Learning objectives At the end of the presentation, participants should

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

Detection

• Detection of antigen-antibody complex

• Antigen-antibody complex requires specific conditions

• temperature
• pH

• Complex may be directly visible or invisible

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

Methods for Ag-Ab detection

• Precipitation

• Agglutination

• Hemagglutination and hemagglutination inhibition

• Viral neutralization test

• Radio-immunoassays

• ELISA

• Immunoflourescence

• Immunoblotting

• Immunochromatography

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)

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

Flocculation test (A precipitation reaction)

Precipitation: Performance, applications

• Advantages

• sensitive for antigen detection

• Limited applications

• Time taken - 10 minutes

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

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)

Reverse passive agglutination

• Principle

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

• Example

• detecting cholera toxin

Reverse passive agglutination

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

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

Hemagglutination 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

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

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)

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

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

• HBsAg
• Thyroid function test

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

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)

ELISA

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)

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

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

Performance comparison of various ELISAs for antibody detection

Immuno-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

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

Types of immuno-fluorescence

• Direct immuno-fluorescence

• Used to detect antigen

• Indirect and sandwich immuno-fluorescence

• Antigen detection
• Antibody detection

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

Western-blot analysis (2)

• Serum, saliva, urine can be tested

• Kits are commercially available

• Recombinant immuno-blotting assays (RIBA) uses recombinant proteins

Immunoblot: Performance, applications

• Advantages

• Used for discrepant analysis
• Highly specific
• Rapid kits available

• Limitations

• Cost
• Concern validated data

• Time taken

• 1 day

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

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

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

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

Interpretation of a single, acute IgM test

Interpretation of two, acute and convalescent IgG tests *

Interpretation of a single IgG test

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