A "biochemist" teaching an Immunology lab. Still trying to learn how to use ELISA results without a KIT!
What is the end-point in the sigmoidal semi-log plot of the detection absorbance vs. log of ug protein in the well?
How is the ELISA data on the pure antigen used to get the amount of antigen in the unknown?
I really need help!
Hi Mary,
Without more information about your assay, the answers you might get will be as vague as the question. For example i would think that a biphasic curve indicates an interaction, and how this interaction relates to the antigen concentration you want to fit or determin.
I can be wrong, in any case here is a link that might help?
http://www.graphpad.com/guides/prism/6/curve-fitting/index.htm?reg_biphasic_dose_response.htm
Kind regards,
Tom
I will check the reference, thanks, but to describe the problem better:
In a plot of the absorbance (detection of the Ag by the Ab) vs. the log ( ug protein in the well of the 96-well plate) into which one has loaded serial dilutions of the unknown antigen, two discrete peaks show up. I can rationalize one as Antigen excess, but the second one?? This is reproducible, not noise, and frustrating if you are teaching(?) a course about a technique that you have never used routinely. PS we do not use a kit!
In order to solve the problem one needs to know the proces.
My Guessing or hinting is not going to help you. if you can share the assay protocol and the data i might be able to help, but i can't promise.
Don't you have any colleguas around that are good at statistics, I am sure the can help too.
I guess your antigen shows dimerization with a different affinity to the antibody compared to the monomers (similar to haemoglobin and its binding to oxygen).
could you provide us with a link to the curve ? And some information about your Ag?
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