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.

Overview Immunization

Bleed

Purification of IgG (Protein A Chromatography)

Western Blot

Analysis of Results

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.

Injection site

SubcutaneousIntramuscular

Active ImmunizationDay 0

Bleed 3 ml (ear vein)to obtain pre–immune serum from a rabbitImmunize rabbit with 100g Brucella antigen

Day 7Bleed 10 ml (either jugular or cephalic vein) to obtain antiserum

Day 10Bleed 10 ml

Day 28Immunize with 100g of Brucella antigen (Booster)

Day 38Bleed 10 ml

Example of Response of antibody-formation to an antigen

bleed bleed

Preparation of blood

Clot blood at room temperature

Remove all unwanted blood components by centrifugation

Supernatant diluted with PBS

Store at 4oC

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.

Extinction coefficient:E280(1%) = 1.4

Stability range: pH 1.0-12.0 Isoelectric point: 4.85-5.10

CHARACTERISTICS

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)

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

Elution ProcedureEquilibrate 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

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.

Western blot overview

Antigen electrophoresis

Transfer to membrane

Staining Detection with 1° (control) and 2° Ab

Comparison

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

Expected results

Br

Br

Br

Br – Brucella

bleed bleed

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.