Investigation strategies and methods

Antigen and antibody detection

May 2007
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 or antibodies

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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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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Methods for Ag-Ab detection

Precipitation Agglutination Hemagglutination and hemagglutination inhibition Viral neutralization test

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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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

Kahns test for syphilis

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Flocculation test (A precipitation reaction)

(1) Non Reactive

(2) Weakly Reactive RPR card test

(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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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
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Positive

Negative

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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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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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

detecting antibodies to influenza and dengue viruses

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Hemagglutination inhibition for detection of Dengue antibodies

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 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
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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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Labeling technique Enzyme-linked immunosorbant

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
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Types of ELISA (Ag Ab tests)

Competitive
Antigen

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

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)

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Labeling technique Types of ELISA used in the detection 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

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Time taken - 1 day

Performance comparison of various ELISAs for antibody detection

Performance characteristic Noncompetitive ELISA Competitive ELISA Capture ELISA

Purpose

Antibody

Antibody

Best for class specific antibody ++ +++

Sensitivity Specificity

++ ++

++ ++

Cost
Ease of performance 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

Cell infected with Dengue virus

Herpes virus IgM

Dengue virus
Rabies virus Scrub and murine typhus
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V. Cholerae

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 1 day Laboratory for Field Epidemiologists

Types of immuno-fluorescence
Direct

Labeling technique

immunofluorescence Used to detect antigen

Indirect

and sandwich immuno-fluorescence Antigen detection

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 crossreacting antibodies identified more specifically

Examples
T. pallidum, B.burgdorferi,

Herpes simplex virus types 1 and 2

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Anti HIV-1

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
Bound AB Free labled AB Nitrocellulose strip Lysing agend Labled AB. Test band Control band (bound AB) (bound AB)

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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-AGcomplex Captured by bound AB of test band

Labelled AB-AGcomplex Captured by bound AB of 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

Laboratory Training for Field Epidemiologists

Time taken - 1 hour

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 Positive Negative Interpretation Current or recent infection

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 Negative Positive (Newborn) Positive (Adult)

Interpretation
No

current infection

Congenital

infection
Primary

or current

infection

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Interpretation of two, acute and convalescent IgG tests *

Test Negative

Interpretation 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

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Interpretation of a single IgG test

Test Negative Positive (Newborn)

Interpretation
No

exposure or immuno-suppression antibodies crossed the placenta

Maternal

Positive (Adult)

Evidence
Infection May

of infection at some undetermined time

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

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 Field Epidemiologists