I am trying to measure IgG1 from purified mouse B cell culture supernatant by ELISA. I have an rat anti-mouse Ab which I am testing for its B cell activating abilities in general and in vitro Ig class switching in particular. When I add this Ab (over a wide range of dilutions) to B cell cultures with or without LPS or CD40 or LPS/IL4, the color development is very strong. I suspected that my Ab (purified via ascitis over protein G column) may have traces of mouse Ig (via blood). However, when adsorbed my Ab with anti-mouse IgG-agarose beads, the result was still the same. I am now guessing that my secondary Ab (goat anti-mouse IgG1-HRP from Southern Biotech) may be cross reacting with the the Rat IgG (from added Ab) in the culture supernatant and could be the culprit. Here is where I need your help. I have looked up Biocompare.com without luck to see if there are any vendors who have rat adsorbed anti-mouse IgG1-HRP. I shall appreciate if any of you could help me locating such vendor.
Thank you.
-vinayadass-
vinayadass on Mar 24 2010, 05:05 AM said:
I am trying to measure IgG1 from purified mouse B cell culture supernatant by ELISA. I have an rat anti-mouse Ab which I am testing for its B cell activating abilities in general and in vitro Ig class switching in particular. When I add this Ab (over a wide range of dilutions) to B cell cultures with or without LPS or CD40 or LPS/IL4, the color development is very strong. I suspected that my Ab (purified via ascitis over protein G column) may have traces of mouse Ig (via blood). However, when adsorbed my Ab with anti-mouse IgG-agarose beads, the result was still the same. I am now guessing that my secondary Ab (goat anti-mouse IgG1-HRP from Southern Biotech) may be cross reacting with the the Rat IgG (from added Ab) in the culture supernatant and could be the culprit. Here is where I need your help. I have looked up Biocompare.com without luck to see if there are any vendors who have rat adsorbed anti-mouse IgG1-HRP. I shall appreciate if any of you could help me locating such vendor.
Thank you.
Are u talking abt this??
http://products.invitrogen.com/ivgn/product/046120 -Pradeep Iyer-
Pradeep Iyer on Mar 23 2010, 08:24 PM said:
vinayadass on Mar 24 2010, 05:05 AM said:
I am trying to measure IgG1 from purified mouse B cell culture supernatant by ELISA. I have an rat anti-mouse Ab which I am testing for its B cell activating abilities in general and in vitro Ig class switching in particular. When I add this Ab (over a wide range of dilutions) to B cell cultures with or without LPS or CD40 or LPS/IL4, the color development is very strong. I suspected that my Ab (purified via ascitis over protein G column) may have traces of mouse Ig (via blood). However, when adsorbed my Ab with anti-mouse IgG-agarose beads, the result was still the same. I am now guessing that my secondary Ab (goat anti-mouse IgG1-HRP from Southern Biotech) may be cross reacting with the the Rat IgG (from added Ab) in the culture supernatant and could be the culprit. Here is where I need your help. I have looked up Biocompare.com without luck to see if there are any vendors who have rat adsorbed anti-mouse IgG1-HRP. I shall appreciate if any of you could help me locating such vendor.
Thank you.
Are u talking abt this??
http://products.invitrogen.com/ivgn/product/046120
........
The Ab apparently has been pre-adsorbed to remove mouse non-IgG1, swine, and human cross reactivity but doesn't say whether rat IgG cross reactivity has been taken care of. I do not think it will serve my purpose.
Thank you for the help.
-vinayadass-
