I am planning to immunize the Balb/c mice using the recombinant protein for generation of antigen specific antibody used for ELISA assay. The his-tagged recombinant protein was purified using Ni-NTA resin and refolded by dialysis using decreasing urea concentration, followed by final dialysis using PBS. However, protein aggregation was observed after dialysis. Based on the literature, the addition of glycerol is good for protein aggregation prevention during protein refolding. My question is, may the presence of glycerol cause any harm in the mouse model when it is administered together with the antigen? If it doesn't, what is the recommended concentration of glycerol which is normally used in the antigen preparation? Appreciate if anyone could help me on this.
Glycerol is commonly if not routinely used additive as preservative and stabilizing agent in vaccines. Although its toxicity is disputed, for this reason patients receiving vaccines must be informed of its presence. The following link will give you and idea of other more toxic additives that used in vaccines:
http://realhealthtalk.com/Vaccines_Additives.html
...and the next link will take you to Vaccine journal's research article where glycerol is used as fromulation for creating stable vaccines
http://www.path.org/publications/files/TS_freeze-stable_vac_alum_salt.pdf
Development of a freeze-stable formulation for vaccines containing
aluminum salt adjuvants
WHO recommends addition of glycerol with or without antibacterial agents ie antibiotics
http://books.google.co.in/books?id=7fgy25Hr6Z0C&pg=PA20&lpg=PA20&dq=glycerol+as+additive+in+vaccines&source=bl&ots=6E0yx-s177&sig=Dn0GUFI_L2jbfxSadI7sZ9RbgpQ&hl=en&sa=X&ei=0g7IUqfLAYOWrAe16oCwCg&ved=0CG0Q6AEwBw#v=onepage&q=glycerol%20as%20additive%20in%20vaccines&f=false
The following link will give you an idea of vaccine excepients and media which are part of numerous vaccines.
There is no prescribed safe limit for glycerol percentage 20-50% should suffice for your protein stability.
Hope this piece of information suffices for your query.
Glycerol is commonly if not routinely used additive as preservative and stabilizing agent in vaccines. Although its toxicity is disputed, for this reason patients receiving vaccines must be informed of its presence. The following link will give you and idea of other more toxic additives that used in vaccines:
http://realhealthtalk.com/Vaccines_Additives.html
...and the next link will take you to Vaccine journal's research article where glycerol is used as fromulation for creating stable vaccines
http://www.path.org/publications/files/TS_freeze-stable_vac_alum_salt.pdf
Development of a freeze-stable formulation for vaccines containing
aluminum salt adjuvants
WHO recommends addition of glycerol with or without antibacterial agents ie antibiotics
http://books.google.co.in/books?id=7fgy25Hr6Z0C&pg=PA20&lpg=PA20&dq=glycerol+as+additive+in+vaccines&source=bl&ots=6E0yx-s177&sig=Dn0GUFI_L2jbfxSadI7sZ9RbgpQ&hl=en&sa=X&ei=0g7IUqfLAYOWrAe16oCwCg&ved=0CG0Q6AEwBw#v=onepage&q=glycerol%20as%20additive%20in%20vaccines&f=false
The following link will give you an idea of vaccine excepients and media which are part of numerous vaccines.
There is no prescribed safe limit for glycerol percentage 20-50% should suffice for your protein stability.
Hope this piece of information suffices for your query.
You can dialyse the current protein which is in glycerol in PBS before immunization, which could remove the glycerol, but the volume would increase. Further you concentrate the protein by centricon or lyophilization and can quantify the protein and immunize accordingly. Based on my experience I have always avoided Gycerol when immunization was to be given, but for long term storage of the antigen we would dialyse the proteins in 50% glycerol.
we used glycerol for immunization in mice and no adverse effects we seen. you want to see the raise of Ab. thus it can't have any effect i guess.
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