experimental systems in immunology sadegh babashah, phd department of molecular genetics, faculty of...
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Experimental systemsin Immunology
Sadegh Babashah, PhDDepartment of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University
• Experimental animal models
• Adoptive transfer system
• SCID mice
• Cell culture system
• Protein biochemistry
• Immunoscreening
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Production of SCID human mouse
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Production of B cell and T cell hybridomas by somatic-cell hybridization
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PAGE apparatus
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Isoelectric focusing9
Two-dimentional gel electrophoresis of 35S-metionin labeled total cell protein from murine lymphocyte (red arrow=IEF, blue arrow=SDS-PAGE) 10
Two-dimentional gel electrophoresis
X-ray crystalography11
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Antigen-Antibody Interactions
- Properties
- Applications of in laboratory tests
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• Bonds:
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Properties of Antigen-Antibody Interactions
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• Affinity:
Properties of Antigen-Antibody Interactions
K1 K-1 Ka Kd
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• Avidity IgM: high avidity, IgG: low avidity
• Cross-reactivity ABO blood groups & normal flora Streptococcus & heart antigens
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Properties of Antigen-Antibody Interactions
Applications of Ag-Ab interactions in laboratory tests
• Precipitation reactions• Agglutination reactions• Radio Immuno Assay• ELISA• Chemiluminescence• ELISPOT• Western Blot• Immunofluorescence• Immunoprecipitation• Immunoelectron Microscopy
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Precipitation reactions
• Definition Precipitation from soluble Ag by Ab
• Lattice formation Ag and Ab must be bivalent (at least)
• Precipitation curve Equivalence zone
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Precipitation reactions
• Precipitation in gel (immunodifusion reactions) Radial immunodifusion Double immunodifusion
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Precipitation reactions
• Immunoelectrophoresis
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• Immunoelectrophoresis
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Agglutination Reactions
Interaction of particulate Ag with Ab
• Hemagglutination: blood grouping
• Passive agglutination: Ag coated on RBC or synthetic beads (Latex particles)
• Bacterial agglutination: Salmonella typing
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Agglutination Reactions
Agglutination inhibition• Pregnancy test kits • Drug addiction detection• Rubella Ab detection in premarital testing
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Pregnancy test kit based on agglutination inhibition 29
Radioimmunoassay (RIA)
• History
• High sensitivity
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Enzyme-Linked Immunosorbent Assay (ELISA)
• Mechanism
• High sensitivity
• Safer & cheaper than RIA
• Types: Indirect: for detection of Ab (Ag is coated)
Sandwich: for detection of Ag (Ab is coated)
Competitive: for detection of Ag (Ag is coated)
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Chemiluminescence
• Definition: Luminescence produced by direct transformation of chemical energy into light
energy
Ab-HRP + Ag Ab-HRP-Ag + H2O2 + Luminol light
• Chemiluminescence uses light-generating substrate (ELISA uses
chromogenic substrate)
• Sensitivity is 10-200 times higher than ELISA
• Needs luminometer
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ELISPOT
• A modification of ELISA• Determines the number of cells producing Ab or Ag• Capture Ab or capture Ag is coated in well and
recognizes the Ag or Ab secreted from cell
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S = Secretor
NS = Non-secretor
E = Enzyme
CS = Chromogenic substrate
CP = Colored product
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Western blot
• Identification of a specific protein in a mixture of proteins
• Mechanism: Separation of proteins by electrophoresis
Transfer of protein to membrane
Identification of protein band by labeled antibody
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Immunofluorescence
• Fluorochromes: Absorb one wavelength and emit a different wavelength
Fluorescein: absorbs at 490 nm, emits at 517 nm
• Mechanism: Identification of Ag by fluorochrome conjugated Ab
• Use of fluorescence microscope or FACS
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Ab conjugated magnetic beads: use of Magnet for isolation of Ag
Immunoelectron microscopy
• Identification of Ag by electron-dense labeled Ab
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Thank you for listening, any comments?