Purificiation of Salmonella antibodies - (Oct/17/2008 )
I'm working on Salmonella infection in pigs.
For an upcoming experiment, I need a relatively high concentration of Salmonella antibodies.
Therefore, I collected about 1L of serum from pigs that were infected with Salmonella Typhimurium.
Now I would like to purify the Salmonella antibodies (and only these!) from this serum.
Does anyone know how to do this?
Very basic steps:
1. You have collected the whole blood and removed the cells
2. Filter out any debris...glass wool in the bottom of syringe and push the serum/plasma thru
3. Do an ammonium sulfate cut using maybe 50% SAS (check on this concentration?)
4. After the cut is made centrifuge to pellet the white ppt.
5. Discard supernatant
6. Resuspend pellet in buffer...maybe tris, hepes etc.
7. Dialyze out the ammonium sulfate.
8. You now have ab, albumin and transferrin in buffer (you may have to concentrate the product)
9. You will have to make a column with your specific protein attached to the gel matrix to now isolate the specific abs to your ag. (Do you have Salmonella ag?...this could be a very expensive protein).
10. Run the solution over the gel and then elute the specific abs by changing pH or ionic concentration. Note: The column is re-usable.
Take samples along the way to check concentration and purity
Step 6 - phosphate buffered saline works fine with antibodies.
Step 9 - just a thought - couldn't he/she just use those bacterias used for infection? I mean like - grow them in liquid culture, kill, disintegrate, extract all proteins and use this mix as antigen for affinity column?
I don't know if a protein mixture on the gel will work for isolating the specific abs...
We were not told what the application is for the purified product. If the titer is high enough non-specific abs may have no bearing in the final application. Thus, the affinity chromatography step may not be needed.