I've been trying to adapt the widely published MPO-DNA ELISA used to detect human NETs for dog plasma for quite a while without much success and was wondering if anyone had any advice?
I initially used the Roche cell death ELISA plates with the anti-histone antibody from that kit, but with an anti-MPO or anti-elastase as the detector. I got a lot of background, plus marked interference from hemolysis or icterus (which is a problem because I need to able to use this on plasma from sick patients).
I switched to using the same anti-histone clone but without the biotin conjugation, coated overnight onto NUNC ELISA plates. This works ok with the anti-DNA antibody from the Roche Cell death kit, so I can detect nucleosomes and the interference problem is greatly reduced. However, using anti-MPO, or an anti-elastase (sold as cross-reactive with dog, works on immunofluorescence) detected with an appropriate HRP-conjugated secondary and TMB I get a low signal for plasma (about 0.2AU) and even lower for positive controls from canine neutrophils stimulated with PMA (about 0.1AU). I have tried titrating antibodies over a wide range of concentrations and varying incubation times and wash steps. All the buffers are commercial and pH is correct.
Does anyone with experience of running this ELISA in other species have any advice? Thank you
Signaling elements associated with the extrusion of NETs are significantly enhanced to promote NETosis in RA compared with healthy controls. NETosis depended on the presence of ACPA in ACPA-positive RA serum. The quantitation of NETosis-derived products, such as cell-free nucleosomes in serum, may be a useful complementary tool to discriminate between healthy controls and RA cases.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques