experimental systems in immunology sadegh babashah, phd department of molecular genetics, faculty of...

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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