I work with the T47D-KBluc cell line, which is stably transfected with a triplet ERE (estrogen-responsive elements)–promoter–luciferase reporter gene construct. Therefore the cell line is sensitive to estrogens, and by performing a luciferase assay, the estrogen level of a sample can be determined.
As standard substance I picked 17-beta-estradiol (E2), as a log 3 serial dilution with 1000pM initial concentration. The EC50 I found in the literature was around 5pM.
The result was, that the two highest E2 went totally out of order, the rest was acceptable for a preliminary test (compare figure).
So I'm wondering if this could be due to a kind of High-Dose-Hook-Effect?
Looking at your graph I noticed a few things. Firstly, dependent on your constitutive background response you may already have substantial induction at 0.5 pM. Where is your zero pM? Secondly, the highest two concentrations are physiologically speaking well beyond relevant and needn't be your concern - the system has been pushed to its limits (receptor- and cofactor activation-wise). I would suggest redoing a standard curve from about 30 pM down to 0.1 pM and include zero. Be careful when using this curves, as it is now you really only have one concentration that responds differently from the others (the 0.5 pM). Good luck.
The high dose hook effect usually refers to antibody-antigen interactions, as you know. It does not appear that you are seeing a 'classic' antibody-antigen hook effect in this study. Rather, it seems that you are observing a dose related E2 effect on your responding cells. At high and low E2 doses, the physiological responses of hormone sensitive cells are diminished as compared to the optimal E2 dose, so you see a bell shaped response curve. We examined this dose-response effect of E2, progesterone, and testosterone in macrophages and reported it in a series of papers authored by T.C. Chao in my lab. These were published in Amer. J. Reprod. Immunol. and Cell. Immunol. from 1996 to 2000. There are PDFs available at my RG profile page (Robert J Walter). Here is a link to one of these: https://www.researchgate.net/publication/227971155_Female_Sex_Hormones_Modulate_the_Function_of_LPStreated_Macrophages
Article Female Sex Hormones Modulate the Function of LPS‐treated Macrophages
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