Hello everyone,
I want to do ELISA to detect IgE in serum against an antigen I an working with and some one indicated that abundant proteins like IgG and HSA could either block or lead to unspecificity so there is need for depletion.
Does anyone has any experience in this IgE ELISA?
I suggest to use 10ug/ml antigen and 1:100 diluted sera for 96 well plates. You may read our paper for more detail...
http://journal.frontiersin.org/article/10.3389/fcimb.2017.00038/full
Since other antibody classes are generally much more abundant in plasma than IgE it is possible they may block IgE antigen binding if there is any overlap on the epitopes to which the immunoglobulins bind. Depleting with IgG protein A or other such protein may give you an indication. You will need to carefully take into account any changes in volume and concentration throughout the depletion procedures to determine if there is any blocking going on.
Some assays are working with a huge excess of immobilized antigen to avoid this unwanted competition effect.
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