Serum ELISA IgG titer quantitation - How to determine IgG titer without known sample? (Feb/04/2007 )
I'm looking to quantitate mouse serum for salmonella specific IgG. I have designed an ELISA using salmonella lysate as the coating antigen, infected mouse serum as the primary antibody, and a polyclonal anti-mouse IgG-HRP secondary antibody. I'm trying to figure out how I can determine my titers without using known concentration of serum IgG specific for my antigen. I've been told to use the highest dilution of serum to give an OD above background, but I'm not sure what my background is.
My controls are:
1: Blocking buffer alone (no serum)
2: Pre-immune serum
3: BSA coated wells instead of salmonella lysate, with a positive control serum
4: Positive control serum from 3 week old infected mice
Can anyone help me determine how to quantitate my titers?
Thanks in advance.
I've seen that protocol. None of my data has an inversion point.
It looks more like this...
Do I need to do more dilutions? I can't fit more than 9 dilutions on a plate with controls. I only have 6 dilutions right now.
I have lots of samples, I have graphs like the above for all of them...I'm hoping there's a way to figure this out without doing more experiments.
If you wanted to know how to do this...I finally figured it out. You take the linear part of the graph (at least 3 points, hopefully 4) and use the equation of the line to get your unknown titer.
In order to straighten out your line, you may want to try playing with the HRP incubation time. Also, your amount of coating lysate.