Self versus Non self: Mechanisms of Self tolerance

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5 types of Ig IgG IgM IgA IgE IgD
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• Professor Nelson Fernandez
• Research
• Immunogenetics in mice and humans
• Cell-surface receptors
• Laboratory
• Lab 4.13 and 4.17

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The electron micrograph shows this large molecule
attached in spider form to its antigen, a
bacterial flagellum (b). The five subunits of IgM
are held by a J chain (a).
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IgA and its transport across epithelial surfaces.

• Two (sometimes more) IgA molecules are held
  together by a J chain. These attach to poly Ig
  receptors on the abluminal epithelial surface

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• are transported via endosomes and are released
  together with a piece of the receptor (secretory
  piece) into the lumen.

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How do we raise antibodies in the
laboratory?Experiment inject antigen or
immunogeninto a laboratory mouse.

• i.v- intravenous (parenteral)
• i.p- intraperitoneally
• i.s- intra-splenic (expose spleen and microinject
  antigen)
• o- oral immunisation.

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Use of antibodies in passive immunotherapy

• Replacement therapy in primary and secondary
  antibody deficiencies, AIDS, Leukaemia
• BMT (bone marrow transplantation)
• Adjuvant therapy in infection
• septic shock
• malaria
• TBC
• Immunomodulators in chronic inflammatory
  conditions and
• autoimmunity

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How to raise polyclonal antibodies for therapy

• Identify donors and bleed them aseptically
• screen for infections HIV, Hepatitis A, B and C
• isolate serum and pool serum
• conc. IgG fraction by ethanol precipitation
• solvent and detergent treat preparation
• Pasteurization
• Route of inoculation
• Intramuscular e intravenous

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Rietschel,
E.T. and Brade, H. Scientific American 267, 2,
54-61 (1992)
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Use of antibodies in health monitoring and home
diagnosis.
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Use of antibodies in antenatal prophylaxis for
the treatment of hemolytic disease of the newborn
(HDN)
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HDFN is a condition of pregnancy whereby the
foetal or newborns infant red cell lifespan is
adversely affected by maternal antibodies.
EXPLANATION In humans, in addition to the ABO
blood group there is the Rhesus (Rh) blood group.
Rh is a sugar moiety expressed on the
cell-surface of red blood cells. 85 of the
population are Rh 15 of the population are Rh-
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Hemolytic disease of the fetus and the newborn
(HDFN)
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Treatment protocols In the 60s a single dose
of anti-D antibody given post-delivery. Cause a
fall in the incidence of HDFN. In 1999 the Royal
College of Obstetricians and Gynecologists
recommended 500 IU of anti-D antibody to all Rh-
mothers at 28-week and 34-week of pregnancy
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Polyclonal nature of antibody
response ANTIBODIES CAN BE EITHER POLYCLONAL OR
MONOCLONAL
THE IN VIVO IMMUNE RESPONSE LEADING TO THE
GENERATION OF ANTIBODIES IS ALWAYS
POLYCLONAL IN NATURE!
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IDEALLY WE WANT TO MAKE AN in vitro MONOCLONAL
ANTIBODY DIRECTED AGAINST A SINGLE PATHOGEN OR
CANCER MARKER. MILSTEIN AND KHOLER (1974)
SOLVED THIS PROBLEM AT CAMBRIDGE (UK) BY USING
THE TECHNIQUE OF SOMATIC CELL HYBRIDISATION.
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Monoclonal antibodies

• Are homogenous antibodies produced by a single
  B-cell clone.
• They are usually made from hybridomas, which are
  prepared by fusing immunized mouse or rat spleen
  cells with non-secretor myeloma using fusogenic
  agents.
• The fusion mixture is plated out in HAT medium
  HAT medium contains hypoxanthine, aminopterin and
  thymidine.

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Hybridomas

• Are cells produced by the physical fusion of two
  different cells.
• Polyethylene glycol (PEG) and Sendai virus are
  often used to promote fusion between 2 cell
  types.
• A hybridoma cell and its progeny contain some
  chromosomes from each fusion partner, although
  some others are usually lost.
• Aminopterin blocks a metabolic pathway which can
  be bypassed if hypoxanthine and thymidine are
  present,
• but the myeloma cells lack this bypass and
  consequently die in HAT medium.
• Spleen cells also die naturally in culture after
  1-2 weeks, but fused cells survive because they
  have the immortality of the myeloma and metabolic
  bypass of the spleen cells.
• Some of the fused cells secrete antibody, and
  supernatants are tested in a specific assay e.g.,
  ELISA technique.
• Wells that produce the desired antibody are then
  cloned by limiting dilution techniques and
  preserved for further cell expansion.

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Monoclonal antibody production