Investigation strategies and methods

Antigen and antibody detection

May 2007

Laboratory Training for FieldEEpidemiologists

P I D E M I C A L E 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

Laboratory Training for FieldEEpidemiologists

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

• Detection of antigen-antibody complex

• Antigen-antibody complex requires specific conditions
• temperature
• pH
• Complex may be directly visible or invisible

Laboratory Training for FieldEEpidemiologists

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

equipment e.g. in ELISA, RIA, IFA

Laboratory Training for FieldEEpidemiologists

P I D E M I C A L E R T A N D R E S P O N S E
 Methods for Ag-Ab detection
• Precipitation
• Agglutination
• Hemagglutination and hemagglutination inhibition
• Viral neutralization test
• Radio-immunoassays
• ELISA
• Immunoflourescence
• Immunoblotting
• Immunochromatography
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P I D E M I C A L E R T A N D R E S P O N S E
 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)

Laboratory Training for FieldEEpidemiologists

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 Flocculation test
(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

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 Flocculation test
(A precipitation reaction)

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

RPR card test

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 Precipitation:
Performance, applications
• Advantages
• sensitive for antigen detection
• Limited applications
• Time taken - 10 minutes

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 Direct agglutination

Principle
• combination of an insoluble
particulate antigen with its soluble
antibody
• forms antigen-antibody complex
• particles clump/agglutinate Ag-Ab complex

• used for antigen detection

Examples
• bacterial agglutination tests for
sero-typing and sero-grouping
e.g., Vibrio cholerae,
Positive Negative
Salmonella spp
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 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)
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 Reverse passive agglutination

Principle
• antigen binds to soluble antibody coated on carrier
particles and results in agglutination
• detects antigens

Example
• detecting cholera toxin

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 Reverse passive agglutination

Positive

Negative

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P I D E M I C A L E R T A N D R E S P O N S E
 Agglutination:
Performance, 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

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P I D E M I C A L E 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 fowl’s 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
• detecting antibodies to Hemagglutination inhibition
for detection of Dengue
influenza and dengue
antibodies
viruses
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P I D E M I C A L E R T A N D R E S P O N S E
 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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 Neutralization assays
Principle
• 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)

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 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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 Radio-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

Response
• HBsAg
• Thyroid function test

Antibody

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 Positive Neutralization Assay

Negative

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 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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 Enzyme-linked immunosorbant
Labeling technique
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
Response

Micro-plate reader
Antibody

Positive result

96-well micro-plate
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P I D E M I C A L E R T A N D R E S P O N S E
 Labeling technique
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
•Hydrolysissignal 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)

Laboratory Training for FieldEEpidemiologists

P I D E M I C A L E R T A N D R E S P O N S E
 Types of ELISA used in the Labeling
detectiontechnique of
antigens and antibodies
•Non-competitive

•must remove excess/unbound Ag

or 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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 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
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P I D E M I C A L E R T A N D R E S P O N S E
 Performance comparison of
various ELISAs for antibody
detection
Performance Non- Competitive Capture ELISA
characteristic competitive ELISA
ELISA
Purpose Antibody Antibody Best for class
specific
antibody
Sensitivity ++ ++ ++

Specificity ++ ++ +++
Cost + ++ +++

Ease of ++ +++ ++
performance
Time taken ++ + +++
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 Labeling technique
Immuno-fluorescence
•Principle

• Use fluorescein
isothiocyanate labeled-
immunoglobulin to detect
antigens or antibodies
according to test systems
Cell infected with Dengue virus
• Requires a fluorescent
microscope
•Examples

• Herpes virus IgM

• Dengue virus
• Rabies virus
• Scrub and murine typhus V. Cholerae
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P I D E M I C A L E R T A N D R E S P O N S E
 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
• Training
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P I D E M I C A L E R T A N D R E S P O N S E
 Labeling technique
Types of immuno-fluorescence

•Directimmuno- Steps Direct FA Indirect FA Sandwich FA

fluorescence 1st

• Used to detect 2nd

antigen
•Indirect
and sandwich
3rd

Legend
Ag=

immuno-fluorescence
=FITC-conjAnti-Ig

Ab=
=FITC-conjugated Ab

• Antigen detection 4th

• Antibody detection

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 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 2
Anti HIV-1
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P I D E M I C A L E R T A N D R E S P O N S E
 Western-blot analysis (2)

• Serum, saliva, urine can be tested

• Kits are commercially available
• Recombinant immuno-blotting assays (RIBA) uses
recombinant proteins

Anti HIV-2

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 Immunoblot:
Performance, applications
• Advantages
• Used for discrepant analysis
• Highly specific
• Rapid kits available
• Limitations
• Cost
• Concern validated data
• Time taken
• 1 day
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 Immuno-chromatography:
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 agend Test band Control band
Bound Labled AB. (bound AB) (bound AB)
AB

Free labled
AB

Nitrocellulose strip

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 Immuno-chromatography:
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 Captured labelled
Ab-complex Ab

Labelled AB-AG- Labelled AB-AG-

complex complex
Captured by Captured by
bound AB of bound AB of
test band control band

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 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
Laboratory Training for FieldEEpidemiologists
P I D E M I C A L E R T A N D R E S P O N S E
 Interpretation of antigen detection
tests
• 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)

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 Interpretation of a single, acute
IgM test

IgM test Interpretation

Negative •No current infection
Positive (Newborn) •Congenital
infection
Positive (Adult) •Primary or current
infection

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 Interpretation of two, acute and
convalescent IgG tests *

Test Interpretation
Negative •No current infection
•Past infection
•Immuno-
suppression

Positive •Recent infection

(4-fold rise or fall in
titer)
* Convalescent serum collected 2-4 weeks after onset

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 Interpretation of a single IgG test

Test Interpretation
Negative •No exposure or immuno-suppression
Positive •Maternal antibodies crossed the placenta
(Newborn)
Positive (Adult) •Evidenceof infection at some un-
determined time
•Infectionin some cases (e.g., rabies,
legionella, Ehrlichia)
•May
be significant if immuno-suppression
(e.g., AIDS)
* Collected between onset and convalescence

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 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 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

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 Investigation strategies and methods

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

Laboratory Training for FieldEEpidemiologists

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