Intended use
This CD8 ELISA kit is intended Laboratory for Research use only ａnd is not for use in diagnostic or therapeutic procedures. The Stop Solution changes the color from blue to yellow ａnd the intensity of the color is measured at 450 nm using a spectrophotometer. In order to measure the concentration of CD8 in the sample, this CD8 ELISA Kit includes a set of calibration standards. The calibration standards are assayed at the same time as the samples ａnd allow the operator to produce a standard curve of Optical Density versus CD8 concentration. The concentration of CD8 in the samples is then determined by comparing the O.D. of the samples to the standard curve.
Sample collection 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 shoule be centrifugated dequately 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.  Do not substitute reagents from one kit to another. Standard, conjugate ａnd microplates are matched for optimal performance. Use only the reagents supplied by manufacturer.
2.  Do not remove microplate from the storage bag until needed. Unused strips should be stored at 2-8°C in their pouch with the desiccant provided.
3.  Mix all reagents before using.
Remove all kit reagents from refrigerator ａnd allow them to reach room temperature ( 20-25°C)
Materials supplied
Name 96 determinations 48 determinations
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:
24、12、6、3、1.5、0 ng/mL.
Reagent preparation
20×wash solution:Dilute with Distilled or deionized water 1:20.

加样：加测试样品10μL然后加样品稀释液40μl测试样品；空白不添加任何
4。加入HRP 100μl共轭试剂对每口井用胶条覆盖，放置60分钟在37°C.
5。像每个好洗，这个过程重复四次共五洗。通过填充每个与洗涤液洗（400μL）使用一个喷瓶，管机或自动洗涤机。在每个步骤中完全除去液体是良好性能的关键。*后一次洗涤后，抽吸或过滤去除任何剩余的洗涤液。把盘子倒置，用干净的纸巾吸干.
6。加色溶液50μL和色溶液B 50μl每。轻轻混匀，放置15分钟在37°C.避光
7。添加50个每井的解决方案。威尔斯的颜色应该由蓝色变为黄色。如果威尔斯中的颜色是绿色或颜色变化不
出现均匀，轻轻拍打板，以确保彻底混合。
8。读取光密度（OD）在450 nm的15分钟内，用酶标仪。
 

Assay procedure
1.  Prepare all reagents before starting assay procedure. It is recommended that all Standards ａnd Samples be added in duplicate to the Microelisa Stripplate.
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.  Add 100μl of HRP-conjugate reagent to each well, cover with an adhesive strip ａnd incubate for 60 minutes 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 0.1 ng/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!