Download - Immunodiagnosis
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Immuno-diagnosis
Dr. B.S. VishwanathProfessor, Department of Studies in BiochemistryUniversity of MysoreRegistrar, Karnataka State Open UniversityManasagangotri, Mysore.
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Design of a technique
Physical and chemical properties of a molecule.
• Size and shape.
• Ionic charge.
• Hydrophobic and hydrophilic properties.
• Interaction with an adsorbents.
• Behavior in an electrical field / magnetic field.
• Specific interactions.
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• Enzyme - Substrate / Substrate analogues / Cofactors
• Antigen - Antibody
• Hormone - Receptor
• DNA - Complementary strands
• DNA - mRNA
• Lectins - Carbohydrates
Specific reactions
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Based on Specific reactions
• Immuno assay: RIA, ELISA, Western blot.
• Replication: Southern blot
• transcription: Northern blot
• Complementation: PCR.
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Immunotechniques
• RIA - ELISA
• Direct, Sandwich and competitive ELISA
• Precipitation reaction - Immunodiffusion
• Western blot
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Immunodiagnosis
• ELISA - HIV
• ELISA - HbsAg
• Westernblot for HIV
• TORCH - IgM & IgG
• Competitive ELISA for Hormone analysis
• Agglutination test
• Immunodiffusion
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Structure of an antibody
Primary antibody
Secondary antibody
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Direct ELISA for HIV
Antigen coating
Blocking
Primary Ab
Washing
Secondary Ab
Washing
Substrate addition
Colour development
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Monoclonal antibody binding with antigen
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Sandwich ELISA for HBsAg
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Western blot
• ELISA is similar to Western blot but is more informative.
• HIV detection by ELISA and Western blot
• HIV - I and HIV - II
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Western blot
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Competitive ELISA for T3
Ab-AgEC Ag OD
10 0 1.00
10 2 0.80
10 4 0.60
10 6 0.40
10 10 0.10
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Antibody response to infections
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TORCH - panel (IgM & IgG)
• Toxoplasma
• Rubella
• Cytomegalo virus
• Herpes
• IgM - Acute or Recent infection
• IgG - Chronic infection
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Agglutination reaction
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Structure of an IgM antibody
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Agglutination with IgG antibody using latex beads
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Agglutination with antigen using latex beads
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Agglutination reaction:Concentration dependent
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Immunodiffusion - precipitin formation
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Diagnosis of autoimmune reactions
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A PCR cycle
In vitro enzyme amplification of DNA to million folds
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THANK YOU
Acknowledgements
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