Elisa for total human IgG from culture supernatant - (Apr/15/2008 )
I am a newbie to ELISA. I am trying to develop an ELISA to detect and quantitate total human IgG from cell culture. I am thinking of coating plates with monoclonal antihuman-IgG, and using polyclonal antihuman-IgG secondary antibody conjugated to HRP for detection. Please let me know if I am on the right track, and if I am not, please advise me for the correct procedure.
Thanks in advance.
p.s: Can you also guide me for the companies/brands for ELISA plates, monoclonal antihuman-IgG (Fab or whole molecule specific), polyclonal antihuman-IgG secondary antibody (gamma-chain specific) conjugated to HRP, HRP substrate, human-IgG (for controls), ELISA buffers
Do you need to make a kit and go thru the hassel? There are suppliers of hIgG quantative ELISA and other kits in the market. ICL has kit also, I believe Pierce.
But if you must make a kit:
1. Many sources of abs...Sigma, Meridian, Binding Site....etc (you can search Linscotts.com). You can download ELISA package inserts from the various sites to see the specificities of the abs they use.
2. Use Nunc or other high quality plates.
3. Purchase pre-made coating, blocking buffers (check KPL, Pierce, etc)
4. Note: The ELISA kits (and your kit) will be sensitive in the pg range...cultures will be producing in the ug to mg/ml range