ELISA - how to make a protein lysate? - (Jul/30/2008 )

Hello,


I am looking to measure serum antibody titers using ELISA. I don't have a defined antigen that I am looking for so I would like to coat the ELISA wells with a protein lysate of adipocytes. Can anyone point me towards a protocol on how to generate a protein lysate and how to coat ELISA wells with it? Sorry I'm new to this so any help would be appreciated.

Thanks!!!

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coating with lysate may give a high background; if you have no distinct antigen, I hope that you have distinct and specific antibodies in your serum; I would try to use the antibodies as a coat;

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good point, thanks. unfortunately i dont have a specific antigen or antibody. im trying to determine if there is an autoantibody in the serum of patients with a particular disease. i would probably have to normalize the absorbance values to normal, healthy controls.

do you (or anyone) know how i can coat the wells with protein lysate. is there a general protocol? also, in terms of obtaining the protein lysate, would i just need to do a protein extraction from my cells?

thanks.

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How about this: Get frozen sections of normal adipocytes. Layer on your sera from diseased people. Control slides would be same section with layer from non diseased people. Titer the sera. Use anti human IgG with flurosecent tag and use a flu microscope to determine where the binding is taking place if any.

The more you have to dilute the diseased sera the higher the titer. There are many other steps...

This is similar to anti-nuclear ab testing for autoimmune disease...which is the category of immunology you seem to be working in.

Does this seem to be the right track??

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