The coated well immunoenzymatic assay for the quant
itative measurement of NGF utilizes a polyclonal
anti-NGF antibody and an NGF-HRP conjugate. The as
say sample and buffer are incubated together
with NGF-HRP conjugate in pre-coated plate for one
hour. After the incubation period, the wells are
decanted and washed five times. The wells are then
incubated with a substrate for HRP enzyme. The
product of the enzyme-substrate reaction forms a bl
ue colored complex. Finally, a stop solution is ad
ded
to stop the reaction, which will then turn the solu
tion yellow. The intensity of color is measured
spectrophotometrically at 450 nm in a microplate re
ader. The intensity of the color is inversely
proportional to the NGF concentration since NGF fro
m samples and NGF-HRP conjugate compete for the
anti-NGF antibody binding site. Since the number o
f sites is limited, as more sites are occupied by N
GF
from the sample, fewer sites are left to bind NGF-H
RP conjugate. Calibrators of known NGF
concentrations are run concurrently with the sample
s being assayed and a calibration curve is plotted
relating the intensity of the color (O.D.) to the c
oncentration of NGF. The NGF concentration is
interpolated from this calibration curve.