Hello,
I am trying to adapt the human Calprotectin assay to use in bovines. For background, Calpotectin is a protein released by neutrophils following gastrointestinal inflammation. A couple things I am currently troubleshooting:
1)The extraction buffer I should be using: I am currently considering a citrate urea buffer with neutrophil-specific protease inhibitors. I am seeking advice on any alternative buffers or buffer components I should consider and also how I should select which protease inhibitor to include.
2) After adding the buffer to my fecal samples, I will centrifuge samples to collect supernatant: How do I determine the centrifuge settings that are appropriate for this type of assay?
Thank you in advance for any insight! Any insight is greatly appreciated.
My answer to question 2 is that you want to spin long enough and fast enough to pellet all the solid material, leaving a clear supernatant. If you are using a microcentrifuge and 1.5-ml eppendorf tubes, for example, I would suggest spinning for 10 minutes at the highest speed. The highest speed of some microcentrifuges is in the14,000 to 16,000 g range. This should be more than sufficient, since the protocol I read suggested, but did not require, centrifugation. It just said to set the sample on the bench for a few minutes to let the solids settle, then use the supernatant (see section 7 in the link below).
https://calpro.no/wp-content/uploads/2015/09/PI_CalprotectinElisa-CAL0100.pdf
The problem is that the calprotectin assays seem to be fairly species specific. We have tested the human calprotectin assays against pig and dog samples, but we did not see any reaction. I think it is a fair chance that the antibodies will not crossreact with bovine calprotectin. To increase the chances I would probably try either avian antibodies as it will increase the chance of crossreactivity or assays that do not depend on multiple reaction sites as in turbidmetric/nephelometric assays. Possibly a sandwich assay that only need two binding sites.
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