Note: Standard concentration was followed by: 2400、1200、600、300、150、0 pg/mL. Reagent preparation 20×wash solution:Dilute with Distilled or deionized water 1:20. Assay procedure 1. Prepare allreagentsbeforestartingassayprocedure. ItisrecommendedthatallStandardsand Samplesbe addedin duplicateto the MicroelisaStripplate. 2. Add standard: Set Standard wells, testing sample wells. Add standard 50μl to standard well. 3. Add Sample: Add testing sample 10μl Then add sample diluent 40μl to testing sample well; Blank well doesn’t add anyting. 4. Add100μlofHRP-conjugate reagent to each well,cover with an adhesive stripandincubatefor60 minutes at37°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, manifolddispenseror autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Solution by aspirating ordecanting. 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 toyellow. 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. ReadtheOpticalDensity(O.D.)at450nmusinga microtiterplatereaderwithin15minutes. Calculation of results
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.
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.
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.
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.
The sensitivity by this assay is 10 pg/mL.
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!
