we wan to purify Anti-Camel IgG from Poly-clonal serum using Camel IgG couples sepharose.
The only problem that I see is that is the affinity is high you will have hard time to recover antibodies in a good shape since you'll need harsh conditions to elute them from the column acidic pH could be detrimental to your antibodies. In which polyclonal serum have you generated the Anti Camel IgG ? Horse ? Goat ? Rat ?
hi Prakash
Bioaffinity chromatography is a good option but as Jean-Pierre said, harsh elution conditions are frequently needed for high Ab recovery and in many case both ligand and Ab may be affected. That does not mean it is a bad method!! Highly specific antibodies are obtained in this way. If you get the anti-camel IgG from polyclonal serum from a specie like rabbit, mouse or others that show affinity for Protein A or G , you can use this alternative for purifying IgG with high quality. Other good option is Ion exchange chromatography.
Regards, Oscar
Dear@ Jean-Pierre Dangy thanks for idea, I am working on Goat serum.
My elute is hazy, it means antibody is precipitated? And found no activity in elution fraction. Suggest how to proceed.
Did you dilute a lot the serum before to purify it through the column ? you could try different buffers for the elution and goes with a stepwise elution using different pH buffers and see whether you can elute your specific antibodies . Try to swith your Glycine elution buffer to other using eventually 1M Nacl to increase ionic strenth.
Another important thing is to add Tris buffer pH9 in your elution tube to make sure that you neutralise immediately Abs as soon as they are collected in your elution tube.
https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma-Aldrich/General_Information/1/ge-affinity-chromatography.pdf
Yes, we are using Triss buffer as neutralising buffer.
Will try with increasing concentration of NaCl and update you.
Dear Jean...
Thanks for the information...
I am using GE sepharose Activated 4B beads coupled with camel IgG... I have followed same protocol for conjuation as described in GE manual... And purified the goat anti camel IgG as you have mentioned above(GE reference)... And resoved problem of preciptation.... But now I have some doubts on yield and resin stability...
I got only 1.5mg of specific antibody per ml of immunized serum which is less...
Plz suggest how it can be increased... And how many times we can use these camel IgG coupled resin?
Hi But 1.5mg of Ab / mL of serum is not a negligible amount !!! Please, note that this is only a specific Antibody. For checking the column performance and reaching the maximum yield, you have to do the recovery calculate. Anyway, if you observe that the peak corresponding to the specific Ab goes decreasing during different purification cycles means that your column is loosing in yield.
regards Oscar
The ultimate test to determine if your procedure is successful is to test the antibody to see if it has the binding property you were looking for. I agree that 1.5 mg/ml is a good concentration, but you did not mention how many ml you obtained. If you can dilute the antibody solution a great deal and still detect specific binding to your antigen of interest, then what you have obtained may serve your needs. You can prolong the useful life of the resin if, right after elution, you very quickly equilibate to the buffer you use for binding or for storage.
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