Learning objectives At the end of the presentation, participants should

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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 of antigen-antibody complex

  • Antigen-antibody complex requires specific conditions

    • temperature
    • pH
  • Complex may be directly visible or invisible


  • 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


  • Immunoflourescence

  • Immunoblotting

  • Immunochromatography


  • Principle

  • 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


  • 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


  • 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)


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


  • 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

Kataloq: ihr

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