I am setting up an ELISA based assay for studying the affinity of IgG to Fcg receptors. I am able to measure binding of IgG if i coat with the receptor and incubate with immuno complexes of IgG in serial dilution.
To prove that this affinity is "true" I want to do it the other way around and immobilize the IC or IgG and let the receptors bind in. I have triesd to coat with serial dilutions of both IgG and the immune complexes but I can't get any binding of the receptor.
Does anyone have any ideas of how I can solve this?
Or any other smart way to set up the assay?
I will try with a different type of plate.
Yes it is a sandwich ELISA I am doing and yes my sendoary antibody is HRP. I have checked it and it works fine.
It is not really the specifficity I want to show I want to show that the affinity for the receptor is inedependant of the coating e.i. that no artefact has been introduced during coating which alters the relative affinity between different forms of IgGs to the receptors.
It wouldn't help to include IgM or IgE to show that.
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