人Klotho蛋白（Klotho）試劑盒（ELISA）說明書僅供參考，詳情請咨詢我司的銷售人員。 

Human Klotho ELISA Kit

For the quantitative in vitro determination of Human Klotho concentrations in

 serum - plasma - celiac fluid - tissue homogenate - body fluid 

FOR LABORATORY RESEARCH USE ONLY. 

NOT FOR USE IN DIAGNOSTIC PROCEDURES. 

This package insert must be read in its entirety before using this product.

ELISA

ENZYME LINKED IMMUNOSORBENT ASSAY

INTENDED USE AND TEST PRINCIPLE

This Klotho 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 Klotho in the sample, this Klotho 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 Klotho concentration. The concentration of Klotho 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 2 hours at room temperature or overnight at 4℃ before centrifugation for 20 minutes at approximately 2000×g. Remove serum and assay immediately or aliquot and store samples at -20℃. Avoid repeated freeze-thaw cycles

Plasma - Collect plasma using heparin as an anticoagulant. Centrifuge samples for 30 minutes at 2000×g at 2-8℃ within 30 minutes of collection. Store samples at -20℃. Avoid repeated freeze-thaw cycles.

Cell culture supernates, tissue homogenate and other biological fluids - Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20℃. 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.  37 ℃ incubator

2.  Standard microplate reader capable of measuring absorbance at 450 nm

3.  Precision pipettes, disposable pipette tips and Absorbent paper

4.  Distilled or deionized water

REAGENTS PROVIDED 

All reagents provided are stored at 2-8°C. Refer to the expiration date on the label. 

Name96 determinations48 determinations

MICROTITER PLATE8*12strips8*6strips

STANDARD（6 vial）0.3ml/vial0.3ml/vial

SAMPLE DILUENT6.0ml3.0ml

ENZYME CONJUGATE10.0ml5.0ml

WASH SOLUTION25ml15ml

SUBSTRATE A6.0ml3.0ml

SUBSTRATE B6.0ml3.0ml

STOP SOLUTION6.0ml3.0ml

Closure plate membrane22

User manual11

Sealed bags11

Note: 

1.  Standard concentration was followed by: 16, 8, 4, 2, 1, 0 pg/mL.

2.  If samples generate values higher than the highest standard, please dilute the samples with Sample Diluent and repeat the assay.

PRECAUTIONS

1.Do not substitute reagents from one kit lot to another. Standard, conjugate and microtiter plates are matched for optimal performance. Use only the reagents supplied by manufacturer. 

2.Allow kit reagents and materials to reach room temperature (20-25°C) before use. Do not use water baths to thaw samples or reagents. 

3.Do not use kit components beyond their expiration date. 

4.Use only deionized or distilled water to dilute reagents. 

5.Do not remove microtiter plate from the storage bag until needed. Unused strips should be stored at 2-8°C in their pouch with the desiccant provided. 

6.Use fresh disposable pipette tips for each transfer to avoid contamination. 

7.Do not mix acid and sodium hypochlorite solutions. 

8.Serum and plasma should be handled as potentially hazardous and capable of transmitting disease. Disposable gloves must be worn during the assay procedure, since no known test method can offer complete assurance that products derived from Rat blood will not transmit infectious agents. Therefore, all blood derivatives should be considered potentially infectious and good laboratory practices should be followed. 

9.All samples should be disposed of in a manner that will inactivate viruses. 

10.Liquid Waste: Add sodium hypochlorite to a final concentration of 1.0%. The waste should be allowed to stand for a minimum of 30 minutes to inactivate the viruses before disposal. 

11.Substrate Solution is easily contaminated. If bluish prior to use, do not use. 

12.Substrate B contain 20% acetone, keep this reagent away from sources of heat or flame. 

13.Remove all kit reagents from refrigerator and allow them to reach room temperature ( 20-25°C).

REAGENT PREPARATION AND STORAGE

Wash Solution (1X) - Dilute 1 volume of Wash solution (20X) with 19 volumes of deionized or distilled water. Wash Solution is stable for 1 month at 2-8°C. 

ASSAY PROCEDURE

1.  Prepare all reagents before starting assay procedure. It is recommended that all Standards and Samples be added in duplicate to the Microtiter plate.

2.  Add 50μl of Standard or Sample to the appropriate wells. Blank well doesn’t add anyting.

3.  Add 100μl of Enzymeconjugate to standard wells and sample wells except the blank well, cover with an adhesive strip and incubate for 60 minutes at 37°C.

4.  Wash the Microtiter Plate 4 times.

Manual Washing - Remove incubation mixture by aspirating contents of the plate into a sink or proper waste container. Using a squirt bottle, fill each well completely with Wash Solution (1X), then aspirate contents of the plate into a sink or proper waste container. Repeat this procedure for a total of four times. After final wash, invert plate, and blot dry by hitting plate onto absorbent paper or paper towels until no moisture appears. Note: Hold the sides of the plate frame firmly when washing the plate to assure that all strips remain securely in frame. 

Automated Washing - Aspirate all wells, then wash plates four times using Wash Buffer (1X). Always adjust your washer to aspirate as much liquid as possible and set fill volume at 350μL/well/wash. After final wash, invert plate, and blot dry by hitting plate onto absorbent paper or paper towels until no moisture appears.

5.  Add Substrate A 50μl and Substrate B 50μl to each well. Gently mix and incubate for 15 minutes at 37°C. Protect from light.

6.  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.

7.  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 (X) axis versus the corresponding concentration on the horizontal (Y) 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 balnk well 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.Intra-assay CV(%) is less than 10% and Inter-assay CV(%) is less than 15%.

6.Assay range: 0.5 pg/mL – 16 pg/mL.

7.  Sensitivity: The minimum detectable dose of Human Klotho is typically less than 0.1 pg/mL.

8.  Cross-reactivity: This assay recognizes recombinant and natural Human Klotho. No significant cross-reactivity or interference was observed.

9.  Storage: 2-8℃ (Use frequently); six months (-20℃)。

10.  Standard curve

FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS! PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE BEGINNING!

人Klotho蛋白（Klotho）試劑盒（ELISA）使用說明書

?本試劑盒用于體外定量檢測血清、血漿、組織、細胞上清及相關液體樣本中人Klotho蛋白（Klotho）的含量。

?有效期：6個月

?保存條件：2-8℃

實驗原理

試劑盒采用雙抗體一步夾心法酶聯免疫吸附試驗（ELISA）。往預先包被人Klotho蛋白（Klotho）捕獲抗體的包被微孔中，依次加入標本、標準品、HRP標記的檢測抗體，經過溫育并徹底洗滌。用底物TMB顯色，TMB在過氧化物酶的催化下轉化成藍色，并在酸的作用下轉化成最終的黃色。顏色的深淺和樣品中的人Klotho蛋白（Klotho）呈正相關。用酶標儀在450nm 波長下測定吸光度（OD 值），計算樣品濃度。

樣本處理及要求

1.  血清：全血標本請于室溫放置2小時或4℃過夜后于1000g離心20分鐘，取上清即可檢測，或將標本放于-20℃或-80℃保存，但應避免反復凍融。

2.  血漿：可用EDTA或肝素作為抗凝劑，標本采集后30分鐘內于2 - 8°C 1000g離心20分鐘，或將標本放于-20℃或-80℃保存，但應避免反復凍融。

3.  細胞培養物上清或其它生物標本：1000g離心20分鐘，取上清即可檢測，或將標本放于-20℃或-80℃保存，但應避免反復凍融。

注：標本溶血會影響最后檢測結果，因此溶血標本不宜進行此項檢測。

需要而未提供的試劑和器材

1.酶標儀（450nm）

2.高精度加樣器及槍頭：0.5-10uL、2-20uL、20-200uL、200-1000uL

3.37℃恒溫箱

4.蒸餾水或去離子水

人Klotho蛋白（Klotho）試劑盒（ELISA）試劑盒組成

名稱96孔配置48孔配置備注

微孔酶標板8孔×12條8孔×6條無

標準品0.3mL*6管0.3mL*6管無

樣本稀釋液6mL3mL無

檢測抗體-HRP10mL5mL無

20×洗滌緩沖液25mL15mL按說明書進行稀釋

底物A6mL3mL無

底物B6mL3mL無

終止液6mL3mL無

封板膜2張2張無

說明書1份1份無

自封袋1個1個無

備注：

1.  標準品濃度依次為：16、8、4、2、1、0 pg/mL

2.  經過大量正常標本檢驗，標本的正常濃度值均在試劑盒提供的檢測范圍內，實驗過程中直接取50μL樣本上樣即可。當有部分樣本值超過最大標準品濃度時，可用樣本稀釋液將標本進行適當稀釋后再進行實驗。

注意事項

1.嚴格按照規定的時間和溫度進行溫育以保證準確結果。所有試劑都必須在使用前達到室溫20-25℃。使用后立即冷藏保存試劑。

2.洗板不正確可以導致不準確的結果。在加入底物前確保盡量吸干孔內液體。溫育過程中不要讓微孔干燥掉。

3.消除板底殘留的液體和手指印，否則影響OD值。

4.底物顯色液應呈無色或很淺的顏色，已經變藍的底物液不能使用。

5.避免試劑和標本的交叉污染以免造成錯誤結果。

6.在儲存和溫育時避免強光直接照射。

7.平衡至室溫后再打開密封袋以防水滴凝聚在冷板條上。

8.任何反應試劑不能接觸漂白溶劑或漂白溶劑所散發的強烈氣體。任何漂白成分都會破壞試劑盒中反應試劑的生物活性。

9.不能使用過期產品。

10.如果可能傳播疾病，所有的樣品都應管理好，按照規定的程序處理樣品和檢測裝置。

試劑準備

試劑盒從冷藏環境中取出應在室溫平衡后方可使用。

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

人Klotho蛋白（Klotho）試劑盒（ELISA）操作步驟

1.  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℃。

2.  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；

3.  樣本孔中加入待測樣本50μL；空白孔不加。

4.  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。

5.  棄去液體，吸水紙上拍干，每孔加滿洗滌液（350μL），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）。

6.  每孔加入底物A、B各50μL，37℃避光孵育15min。

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

實驗結果計算

以所測標準品的OD值為橫坐標，標準品的濃度值為縱坐標，在坐標紙上或用相關軟件繪制標準曲線，并得到直線回歸方程，將樣品的OD值代入方程，計算出樣品的濃度。

人Klotho蛋白（Klotho）試劑盒（ELISA）試劑盒性能

1.  檢測范圍：0.5 pg/mL – 16 pg/mL。

2.  靈敏度：最低檢測濃度小于0.1 pg/mL。

3.  特異性：不與其它可溶性結構類似物交叉反應。

4.  重復性：板內變異系數小于10% ，板間變異系數小于15% 。