Folding Forum

Hi there,
N-terminal Pro-BNP is used successfully in humans and dogs for the early diagnosis of heart-related disease (v. simplicistic). In feline patients it is possible to use the parameter as well, but - due to the naturally v. low concentration (10 x lower than in dogs) and the extreme instability, samples need to arrive at the lab frozen, and some labs have started to use anti-protease-coated tubes for the sample collection. While these workarounds make using this parameter possible, the situation is still far from perfect.
I was wondering whether there is a way to make the feline Pro-BNP fold into a less instable form, which would in turn lower the circumstancial PIA of it's collection.
Many thanks in advance!

Hi Al_Bert - Interesting question! My guess is, probably not. If the assay is an immunoassay (e.g. an electrochemiluminescence immunoassay or even a typical ELISA then refolding of the protein into a more stable state might well compromise its reactivity with the antibody in the assay.

Also, the normal physiological configuration of the protein is usually either the global minimum free energy configuration or at least a deep local minimum in free energy. It would be difficult to refold the N-terminal Pro-BNP without making large changes to the temperature, pH, or ionic strength of your sample, or without adding massive amounts of urea or some other chaotropic agent. These changes would of course prompt the more common proteins to re-fold also.

Is it possible that some sample preparation steps beside addition of protease inhibitors would make the assay more effective? If more abundant proteins interfere with the assay you could consider a centrifugal ultrafiltration step to remove them. That might also remove the proteases. Another possibility is to use a larger sample to load more NT-Pro-BNP into the assay and thus get a higher response. Though clearly there are limits here due to the small size of a cat relative to a human or dog.