Professional Documents
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May 2007
P I D E M I C A L E Laboratory Training for FieldEEpidemiologists R T A N D R E S P O N S E
Learning objectives
At the end of the presentation, participants should
Understand direct and indirect antibody detection Understand the different methods for detecting antigens or antibodies
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Detection
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Detection
Directly visible agglutination Invisible
requires specific probes (enzyme-labelled antiimmunoglobulin, 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
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Radio-immunoassays
ELISA Immunoflourescence Immunoblotting Immunochromatography
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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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Examples
VDRL slide flocculation test
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(3,4) Reactive
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Advantages
sensitive for antigen detection
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Direct agglutination
Principle
combination of an insoluble particulate antigen with its soluble antibody forms antigen-antibody complex particles clump/agglutinate
used for antigen detection
Ag-Ab complex
Examples bacterial agglutination tests for sero-typing and sero-grouping e.g., Vibrio cholerae,
Salmonella spp
P I D E M I C A L E Laboratory Training for FieldEEpidemiologists R T A N D
Positive
Negative
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Example
detecting cholera toxin
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Negative
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Limitations
Prozone phenomenon:
Time taken
10-30 minutes
P I D E M I C A L E Laboratory Training for FieldEEpidemiologists R T A N D R E S P O N S E
Hemagglutination
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 fowls red blood cells
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Hemagglutination inhibition
Principle
Positive Negative
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
Limitations
technically demanding time consuming cannot distinguish IgG from IgM
Time taken
1 day
P I D E M I C A L E Laboratory Training for FieldEEpidemiologists R T A N D R E S P O N S E
Neutralization assays
Principle antibodies in serum neutralize antigens on the surface of viruses (neutralizing antibodies) inhibited viruses cannot infect cell lines Example
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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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Radio-immunoassays
Principle
Radioactively labelled-antibody (or antigen) competes with the patients 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 test
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Positive
Neutralization Assay
Negative
P I D E M I C A L E Laboratory Training for FieldEEpidemiologists R T A N D R E S P O N S E
Limitations
expensive requires isotopes
Time taken
1 day
P I D E M I C A L E Laboratory Training for FieldEEpidemiologists R T A N D R E S P O N S E
assay (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)
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ELISA
Micro-plate reader
Positive result
96-well micro-plate
P I D E M I C A L E Laboratory Training for FieldEEpidemiologists R T A N D R E S P O N S E
Labeling technique
or antibody are labelled with enzyme and allowed to compete with unlabeled ones (in patient serum) for binding to the same target
Hydrolysis
Labeling technique Types of ELISA used in the detection of antigens and antibodies
Non-competitive
must
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
Advantages
Automated, inexpensive
Objective Small quantities required
Limitations
Expensive initial investment Variable sensitivity / specificity of variable tests Cross contamination
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Purpose
Antibody
Antibody
++ ++ + ++
++ ++ ++ +++
Time taken
++
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Immuno-fluorescence
Principle
Labeling technique
Use fluorescein isothiocyanate labeledimmunoglobulin to detect antigens or antibodies according to test systems Requires a fluorescent microscope
Examples
Dengue virus
Rabies virus Scrub and murine typhus
P I D E M I C A L E Laboratory Training for FieldEEpidemiologists R T A N D
V. Cholerae
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Advantages
Sensitive and specific
Can be used for discrepant analysis
Limitations
Expensive (Reagents and equipment)
Subjective Cross reactivity Non-specific immuno-fluorescence
Time taken
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Types of immuno-fluorescence
Direct
Labeling technique
Steps
1st
Direct FA
Indirect FA
Sandwich FA
2nd
3rd
Legend
Ag= Ab= =FITC-conjugated Ab =FITC-conjAnti-Ig
4th
Antibody detection
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Principle
Antigens are separated by Poly Acrylomide Gel Electrophoresis (PAGE) and trans-blotted onto nitrocellulose/nylon membranes
Examples
T. pallidum, B.burgdorferi,
Anti HIV-1
Anti HIV-2
P I D E M I C A L E Laboratory Training for FieldEEpidemiologists R T A N D R E S P O N S E
Advantages
Used for discrepant analysis
Highly specific Rapid kits available
Limitations
Cost Concern validated data
Time taken
1 day
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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
Bound AB Free labled AB Nitrocellulose strip Lysing agend Labled AB. Test band Control band (bound AB) (bound AB)
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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 Captured labelled Ab-complex Ab
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Advantages
Commercially available
Single use, rapid test Easy to perform
Limitations
Cost Concern validated data
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In general, detection of the antigen denotes a presence of the pathogen More important in some of parasitic and fungal diseases
Antigen test Positive Interpretation Current or recent infection
Negative
No infection
Insufficient number of organisms Sensitivity of testing is low (Consider test by test)
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Interpretation
No
current infection
Positive (Newborn)
Positive (Adult)
Congenital
infection
Primary
or current
infection
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Test Negative
infection
Immuno-
infection
Test Negative
Interpretation
No
exposure or immuno-suppression
Positive (Newborn)
Positive (Adult)
Maternal
of infection at some undetermined time in some cases (e.g., rabies, legionella, Ehrlichia)
Test format
Precipitation versus IFA, Rapid test versus ELISA
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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
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