大鼠嗜酸粒細胞趨化因子（ECF）ELISA試劑盒●品牌：子科生物ZIKER,美國R&D,美國immonoway,美國sciencell,德國IBL
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大鼠嗜酸粒細胞趨化因子（ECF）ELISA試劑盒樣品收集、處理及保存方法：
1、 血清……操作過程中避免任何細胞刺激。使用不含熱原和內(nèi)毒素的試管。收集血液后，1000×g離心10分鐘將血紅細胞迅速小心地分離。
2、 血漿……EDTA、檸檬酸鹽、肝素血漿可用于檢測。1000×g離心30分鐘去除顆粒。
3、 細胞上清液……1000×g離心10分鐘去除顆粒和聚合物。
4、 組織勻漿……將組織加入適量理鹽水搗碎。1000×g離心10分鐘，取上清液。
5、 保存……如果樣品不立即使用，應(yīng)將其分成小部分-70℃保存，避免反復(fù)冷凍。盡可能的不要使用溶血或高血脂血。如果血清中大量顆粒，檢測前先離心或過濾。不要在37℃或更高的溫度加熱解凍。應(yīng)在室溫下解凍并確保樣品均勻地充分解凍。

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 10.0 pg/ml
6.Standard curve
Storage：  2-8℃.
validity： six months.

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