Elisa using HEK culture medium - (Apr/16/2012 )
Thanks for your kind Suggestion
I dont have blank or negative control supernatant but i will try using this system now.
What about using medium with FBS and medium without FBS and compare results by Elisa??
You may not be able to grow your cells without FBS in your medium. I have run similar assays previously and reduced the level of FBS to 1%, which has successfully lowered the background level in the ELISA and allowed me to detect the response by the cells.
You should definately run "growth media only" as one of your samples to determine how much of your activity is due to the media that the cells grow in. Just take an aliquot out of your bottle at the same time as you collect your cell supernatant and treat it in the same way as you do for the rest of your samples (i.e. if you freeze your samples, then you should freeze your media too). It is likely that your high background is due to your FBS.
If your capture and detection reagents have not been selected to have demonstrated clack of cross reactivity to bovine IgG, then yes, you will get the non-specific results you describe.
consider using a capture reagent that has been preabsorbed against bovine IgG or one that is proven not to cross react with bovine immunoglobulins.
antibodies to consider might include southern biotech 2081-01 or jackson 109-005-088 but browse through their websites and there are many options.
The detection reagent isn't so important, as the serum is washed off before you apply that reagent. Southern biotech 9040-04 is an excellent Fc gamma specific mono that would be a good detector for an anti-Fab specific or H+L (heavy and light chain) bovine preabsorbed poly capture reagent.
Just an offside question...with cell culture can an IgG stripped serum be used as a supplement to grow cells?
Bovine IgG doesn't help cells grow, and Gibco, Thermo and others sell 'ultra low' IgG fetal bovine serum, which they claim is just as good. Looking at the specs, it still has a few micrograms per mL of bovine IgG in it, so for this application, a stripped serum might be preferable if the process can remove all the IgG. Protein A and G won't bind all subclasses, so it would have to be passed through some sort of anti-pan bovine IgG affinity matrix. Conditioning the cells to serum free would be simpler.