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ELISA SETUP,negative control OD is higher than tests - (Sep/22/2012 )

Dear all,
I would be so thankful if you share your personal experience about such a problem if you have had faced ever!
I do run an ELISA set up,to detect Ab raised in mice serum after a vaccine model administration.The protein antigen which has been injected to mice,the same one,has been coated and serums were added to mesure specific IgG against.But the point is that the serums of mice which are non-immunized,show higher OD than the test ones.It's nothworthy to mention that other immunoassay have confirmed the immune induction ability of administrated vaccine,and more over,this test had been set up months ago and it worked well.But now,it doesn't work I have also checked these serums in another ELISA run which an irrelevant Ag had been coated!!! That just drove me crazy when I found the same result in an ELISA run utilizing an irrelevant antigen!
I'm just seeking for an advice to check out how can I solve this problem


Check that your mice don't have some sort of infection (virus, bacteria or eukaryotic), especially something like pinworm, which is very common and leads to a heightened immune response. Also check that they are not on some sort of feed that could cause a response like this - many of the medicated feeds lead to weird immunological responses.


Thnx Global moderator

The point is that I've check cellular immunity as well and control mice didn't responde in any of cell mediated immune responses including,Lymphocyte proliferation assay,Cytokine secretion by spleen cells after invitro recall and CTL activity...


Yeah - that's why I think the problem is with the mice as a whole - so something in the mouse facility, not your experiments individually.


So, if I have understood correctly, you are coating the vaccine antigen on the plate, and then capturing specific mouse anti vaccine antibodies on the immobilized vaccine antigen, and then detecting with a specific species (goat for example) anti-mouse enzyme conjugate. All mice will have an abundance of mouse IgG in their serum, and your vaccinated mice have an abundance of mouse IgG in their serum, a portion of which is specific for the vaccine.

So, the secondary antibody (anti-mouse conjugate) is binding to the contents of all wells somehow. This could be because the reagent has gone off and is binding nonspecifically to the contents of the wells, or alternatively the IgG within the samples is binding to the well contents and the binding is happening by some process independent of the vaccine antigen on the plate.

In assays to detect human IgGs in various species including human (my experience) the sample quality is often very important in getting a clean signal. Several freeze thaws and a few months at -20 will be enough to result in binding of non-specific sample IgG to the plate making it impossible to differentiate signal from noise.

Another possibility could relate to the blocking (and diluent) Has the blocking solution been stored for a while without preservative? If the blocker has gone off, then you might expect the IgG within the samples to bind to the incompletely blocked plastic surface.

So suggestions:

Test fresh blank matrix (mouse serum)
Test fresh blocker and different lot
Test fresh anti mouse detection reagent and different lot
Test diluent only instead of anti mouse detection reagent (to make sure there isn't something else in the assay affecting the chromogen)
Prepare fresh coating buffer in disposable plastic as IgG does have a habit of getting into coating solutions especially when reusable glass is used to prepare them

Please let us know how you get on

good luck

-Ben Lomond-

Dear bob1

I mean that control mice didn't raise any cellular immune response in cell mediated immunity.So,its hard to consider sth is wrong with mice in whole..But that could be possible though..


Ben lomond

I've checked

-Fresh blocker
-different chromogen lot
-different coating buffer
and will check your other commented options for sure.

but I have to mention that,
when I coated an unrelated antigen and ran ELISA,again my control group serums showed highest OD!!
when I ran ELISA without coating the well,but blocked it,the negative serum group showed highest OD again!
the in all cases,negative controls always showe higher ODs than test groups!!

would be so thankful of sharing your own experience regarding such a problem..


OK, just go through the list and identify the problem by a process of elimination. Your experiments demonstrate that the problem is independent of the coating antigen, coating buffer, and chromogen, so these can be taken off the list of reagents causing the excessive background.

-Ben Lomond-