NETs assayed by ELISA
The serum level of NETs was measured using the capture ELISA protocol as
described previously[16, 26]. Briefly, microtiter plates were first
coated with the monoclonal anti-MPO antibody (ab25989, Abcam, UK) and
incubated overnight at 4°C. After blocking with 3% skimmed milk, the
blood sample diluted at 1:50 was added and incubated for 90 minutes.
Then the secondary anti-dsDNA antibody (ab27156, Abcam, UK, 1:2000
dilution) was added into the wells as second antibody. After incubation
for 2 hours and washed 4 times, horseradish peroxidase-conjugated rabbit
anti-mouse IgG H&L antibody
(ab6728,
Abcam, UK) was put in. After incubation again, the substrate chromogenic
agent was added in turn. 10 minutes later, the reaction suspension
solution was mixed and the results was calculated with optical density
(OD) at the wavelength of 450nm on the ELIASA (PRA ELX800, Bio Tek,
Vermont, USA).