Samplecollection and storages Serum - Use a serum separator tube and allow samples to clot for 30 minutes before centrifugation for 10 minutes at approximately 3000×g. Remove serum and assay immediately or aliquot and store samples at -20℃ or -80℃.Avoid repeated freeze-thaw cycles Plasma- Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 30 minutes at 3000×g at 2-8℃ within 30 minutes of collection. Store samples at -20℃or -80℃. Avoid repeated freeze-thaw cycles. Cell culture supernates and other biological fluids - Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20℃or -80℃. Avoid repeated freeze-thaw cycles. Note: The samples should be centrifugated adequately and no hemolysis or granule was allowed. Materials required but not supplied 1. Standard microplate reader(450nm) 2. Precision pipettes and Disposable pipette tips. 3. 37 ℃ incubator Precautions 1. Donot substitutereagentsfromone kit to another.Standard, conjugateandmicroplates are matchedfor optimal performance. Useonly thereagentssuppliedby manufacturer. 2. Donot removemicroplatefrom the storage baguntilneeded. Unusedstripsshouldbe stored at2-8°Cin their pouchwith the desiccantprovided. 3. Mix all reagents before using. Remove allkit reagentsfrom refrigerator and allow them to reachroom temperature( 20-25°C) Materials supplied
Name
96determinations
48determinations
Microelisa stripplate
12*8strips
12*4strips
Standard
0.3ml
0.3ml
Sample diluent
6.0ml
3.0ml
HRP-Conjugate reagent
10.0ml
5.0ml
20X Wash solution
25ml
15ml
Chromogen Solution A
6.0ml
3.0ml
Chromogen Solution B
6.0ml
3.0ml
Stop Solution
6.0ml
3.0ml
Closure plate membrane
2
2
User manual
1
1
Sealed bags
1
1
Note: Standard concentration was followed by: 8、4、2、1、0.5、0 ng/ml. Reagent preparation 20×wash solution:Dilute with Distilled or deionized water1:20. 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 sample10μl Then add sample diluent 40μl to testing sample well; Blank welldoesn’t add anyting. 4. Add100μlofHRP-conjugate reagentto 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 is0.1 ng/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!
