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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 ��nd 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 ��nd extend a horizontal line to the standard curve. At the point of intersection, draw a vertical line to the X-axis ��nd read the corresponding concentration.

4.         Any variation in operator, pipetting ��nd washing technique, incubation time or temperature, ��nd kit age can cause variation in result. Each user should obtain their own standard curve.

5.         The sensitivity by this assay is 10 pg/mL.

6.         Standard curve

Storage�� 2-8��.

validity�� six months.

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