IMMUNOLOGY

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IMMUNOLOGY
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• A rabbit has been immunized with Brucella. The
  rabbit will start producing IgM and then
  converting to IgG. Using Protein A column as a
  platform, describe how you would determine if the
  rabbit has started producing IgG. You would be
  using western blotting as well. Include in your
  discussion, your understanding of antibody
  classes and antibody class switching.

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Overview

• Immunization
• Bleed
• Purification of IgG (Protein A Chromatography)
• Western Blot
• Analysis of Results

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Injection Sites

• For brucella injection, no adjuvant is used. So
  injection can be done subcutaneously or it can be
  intramuscular injection.
• Subcutaneous injection is done just under skin on
  the neck region while intramuscular injection is
  administered on the thigh.

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Injection site
Subcutaneous
Intramuscular
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Active Immunization

• Day 0
• Bleed 3 ml (ear vein)to obtain preimmune serum
  from a rabbit
• Immunize rabbit with 100?g Brucella antigen
• Day 7
• Bleed 10 ml (either jugular or cephalic vein) to
  obtain antiserum
• Day 10
• Bleed 10 ml
• Day 28
• Immunize with 100?g of Brucella antigen (Booster)
• Day 38
• Bleed 10 ml

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Example of Response of antibody-formation to an
antigen
bleed
bleed
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Preparation of blood

• Clot blood at room temperature
• Remove all unwanted blood components by
  centrifugation
• Supernatant diluted with PBS
• Store at 4oC

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Protein A
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Introduction

• Staphylococcal Protein A (SpA)
• is an immunoglobulin (IgG) binding protein,
  which is found in the bacterial cell wall of
  Staphylococcus aureus. SpA is a group specific
  ligand that for detection or purification of IgG
  antibodies. SpA binds to rabbit IgG family
  specifically.

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CHARACTERISTICS

• Extinction coefficient
• E280(1) 1.4
• Stability range
• pH 1.0-12.0
• Isoelectric point
• 4.85-5.10

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Preparation of Protein A column

• Prepare beads
• Bind Protein A ligands to beads
• Wash beads
• Suspend beads in buffer
• Remove beads and add DMP (dimethylpimelimidate)

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Preparation of Protein A column

• Mix on roller for 30 minutes
• Remove beads and wash to remove weakly-bound
  ligands
• Incubate on roller for 2 hours to stop binding
  reaction
• Wash beads with PBS
• Store at 4oC

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Elution Procedure

• Equilibrate column at pH 7.0 and collect
  flow-through
• Add in the diluted serum
• Wash with PBS
• Add glycine
• Collect fractions of elute
• Neutralize fractions with Tris-HCl buffer
• Measure the absorbance of the elution fraction

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Western Blot
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Why western blot?

• Western blot analysis is to separate of one
  protein in a mixture of any number of proteins,
  based on its size. However Southern blot is to
  locate a particular DNA sequence and Northern
  blot is for the detection of RNA sequence.

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Western blot overview

• Antigen electrophoresis
• Transfer to membrane
• Staining Detection with 1
  (control) and 2 Ab
• Comparison

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Antigen electrophoresis
Stain with Coomasie blue dye (control)
transfered to nitrocellulose membrane for
antibody detection
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Transfer of protein to membrane

• The gel is positioned between two electrodes
  that generate an uniform electric field across
  the thickness of the gel
• A membrane is placed on the anode side of the gel
  
• Filter papers are used to maintain buffer contact
  with the gel
• Proteins migrate out of the gel and a replica of
  the gel is formed on the membrane
• The completeness of the transfer can be assessed
  through the use of prestained proteins as size
  markers

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blocking with skim milk incubate with primary
antibody wash unbound antibody incubate with
secondary antibody wash unbound
antibody develop with substrate
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Analysis of Results
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Expected results
IgM to IgG
IgG increased
Day 0 / 7
Day 10
Day 38
Br
Br
Br
Br Brucella
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bleed
bleed
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Conclusion

• The IgG level specific to Brucella increased
  gradually as we bleed the rabbit on Day 0, 7, 10
  and 38.
• After the booster, there is a drastic increase in
  IgG titer due to secondary response against
  Brucella.
• This goes to prove our main point that IgG has
  been produced. This IgG is specific to Brucella.

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THANK YOU