1. IMMUNOLOGY

2. A rabbit has been immunized with Brucella
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.

3. Purification of IgG (Protein A Chromatography)
Overview
Immunization
Bleed
Purification of IgG (Protein A Chromatography)
Western Blot
Analysis of Results

4. 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.

5. Injection site
Subcutaneous
Intramuscular

6. Active Immunization Day 0
Bleed 3 ml (ear vein)to obtain pre–immune 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

7. Example of Response of antibody-formation to an antigen
bleed
bleed

8. Preparation of blood Clot blood at room temperature
Remove all unwanted blood components by centrifugation
Supernatant diluted with PBS
Store at 4oC
Antibodies are present in the serum fraction of blood. Serum should be separated from cells ASAP after the collection of blood; otherwise cells lyze and release contaminating proteins including proteolytic enzymes which will degrade the antibodies

9. Protein A

10. ? 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.
What other mammals IgG SpA binds to? Cow(moderate), guinea pig, hamster, horse(moderate), mouse(all subtypes but diff affinity), pig and sheep(weak), rat (some subtypes only and weakly) ?

11. CHARACTERISTICS Extinction coefficient: E280(1%) = 1.4
Stability range: pH
Isoelectric point:
Extinction coefficient : for calculation of concentration
Large range of stable point, able to eluted proteins over a large range without getting chemically unstable, and interfere in the elution process

12. 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)
Why we choose DEAE-cellulose?
Common, can quite rigid, able to withstand some pressure by the flow, and quite cheap, easily available on the market.
Borate buffer

13. 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
Note the diff between removing weakly-bound and stopping of reaction though using the same solution.
PBS phosphate buffered saline (phosphate buffer solution)

14. 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
How high the absorbance is to be considered too high, thus far too much of Protein might have come unbound
even the elution process starts. pH of glycine 2.0 too low, might denature protein (IgG)

15. Western Blot

16. 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.

17. Antigen electrophoresis
Western blot overview
Antigen electrophoresis
Transfer to membrane
Staining Detection with 1° (control) and 2° Ab
Comparison

18. Antigen electrophoresis
Stain with Coomasie
blue dye (control)
transfered to
nitrocellulose membrane
for antibody detection

19. 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

20. blocking with skim milk incubate with primary antibody
wash unbound antibody
incubate with secondary antibody
develop with substrate

21. Analysis of Results

22. Expected results Day 0 / 7 Day 10 Day 38 Br Br Br Br – Brucella
IgM to IgG
IgG increased
Day 0 / 7
Day 10
Day 38 Br Br Br
Br – Brucella

23. bleed
bleed

24. 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.

25. THANK YOU