替補

• 產品貨號:RF-Z346-B
  檢測范圍:
• 產品規格:96T
  保存溫度:2-8℃
• 目錄價：￥2380
  

37℃恒溫箱

1. 操作注意事項

2.   試劑盒保存在2-8℃，使用前室溫平衡20分鐘。從冰箱取出的濃縮洗滌液會有結晶，這屬于正常現象，水浴加熱使結晶完全溶解后再使用。

3.   實驗中不用的板條應立即放回自封袋中，密封（低溫干燥）保存。

4.   濃度為0的S0號標準品即可視為陰性對照或者空白；按照說明書操作時樣本已經稀釋5倍，最終結果乘以5才是樣本實際濃度。

5.   嚴格按照說明書中標明的時間、加液量及順序進行溫育操作。

6.   所有液體組分使用前充分搖勻。

   試劑盒組成

注：標準品（S0-S5）濃度依次為：0、10、20、40、80、160 pg/mL

試劑的準備

 20×洗滌緩沖液的稀釋：蒸餾水按1：20稀釋，即1份的20×洗滌緩沖液加19份的蒸餾水。

洗板方法

1.   手工洗板：甩盡孔內液體，每孔加滿洗滌液，靜置1min后甩盡孔內液體，在吸水紙上拍干，如此洗板5次。

2.   自動洗板機：每孔注入洗液350μL，浸泡1min，洗板5次。

   操作步驟

3.   從室溫平衡20min后的鋁箔袋中取出所需板條，剩余板條用自封袋密封放回4℃。

4.   設置標準品孔和樣本孔，標準品孔各加不同濃度的標準品50μL；

5.   樣本孔先加待測樣本10μL，再加樣本稀釋液40μL；空白孔不加。

6.   除空白孔外，標準品孔和樣本孔中每孔加入辣根過氧化物酶（HRP）標記的檢測抗體100μL，用封板膜封住反應孔，37℃水浴鍋或恒溫箱溫育60min。

7.   棄去液體，吸水紙上拍干，每孔加滿洗滌液，靜置1min，甩去洗滌液，吸水紙上拍干，如此重復洗板5次（也可用洗板機洗板）。

8.   每孔加入底物A、B各50μL，37℃避光孵育15min。

9.   每孔加入終止液50μL，15min內，在450nm波長處測定各孔的OD值。

   結果判斷

    繪制標準曲線：在Excel工作表中，以標準品濃度作橫坐標，對應OD值作縱坐標，繪制出標準品線性回歸曲線，按曲線方程計算各樣本濃度值。

    

    

    

   試劑盒性能

10.  準確性：標準品線性回歸與預期濃度相關系數R值，大于等于0.9900。

11.  靈敏度：最低檢測濃度小于1.0 pg/mL。

12.  特異性：不與其它可溶性結構類似物交叉反應。

13.  重復性：板內、板間變異系數均小于15%。

14.  貯藏：2-8℃，避光防潮保存。

15.  有效期：6個月

    免責聲明

16.   試劑盒僅供研究使用，不得用于臨床實驗或人體實驗，否則所產生的一切后果，由實驗者承擔，本公司概不負責。

17.   嚴格按照說明書操作，實驗者違反說明書操作，后果由實驗者承擔。

     

    FOR RESEARCH USE ONLY. 

    NOT FOR USE IN DIAGNOSTIC PROCEDURES.

     

    Soil Reactive Oxygen Species (ROS) ELISA Kit instruction

     

    Intended use

    This ROS 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 ROS in the sample, this ROS 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 ROS concentration. The concentration of ROS in the samples is then determined by comparing the O.D. of the samples to the standard curve.

    Sample collection and storages

18. Can’t detect the samples which contain NaN3, because NaN3 inhibits HRP activity of the horseradish peroxidase.

    2.  Extract as soon as possible after Specimen collection, Extracted according to the relevant literature.

    Cell culture supernates and Soil exact fluids - Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20°C or -80°C. Avoid repeated freeze-thaw.

    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

Note: Standard (S0 → S5) concentration was followed by: 0,10,20,40,80,160 pg/mL

Reagent preparation

20×wash solution:Dilute with Distilled or deionized water 1:20.

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 1.0 pg/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!