ELISA set up,negative control OD is higher than test!

[Christ]

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
Regards

[Zagami]

Dear Crist
Q. The protein antigen which has been injected to mice, the same one, has been coated and serums were added to mesure specific IgG against.
A. same protein injected solution is used for coating microplate ? If yes, to verify if adjuvant molecule can be cause to high Abs, coating MTP with only adjuvant. If coating with purified protein, is possible no good saturation of free site.
Q. But the point is that the serums of mice which are non-immunized, show higher OD than the test ones.
A. Cross-reaction, heterophils antibodies (change origin species of the conjugate), no good saturation of the free site
Q. It's noteworthy 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.
A. to check again all ingredients and they stability, and check procedure of made reagents and of assay
Q. 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!
A. to see conjugate solution again

If you would like let me know more on your specific home-made kit