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Antigen and antibody detection

Investigation strategies and methods. Antigen and antibody detection. May 2007. 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.

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Antigen and antibody detection

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  1. Investigation strategies and methods Antigen and antibody detection May 2007

  2. 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

  3. Detection • Detection of antigen-antibody complex • Antigen-antibody complex requires specific conditions • temperature • pH • Complex may be directly visible or invisible

  4. 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

  5. Methods for Ag-Ab detection • Precipitation • Agglutination • Hemagglutination and hemagglutination inhibition • Viral neutralization test • Radio-immunoassays • ELISA • Immunoflourescence • Immunoblotting • Immunochromatography

  6. 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)

  7. 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

  8. Flocculation test (A precipitation reaction) (1) Non Reactive (2) Weakly Reactive (3,4) Reactive RPR card test

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

  10. Direct agglutination Ag-Ab complex Positive Negative 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

  11. 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)

  12. Reverse passive agglutination Principle • antigen binds to soluble antibody coated on carrier particles and results in agglutination • detects antigens Example • detecting cholera toxin

  13. Reverse passive agglutination Positive Negative

  14. 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

  15. 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

  16. 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 Positive Negative Hemagglutination inhibition for detection of Dengue antibodies

  17. 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

  18. 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)

  19. 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

  20. Radio-immunoassays Response Antibody • 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

  21. Positive Negative Neutralization Assay

  22. 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

  23. Enzyme-linked immunosorbant assay (ELISA) Labeling technique 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)

  24. ELISA Micro-plate reader Positive result Response 96-well micro-plate Antibody

  25. Types of ELISA (Ag Ab tests) Labeling technique 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)

  26. Types of ELISA used in the detection of antigens and antibodies Labeling technique • Non-competitive • must remove excess/unbound Agor 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

  27. 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

  28. Performance comparison of various ELISAs for antibody detection

  29. Immuno-fluorescence Labeling technique • 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 Cell infected with Dengue virus V. Cholerae

  30. 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

  31. Types of immuno-fluorescence Labeling technique • Direct immuno-fluorescence • Used to detect antigen • Indirect and sandwich immuno-fluorescence • Antigen detection • Antibody detection

  32. 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

  33. 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

  34. Immunoblot:Performance, applications • Advantages • Used for discrepant analysis • Highly specific • Rapid kits available • Limitations • Cost • Concern validated data • Time taken • 1 day

  35. Immuno-chromatography: Principle (1) Lysing agend Labled AB. Control band (bound AB) Test band (bound AB) Bound AB Free labled AB Nitrocellulose strip • 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

  36. Immuno-chromatography: Principle (2) Captured Ag-labelled Ab-complex Captured labelled Ab Labelled AB-AG-complex Captured by bound AB of test band Labelled AB-AG-complex Captured by bound AB of control band • If antigen is present, some labelled antibody will be trapped on the test line • Excess-labelled antibody is trapped on the control line

  37. 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

  38. 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

  39. Interpretation of a single, acute IgM test

  40. Interpretation of two, acute and convalescent IgG tests * * Convalescent serum collected 2-4 weeks after onset

  41. Interpretation of a single IgG test * Collected between onset and convalescence

  42. 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

  43. 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

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