Hello everybody,

I immunized mice successfully with NP-KLH ( 4-Hydroxy-3-nitrophenylacetyl hapten is conjugated to KLH (Keyhole Limpet Hemocyanin) lysine through amide bonds). I let them sit for four weeks and took a blood sample each week. Now I want to do an ELISA for checking how many high and low affinity antibodies (i.e. NP-specific IgG1) there are in each sample comparing different genotypes. Technically the ELISA is ok, but I attached a protocol anyways.

The problem is that we are running out of standard for NP and the TA who did this is not here anymore and also didn't leave a protocol. With the old standard (where I don't know how it was made, how long the mouse was immunized etc) everything is fine, I see more binding on the NP27 coated site of the plate. But with simply using serum from previously immunized mice as a standard it does not work. I attached an example of this where I used the sera of the same mice and on the plate with St NP (= old standard) everything is fine, but with St Tea (new standard) it does not work.

When I look into the literature the only description I find is that you use serum from previously immunized mice, but that doesn't seem to work. So I think, maybe I need to immunize the multiple times or longer or dilute the standard in some way?

Could anyone please share with me a protocol for making a new standard for NP ELISAs? For other standards you can simply buy them, i.e. IgG standard, but with NP I didn't find anything similar.

Thank you so much for your help in advance!

Best,

Laura