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Antigen and antibody detection PowerPoint Presentation

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Description : Check out this medical presentation on Influenza (Flu), which is titled "Antigen and antibody detection", to understand direct and indirect antibody detection, and the different methods for detecting antigens or antibodies.

Tags : Influenza, Flu, Antigen, antibody detection

Published on : Aug 29, 2021 | Views : 2333 | Downloads : 0

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Slide 1-

Antigen and antibody detection Investigation strategies and methods
Slide 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
Slide 3-

Detection Detection of antigen-antibody complex Antigen-antibody complex requires specific conditions temperature pH Complex may be directly visible or invisible
Slide 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
Slide 5-

Methods for Ag-Ab detection Precipitation Agglutination Hemagglutination and hemagglutination inhibition Viral neutralization test Radio-immunoassays ELISA Immunoflourescence Immunoblotting Immunochromatography
Slide 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)
Slide 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
Slide 8-

RPR card test Flocculation test (A precipitation reaction) (1) Non Reactive (2) Weakly Reactive (3,4) Reactive
Slide 9-

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

Positive Negative 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
Slide 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)
Slide 12-

Principle antigen binds to soluble antibody coated on carrier particles and results in agglutination detects antigens Example detecting cholera toxin Reverse passive agglutination
Slide 13-

Positive Negative Reverse passive agglutination
Slide 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
Slide 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
Slide 16-

Positive Negative Hemagglutination inhibition for detection of Dengue antibodies 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
Slide 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
Slide 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)
Slide 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
Slide 20-

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
Slide 21-

Neutralization Assay
Slide 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
Slide 23-

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)
Slide 24-

ELISA
Slide 25-

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 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)
Slide 26-

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
Slide 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
Slide 28-

Performance comparison of various ELISAs for antibody detection
Slide 29-

Labeling technique Cell infected with Dengue virus V. Cholerae 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
Slide 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
Slide 31-

Labeling technique Types of immuno-fluorescence Direct immuno-fluorescence Used to detect antigen Indirect and sandwich immuno-fluorescence Antigen detection Antibody detection
Slide 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
Slide 33-

Anti HIV-2 Western-blot analysis (2) Serum, saliva, urine can be tested Kits are commercially available Recombinant immuno-blotting assays (RIBA) uses recombinant proteins
Slide 34-

Immunoblot: Performance, applications Advantages Used for discrepant analysis Highly specific Rapid kits available Limitations Cost Concern validated data Time taken 1 day
Slide 35-

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
Slide 36-

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
Slide 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
Slide 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
Slide 39-

Interpretation of a single, acute IgM test
Slide 40-

Interpretation of two, acute and convalescent IgG tests * * Convalescent serum collected 2-4 weeks after onset
Slide 41-

Interpretation of a single IgG test * Collected between onset and convalescence
Slide 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
Slide 43-

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 Investigation strategies and methods