Coudl your antigen it be sticking to the container surfaces? See attached for useful tips?

Thank you, John! I'll check it

Hello,

What is your antigen? Is it a protein? Usually proteins dislike freezing/thawing cycles, even in plasma, but in your case, they mey be stable enough in plasma while not in urine. So if you're antigen is a protein, it's possibly denatured by long-storage conditions in a matrix which is not suitable for it i.e. the urine. What about the pH of your urine sample vs. the pI of your protein? What about the bacterial activity in this urine? Because bacterial flora could also have an enzymatic activity (proteolysis) in between the freezing/thawing cycles which could lead to the degradation of your antigen..Have you tried to use a cocktail of protease inhibitor or aprotinin?

Thank you, Cecile, for your answer!

The antigen is a protein (soluble endoglin). Testing a collection of urea samples (stored in -80 deg C) derived from cancer patients I found that concentration of the antigen was less than in fresh samples obtained from healthy donors. A little experiment showed that freezing-thawing procedure is the major cause of loss of the antigen.

Now I'm thinking about  improving of the protocol of urea sample collection.

- Yes, we know that the protein is very sensitive to pH, but it retains its antigenicity in pH as little as 3.6. So, I should try to add some buffering compounds like (HEPES or something like that) to urine.

- I don't think that bacterial flora cause antigen proteolysis because all samples were frozen immediately after obtaining. In our recent publication we showed that soluble endoglin purified from blood plasma consists of several peptides of variable molecular weights. It seems these fragments are natural, i.e. they don't form as a result of inadequate storage conditions. Tests show that protein inhibitors (Pepstatin + PMSF) don't have any impact on antigen. Maybe other inhibitors will...

- I can say nothing about antigen's pI because in blood plasma it is complexed by other proteins. Maybe I should consider this question more precisely.

Thank you, Cecile!

I would be very pleased if you share you thoughts about the problem.

Hi Ilya,

Thank you for your answer. It's indeed really interesting. I think adding buffering compounds to urine before testing the effect of thawing/freezing cycles is an easy and relevant thing to try first. Why not just PBS instead HEPES or directly your assay buffer?

Also, reading your description of endoglin, I'm wondering about the capabilty of your ELISA to recognize in an equivalent way all the peptides... I mean, would it be possible that endoglin from fresh urine sample displays different molecular forms than urine stored @-80°C? And those various molecular forms aren't recognized equivalently by the atibodies of ELISA so that you may face discrepancies in the measurement. For instance, I observed the same problematic from my work on Fel d 1, which is a very polymorphic allergen from cat: see my article enclosed.

Consequently, you could try to test the parallelism of your samples in your ELISA with serially diluted fresh and frozen urine samples vs. the standard curve to check if all antigens are recognized in the same way by the antibodies and so measured equivalently, ensuring a right determintation of the concentration. Also it's possible that the antigen molecular forms are different in plasma and urine so again not measured equivalently. This phenomenon is well-knwon for TSH hormone for example, displaying different molecular forms in pituitary extract and plasma, due to different glycosylation pattern in this case notably (see file enclosed).

Good luck again!

Best wishes

Thank you Cecile for your answer!

PBS has a very limited buffering capacity I believe.

Yes, you're right. Our recent publication is called "Different pairs of monoclonal antibodies detect variable amounts of soluble endoglin in human blood plasma". One of our ELISA system is capable to detect about 100 more antigen than widely used commercial kit.

Thank you for the publications and thoughts!