at 37°C.
5. Aspirate each well and wash, repeating the process four times for a total of five washes. Wash by filling each
well with Wash Solution (400μl) using a squirt bottle, manifold dispenser or autowasher. Complete removal of
liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Solution by
aspirating or decanting. Invert the plate and blot it against clean paper towels.
6. Add chromogen solution A 50μl and chromogen solution B 50μl to each well. Gently mix and incubate for 15
minutes at 37°C. Protect from light.
7. Add 50μl Stop Solution to each well. The color in the wells should change from blue to yellow. If the color in
the wells is green or the color change does not
appear uniform, gently tap the plate to ensure thorough mixing.
8. Read the Optical Density (O.D.) at 450 nm using a microtiter plate reader within 15 minutes.
Calculation of results
1. This standard curve is used to determine the amount in an unknown sample. The standard curve is generated
by plotting the average O.D. (450 nm) obtained for each of the six standard concentrations on the vertical (Y)
axis versus the corresponding concentration on the horizontal (X) axis.
2. First, calculate the mean O.D. value for each standard and sample. All O.D. values, are subtracted by the
mean value of the zero standard before result interpretation. Construct the standard curve using graph paper
or statistical software.
3. To determine the amount in each sample, first locate the O.D. value on the Y-axis and extend a horizontal
line to the standard curve. At the point of intersection, draw a vertical line to the X-axis and read the
corresponding concentration.
4. Any variation in operator, pipetting and washing technique, incubation time or temperature, and kit age can
cause variation in result. Each user should obtain their own standard curve.
5. The sensitivity by this assay is 1.0 pg/ml
6. Standard curve
Storage: 2-8℃.
validity: six months.
FOR RESEARCH USE ONLY;
NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS!
PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE BEGINNING!