1、,Antigen and antibody detection,Investigation strategies and methods,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,Detection of antigen
2、-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-A
3、b 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 Immunoblot
4、ting 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 rea
5、ction),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,RPR card test,Flocculation test (A precipi
6、tation reaction),(1) Non Reactive (2) Weakly Reactive (3,4) Reactive,Precipitation: Performance, applications,Advantages sensitive for antigen detection Limited applications Time taken - 10 minutes,Positive Negative,Direct agglutination,Principle combination of an insoluble particulate antigen with
7、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 a
8、gglutination - 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 leptos
9、pirosis Widal test (typhoid fever),Principle antigen binds to soluble antibody coated on carrier particles and results in agglutination detects antigens Example detecting cholera toxin,Reverse passive agglutination,Positive,Negative,Reverse passive agglutination,Agglutination: Performance, applicati
10、ons,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 su
11、rface structures on red blood cells from different species thereby causing agglutination Example influenza virus binds to fowls red blood cells,Positive,Negative,Hemagglutination inhibition for detection of Dengue antibodies,Hemagglutination inhibition,Principle Antibodies to the virus in the patien
12、t 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 techn
13、ically 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
14、, 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
15、 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 patients unlabelled antibody (or antigen) for binding sites on a known
16、 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,Neutraliza
17、tion Assay,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,Labeling technique,Enzyme-linked immunosorbant assay (ELISA),Principle use of enzyme-labe
18、lled 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,Labeling technique,Types of ELISA (Ag Ab tests),Competitive A
19、ntigen 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 fr
20、om the reaction plate or tubes),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
21、, 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
22、 sensitivity / specificity of variable tests Cross contamination Time taken - 1 day,Performance comparison of various ELISAs for antibody detection,Labeling technique,Cell infected with Dengue virus,V. Cholerae,Immuno-fluorescence,Principle Use fluorescein isothiocyanate labeled-immunoglobulin to de
23、tect 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 Expensi
24、ve (Reagents and equipment) Subjective Cross reactivity Non-specific immuno-fluorescence Time taken 1 day,Labeling technique,Types of immuno-fluorescence,Direct immuno-fluorescence Used to detect antigen Indirect and sandwich immuno-fluorescence Antigen detection Antibody detection,Western-blot anal
25、ysis (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 cr
26、oss-reacting antibodies identified more specifically Examples T. pallidum, B.burgdorferi, Herpes simplex virus types 1 and 2,Anti HIV-1,Anti HIV-2,Western-blot analysis (2),Serum, saliva, urine can be tested Kits are commercially available Recombinant immuno-blotting assays (RIBA) uses recombinant p
27、roteins,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
28、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
29、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 fiel
30、d 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, ac
31、ute and convalescent IgG tests *,* Convalescent serum collected 2-4 weeks after onset,Interpretation of a single IgG test,* Collected between onset and convalescence,Elements influencing the sensitivity and specificity of a given test kit,Test format Precipitation versus IFA, Rapid test versus ELISA
32、 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
33、reactivity,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,
