IMMUNOLOGY
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.
Purification of IgG (Protein A Chromatography) 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 Subcutaneous Intramuscular
Active Immunization Day 0 Bleed 3 ml (ear vein)to obtain pre–immune serum from a rabbit Immunize rabbit with 100g Brucella antigen Day 7 Bleed 10 ml (either jugular or cephalic vein) to obtain antiserum Day 10 Bleed 10 ml Day 28 Immunize with 100g of Brucella antigen (Booster) Day 38
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 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
Protein A
? 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) ?
CHARACTERISTICS Extinction coefficient: E280(1%) = 1.4 Stability range: pH 1.0-12.0 Isoelectric point: 4.85-5.10 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
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
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)
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)
Western Blot
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.
Antigen electrophoresis Western blot overview Antigen electrophoresis Transfer to membrane Staining Detection with 1° (control) and 2° Ab Comparison
Antigen electrophoresis Stain with Coomasie blue dye (control) transfered to nitrocellulose membrane for antibody detection
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
blocking with skim milk incubate with primary antibody wash unbound antibody incubate with secondary antibody develop with substrate
Analysis of Results
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
bleed bleed
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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