Measurement of mMCP-1 and mMCP-7 by ELISA.
96-well plates were coated overnight at 4°C with anti-mouse MCPT-1
(eBioscience) or MCPT-7 (R&D systems). Plates were washed in PBS
1X-Tween® 0.05% and blocked 1 hour at RT with
blocking buffer 1X (eBioscience). Plates were incubated overnight at 4°C
with plasma samples or recombinant MCPT-1 (eBioscience) or MCPT-7 (R&D
systems) as standards. Following washing, plates were incubated 1 hour
at RT with biotinylated anti-mouse MCPT-1 (eBioscience) or MCPT-7 (R&D
systems), then 30 minutes at RT with HRP-conjugated avidin
(eBioscience). HRP was developed using TMB substrate (eBioscience), for
15 minutes at RT. The reaction was stopped with 1 N chloride acid and
O.D. at 450 nm was recorded. Concentrations were calculated with
Boltzmann sigmoidal equation using O.D. obtained from standards
(GraphPad Prism®), and after subtracting O.D. obtained
from non-specific points.