Experimental systems in Immunology Sadegh Babashah, PhD Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University

Experimental animal models Adoptive transfer system SCID mice Cell culture system Protein biochemistry Immunoscreening 2

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Production of SCID human mouse 4

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Production of B cell and T cell hybridomas by somatic-cell hybridization 6

PAGE apparatus 7

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Isoelectric focusing 9

Two-dimentional gel electrophoresis of 35 S-metionin labeled total cell protein from murine lymphocyte (red arrow=IEF, blue arrow=SDS-PAGE) 10 Two-dimentional gel electrophoresis

X-ray crystalography 11

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Antigen-Antibody Interactions - Properties - Applications of in laboratory tests 13

Bonds: 14 Properties of Antigen-Antibody Interactions

15 Affinity: Properties of Antigen-Antibody Interactions K 1 K -1 K a K d

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Avidity IgM: high avidity, IgG: low avidity Cross-reactivity ABO blood groups & normal flora Streptococcus & heart antigens 17 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 18

Precipitation reactions Definition Precipitation from soluble Ag by Ab Lattice formation Ag and Ab must be bivalent (at least) Precipitation curve Equivalence zone 19

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Precipitation reactions Precipitation in gel (immunodifusion reactions) Radial immunodifusion Double immunodifusion 22

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Precipitation reactions Immunoelectrophoresis 24

25 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 27

Agglutination Reactions Agglutination inhibition Pregnancy test kits Drug addiction detection Rubella Ab detection in premarital testing 28

Pregnancy test kit based on agglutination inhibition 29

Radioimmunoassay (RIA) History High sensitivity 30

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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) 32

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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 times higher than ELISA Needs luminometer 34

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 35

S = Secretor NS = Non-secretor E = Enzyme CS = Chromogenic substrate CP = Colored product 36

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 37

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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 40

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43 Ab conjugated magnetic beads: use of Magnet for isolation of Ag

Immunoelectron microscopy Identification of Ag by electron-dense labeled Ab 44

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