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post-immunization ELISA standards question - (Jan/30/2007 )

Hello, I'm trying to set up a sandwich ELISA to detect antibodies in mice to the hapten NP (4-Hydroxy-3-nitrophenylacetyl). I immunized mice with NP-ficoll, and am plating my ELISA plates with NP-BSA as the capture antigen. My question is regarding the standards. I'm having trouble locating a source of mouse anti-NP antibody to use a standard in order to quantitate my experimental O.D.s... Can I use a substitute? Any suggestions on what I should do? Any help would be much appreciated, thanks!

I'm using the mouse IgG and IgM ELISA kits from Bethyl Laboratories, which include anti-mouse IgG (IgM) coating antibody, and a goat-anti-mouse secondary antibody.


Isn't your anti-NP antibody coming from the mice you are immunizing?


The anti-NP antibody from the mice I immunnize is what I want to measure. The problem is I don't know how to exactly quantitate it--I don't have any anti-NP antibody of known concentration to use to create my standard curve. I only have regular mouse serum to use for my standard curve, and I doubt that serum contains anti-NP antibodies (and even if it does, I don't know in what concentration).

So then, do I need to purchase anti-NP antibody for my standards? Or is there another way? Someone else mentioned that I may be able just to do a more qualitative analysis using anti-NP antibody titers? Is that true? And would I need to set up some sort of standards/controls for that?

Sorry for all the confusion, not very familiar with ELISAs.



If you take for granted that 1 HRP labeled antibody binds 1 anti-NP antibody, then you can deduce the amount of anti NP antibody that way, right?