antigen – antibody reactions
TRANSCRIPT
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Antigen – Antibody reactionsPart II
Dr.M.MalathiFinal year PG
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Time to recollect !!!
• Antigen?
• Antibody?
• Antigen antibody reactions?
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Complement Fixation Test (CFT)
• What is a complement ?
• Antigen antibody complex – bound by complement – mediates bacterial clearance, viral neutralisation, lysis of RBC
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Im the source of complement
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Procedure
• Heat inactivation of the antiserum – to inactivate the complementary activity of serum
• The source of the complement is guinea pig serum.
• Complement – heat labile
• Freshly draw the serum
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Ingredients
• CFT has two steps and five reagents• Antigen• Antibody• Complement• Sheep erythrocytes• Amboceptor ( Rabbit antibody to sheep red
cells)
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Amboceptor ( Rabbit antibody to sheep red cells)
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Wassermann reaction
• Formerly used for the serodiagnosis of syphilis• Procedure
1. Lysis of erythrocytes – Negative CFT2. No lysis of erythrocytes – Positive CFT• For every test, appropriate controls should be
added.
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Other complement dependent tests:
• Immune adherence-T.pallidum, V.cholerae
• Immobilisation test – Treponema pallidum
• Cytolytic or cytocidal test- V.cholerae
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Conglutination
• Some donot fix the guinea pig serum complement
• Use indirect CFT or conglutination test• Use sheep RBC with bovine serum• Bovine serum – conglutinin – beta globulin –
antibody to complement
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NEUTRALISATION TESTS
• Virus neutralisation tests:1. Neutralisation of viruses by their antibodies
and are demonstrated in cell cultures, eggs and animals
2. Neutralisation of bacteriophages by the plaque inhibition test
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Toxin neutralisation tests
• Bacterial exotoxins - induce neutralising antibodies(antitoxins) protection against diseases such as diphtheria and tetanus.
• In vivo tests:1. Toxigenicity test-detection of toxin of C.diphtheriae2. Schick test
• In vitro tests:1. Anti streptolysin O (ASO) test2. Virus neutralisation test3. Nagler reaction-rapid detection of C.welchii
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OPSONISATION TEST
• Opsonisation - process by which a particulate antigen becomes more susceptible to phagocytosis.
• Opsonin – is then identified as a complement.• Bacteriotrophin – heat stable serum factor
with similar activity Specific antibody
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• Opsonic index ratio of the phagocytic activity of the patients blood for a given bacterium, to the normal individual.
• Measured by incubating fresh citrated blood with the bacterial suspension at 37deg C for 15 minutes
• Phagocytic Index: Estimating the average number of phagocytosed bacteria per PMN from stained blood films
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Immunofluorescence
• Fluorescence ?• Immuno fluorescence ?• Types – Direct and indirect
• The commonly used fluorescent dyes are:i) Fluorescin isothiocyanate (Blue green
fluorescence)ii) Lissamine rhodamine (orange red fluorescence)
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Direct immunofluorescence test
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Uses
1. It is commonly employed for detection of bacteria, viruses or other antigens in blood, CSF, urine, faeces, tissues and other specimens
2. It is a sensitive method to diagnose rabies by detection of the rabies virus antigens in brain smears
Disadvantage
• Separate specific fluorescent labelled antibody has to prepared against each antigen to be tested
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Indirect immunofluorescence test
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RADIOIMMUNOASSAY (RIA)
• Berson and Yallow (1959) first described RIA• Radioimmunoassay is widely-used because of
its great sensitivity• RIA detect antigens upto picogram (10−12 g)
quantities
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RADIOIMMUNOASSAY(RIA)
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Uses of RIA
• RIA have applications in biology and medicine-
1. Quantitation of hormones, drugs, tumour markers,
2. IgE and viral antigens
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Disadvantages
• Radiation hazards: Uses radiolabelled reagent
• Radioactive waste disposal
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ELISA
Micro-plate reader
96-well micro-plate
Positive result
ENZYME LINKED IMMUNOSORBENT ASSAY
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Types of ELISA
– Direct ELISA
– Indirect ELISA
– Competitive ELISA
– Sandwich ELISA
– Capture ELISA
– Cylinder or cassette ELISA
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Types of ELISA
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Immunochromatographic Tests
• A colored band appears at the second window
• Control also can be recorded
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Chemiluminescence immunoassay (CLIA)
• Chemiluminescence - Chemical reaction emitting energy in the form of light
• Chemilumiscence compounds - Luminol or acridinium esters .
• Signal can be amplified, measured, and the concentration of the analyte calculated
• The method is fully automated• Uses - where the volume of work is large - drug sensitivity testing of M.tuberculosis
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CLIA
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Immunoelectroblot/Westernblot
• Immunoblot or westernblot techniques combine the sensitivity of enzyme immunoassay with much greater specificity.
• Western blotting – Proteins• Northern blotting – RNA• Southern blotting - DNA
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Western blotting (Immunoblotting)
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Western Blot for detection of HIV antibody
HIV-1 Western Blot
• Lane1: Positive Control• Lane 2: Negative Control• Sample A: Negative• Sample B: Indeterminate• Sample C: Positive
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Immunoelectronmicroscopic tests
Immunoelectronmicroscopy :viral particles are mixed with specific antisera and observed under the electron microscope which are seen as clumps.
Use: study of some viruses such as hepatitis A and viruses causing diarrhoea
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Flow cytometry
• An instrument capable of analyzing single cells as they pass through an orifice at high velocity.
• Measures the properties of light scattering by the cells.
• Detection by the emission of light from flourescently labelled mAb bound to the surface of the cell
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• ELISA ?• CLIA ?• EIA ?• RIA ?• CFT ?
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What are the types of ELISA?
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Enumerate the antigen antibody reactions?
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Enumerate the antigen antibody reactions?
• Precipitation• Agglutination• Complement fixation test• Neutralisation test• Opsonisation • Radioimmunoassay (RIA)• Enzyme immunoassay (EIA)
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• Chemiluminescence immunoassay (CLIA)• Immunoelectroblot / western blot• Immuno chromatographic test• Immuno electron microscopic tests• Immunoflourescence
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THANK YOU