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High sample readings in serum ELISA - Please help... (Jun/28/2008 )

Hi all,

I am doing sandwich ELISA for a serum protein. Secondary antibody is a HRP conjugate and TMB/H2O2 is the detection system. I have used serum dilutions from 1:2000 upto 1:10,000. The problem is that all the dilutions in all samples are giving the same readings inspite of the dilutions. The absorbance readings are higher (1-1.2) than the highest standard reading (1.0). As a result I am not able to calculate the protein concentrations.

The blank and the standard readings are perfectly fine. I have tried using upto 7 washes for each step. Any changes I do, the blank and the standard graph always comes out well, except the sample readings which are always high.

Can OPD substrate be used for serum samples?

Kindly give your suggestions.



Your dose response curve and blanks are fine; thus your conjugate dilution and washes are fine also. Your samples read higher than the curve. Make higher dilutions of your samples until they fall on the linear portion of the dose response curve. Your sample could be in the high mg/ml range and the range of your curve in the ng/ml range.

Other things to consider:

You may also have something in the sample which not only is bound to the ab on the plate but also binds to the ab portion of the conjugate. A heterophile positive sample(s)?

Do you have a true negative sample? You did not mention if you had a true negative and if that was also reading high.


Change a secondary antibody.
I met this problem before, after we used another secondary antibody (from another company), the problem solved.