SIB assays - how can we quantitate ab conc. befroe SIB assay (Jan/19/2010 )
We are devel :Do ping monoclonal antibodies. We would like to quantitate the supernatant before doing the SIB assay as some of the sups turn up negative due to low concentration, but I fear this isn't an option. Does anyone have any ideas??
dmartin on Jan 19 2010, 11:51 PM said:
You could establish an immunometric (eg sandwich ELISA) assay for mouse immunoglobulin:
Solid phase: goat anti-mouse light chains
Tracer: HRP goat anti-mouse Fc
Calibrate using standards made from a mouse monoclonal IgG.
You could use polyclonal goat anti-mouse as both solid phase and tracer. Crude but should work fine for your purpose.
Hope this helps
If possible why not adsorb your ag onto wells, block. Add the supernatnats, incubate, wash then add enzyme anti mouse conjugate? You will need a purified control/supernatant. This assay will tell you if your clones are producing specific ab and you can directly compare concentrations to your control. If your ag is small MW you will need to couple to protein to adsorb. You will be measuring specific ab here rather than total which may not have any reactive ab.