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Antigen – Antibody reactionsPart II
Dr.M.MalathiFinal year PG
Time to recollect !!!
• Antigen?
• Antibody?
• Antigen antibody reactions?
Complement Fixation Test (CFT)
• What is a complement ?
• Antigen antibody complex – bound by complement – mediates bacterial clearance, viral neutralisation, lysis of RBC
Im the source of complement
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
Ingredients
• CFT has two steps and five reagents• Antigen• Antibody• Complement• Sheep erythrocytes• Amboceptor ( Rabbit antibody to sheep red
cells)
Amboceptor ( Rabbit antibody to sheep red cells)
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.
Other complement dependent tests:
• Immune adherence-T.pallidum, V.cholerae
• Immobilisation test – Treponema pallidum
• Cytolytic or cytocidal test- V.cholerae
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
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
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
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
• 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
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)
Direct immunofluorescence test
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
Indirect immunofluorescence test
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
RADIOIMMUNOASSAY(RIA)
Uses of RIA
• RIA have applications in biology and medicine-
1. Quantitation of hormones, drugs, tumour markers,
2. IgE and viral antigens
Disadvantages
• Radiation hazards: Uses radiolabelled reagent
• Radioactive waste disposal
ELISA
Micro-plate reader
96-well micro-plate
Positive result
ENZYME LINKED IMMUNOSORBENT ASSAY
Types of ELISA
– Direct ELISA
– Indirect ELISA
– Competitive ELISA
– Sandwich ELISA
– Capture ELISA
– Cylinder or cassette ELISA
Types of ELISA
Immunochromatographic Tests
• A colored band appears at the second window
• Control also can be recorded
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
CLIA
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
Western blotting (Immunoblotting)
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
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
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
• ELISA ?• CLIA ?• EIA ?• RIA ?• CFT ?
What are the types of ELISA?
Enumerate the antigen antibody reactions?
Enumerate the antigen antibody reactions?
• Precipitation• Agglutination• Complement fixation test• Neutralisation test• Opsonisation • Radioimmunoassay (RIA)• Enzyme immunoassay (EIA)
• Chemiluminescence immunoassay (CLIA)• Immunoelectroblot / western blot• Immuno chromatographic test• Immuno electron microscopic tests• Immunoflourescence
THANK YOU
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