Hi all,
I'm doing an ELISA using an anti-c-myc HRP as the secondary antibody. It recognises the amino acid sequence EQKLISEEDL on my primary antibody.
I am getting quite a high background from this assay, and I looked into the amino acid sequence of the antigen which I am coating the plates with, and my antigen contains the sequence HQFVHGEEDL.
In bold italics I have highlighted a similarity shared between my coating antigen and my primary antibody. Is there any chance the secondary antibody will therefore bind to the antigen? I am not sure how sensitive antibody binding is to certain epitopes.
Any input would be very useful. Many thanks.
Yes, it is possible. You should do a blank control, which is a well that contains only the antibodies and coating antigen, without your samples. This should represent the background readings. You can then deduct your sample readings with the blank control readings.
Yes that is what I do, and the background is very high(higher than my positives in many cases) which led me to this question.
Thank you for your response.
Simple solution is to label your antibody directly with hrp using simple commercial kit. This will reduce the background
Thank you for the suggestion Oded. Unfortunately I have over 100 different antibodies that need to be screened for antigen binding, and so it might not be appropriate to directly conjugate them all with HRP :(
If you are using secondary which is goat origin it can cause background, try using Secondary which is monoclonal or pre-absorbed.
Regarding the HRP there are kit for 10ug labeling
Lightning-Link® Horseradish Peroxidase
15min protocol
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