HELP ! Need some Endotoxin-free OVA: where/how to get it ? - (Oct/11/2006 )
Hi,
I´m trying to establish vaccinations with BMDCs which are loaded with OVA-protein before (1mg/mL for 4hours, then LPS). The problem is that I´ve used so far the Sigma-Aldrich OVA, Grad V, which seems to be highly contaminated with endotoxins.( I realized this finally when I made an IL12-p40 ELISA with the OVA BMDCs: it showed that with OVA solely the BMDCS produced as much as IL12-p40 as the ones with 1ug/mL LPS ). 
Since I want to generate a measurable number of OVA-specific T-cells with BMDC vaccination ( with intracellular INFgamma staining) and because I thought that only immature BMDCs are really good at pinocytosis and cross-presentation whereas mature BMDCs lack this abilities , I want to ask, if somebody out here has a tip where to get really endotoxin-free OVA ?
Thanks for your help !
alex
By the way: Is somebody doing similar vaccinations with OVA-BMDCs ?If so, I would appreciate to know how strong the immune response against the SIINFEKL epitope is, you get !
Maybe you could use column form Pierce to remove the endotoxin from your OVA solution.
Detoxi-Gel Endotoxin Removal Gel
http://www.piercenet.com/Objects/View.cfm?...amp;ID=02050101
Detoxi-Gel Endotoxin Removal Gel
http://www.piercenet.com/Objects/View.cfm?...amp;ID=02050101
BUT:
Several reports showed that products based on polymyxin B have limitation in the recognition of different types of LPS and are unable to remove the biologic activity from Salmonella LPS.
Low endotoxin Ovalbumin
Commercially available stocks of OVA are heavily contaminated with LPS.
Ovalbumin is used in conjugo-immuno-determinations and drug and pharmaceutical processing. Also it is often used as a non-relevant carrier protein in ELISA antibody titer screens or as blocking buffer. “However, … , commercially available stocks of proteins, such as HEL, BSA, or OVA, are heavily contaminated with LPS, … .”
[J. Immunol., Jun 1999; 162: 6552 - 6561.]
Therefore not only in biopharma production but also in R&D the removal of endotoxin from ovalbumin solution is of great importance.
Profos offers an application note which summarized general information on ovalbumin. Answers are given to questions like:
Posttranslational Modifications
Physical and chemical properties
detailed protocol for removal of endotoxin from ovalbumin with EndoTrap
Profos offers also endotoxin removal service (e.g. from your ovalbumin sample).
www.profos.de
I´m trying to establish vaccinations with BMDCs which are loaded with OVA-protein before (1mg/mL for 4hours, then LPS). The problem is that I´ve used so far the Sigma-Aldrich OVA, Grad V, which seems to be highly contaminated with endotoxins.( I realized this finally when I made an IL12-p40 ELISA with the OVA BMDCs: it showed that with OVA solely the BMDCS produced as much as IL12-p40 as the ones with 1ug/mL LPS ).
Since I want to generate a measurable number of OVA-specific T-cells with BMDC vaccination ( with intracellular INFgamma staining) and because I thought that only immature BMDCs are really good at pinocytosis and cross-presentation whereas mature BMDCs lack this abilities , I want to ask, if somebody out here has a tip where to get really endotoxin-free OVA ?
Thanks for your help !
alex
By the way: Is somebody doing similar vaccinations with OVA-BMDCs ?If so, I would appreciate to know how strong the immune response against the SIINFEKL epitope is, you get !
In the meantime we have launched EndoGrade Ovalbumin with an endotoxin concentration of < 1 EU/mg (1 mg/ml). More information you find on www.endograde.de