ELISA (Ag Ab tests)
Competitive
•Antigen or antibody are labelled with enzyme and allowed to compete with unlabeled ones (in patient serum) for binding to the same target
•Hydrolysis signal from Ag-Ab complex (enzyme-labelled) is measured
•Antigen or antibody in serum is then calculated
•No need to remove the excess/unbound Ag or Ab from the reaction plate or tubes)
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 used in the detection of Types of ELISA used in the detection of antigens and antibodiesantigens 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
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:ELISA:Performance, applicationsPerformance, 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
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
Performance characteristic
Non-competitive
ELISA
Competitive ELISA
Capture ELISA
Purpose Antibody Antibody Best for class specific antibody
Sensitivity ++ ++ ++
Specificity ++ ++ +++
Cost + ++ +++
Ease of performance
++ +++ ++
Time taken ++ + +++
Performance comparison of various Performance comparison of various ELISAs for antibody detectionELISAs for antibody detection
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-fluorescenceImmuno-fluorescence•Principle
• Use fluorescein isothiocyanate labeled-immunoglobulin to detect antigens or antibodies according to test systems
• Requires a fluorescent microscope
•Examples
• Herpes virus IgM
• Dengue virus
• Rabies virus
• Scrub and murine typhus
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:Immuno-fluorescence:Performance, applicationsPerformance, 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
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 immuno-fluorescenceTypes of immuno-fluorescence
•Direct immuno-fluorescence
• Used to detect antigen
•Indirect and sandwich immuno-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
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)Western-blot analysis (1)• Principle
• Antigens are separated by Poly Acrylomide Gel Electrophoresis (PAGE) and trans-blotted onto nitrocellulose/nylon membranes
• Antibodies in serum react with specific antigens
• Signals are detected according to the principles of test systems
• Antibodies against microbes with numerous cross-reacting antibodies identified more specifically
• Examples• T. pallidum, B.burgdorferi,
• Herpes simplex virus types 1 and 2Anti HIV-1
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)Western-blot analysis (2)
• Serum, saliva, urine can be tested
• Kits are commercially available
• Recombinant immuno-blotting assays (RIBA) uses recombinant proteins
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:Immunoblot:Performance, applicationsPerformance, applications
• Advantages
• Used for discrepant analysis
• Highly specific
• Rapid kits available
• Limitations
• Cost
• Concern validated data
• Time taken
• 1 day
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: Immuno-chromatography: Principle (1)Principle (1)
• Dye-labelled antibody, specific for target antigen, is present on the lower end of nitrocellulose strip or in a plastic well provided with the strip.
• Antibody, also specific for the target antigen, is bound to the strip in a thin (test) line
• Either antibody specific for the labelled antibody, or antigen, is bound at the control line
Lysing agendLabled AB.
Test band(bound AB)
Control band(bound AB)
Nitrocellulose strip
BoundAB
Free labled AB
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: Immuno-chromatography: Principle (2)Principle (2)
• If antigen is present, some labelled antibody will be trapped on the test line
• Excess-labelled antibody is trapped on the control line
Captured Ag-labelled Ab-complex
Captured labelled Ab
Labelled AB-AG-complexCaptured by bound AB of test band
Labelled AB-AG-complexCaptured by bound AB ofcontrol band
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:Immuno-chromatography: Performance, applications 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
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 Interpretation of antigen detection teststests
• In general, detection of the antigen denotes a presence of the pathogen
• More important in some of parasitic and fungal diseases
Antigen test Interpretation
Positive •Current or recent infection
Negative •No infection
•Insufficient number of organisms
•Sensitivity of testing is low (Consider test by test)
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 Interpretation of a single, acute IgM testtest
IgM test Interpretation
Negative •No current infection
Positive (Newborn) •Congenital infection
Positive (Adult) •Primary or current infection
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 Interpretation of two, acute and convalescent IgG tests *convalescent IgG tests *
Test Interpretation
Negative •No current infection
•Past infection
•Immuno-suppression
Positive (4-fold rise or fall in titer)
•Recent infection
* Convalescent serum collected 2-4 weeks after onset
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 testInterpretation 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)
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 Elements influencing the sensitivity and specificity of a given test kitspecificity of a given test kit
• Test format • Precipitation versus IFA, Rapid test versus ELISA
• Purity of the antigen used • Crude versus purified antigen versus synthetic
peptides• Type of the antibody used
• Polyclonal versus monoclonal antibodies• Interfering substances in the sample
• Presence of rheumatoid factor in the serum of the patient
• Similarity in antigenic composition of pathogens • Cross reactivity
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
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