本試劑盒只能用于科學(xué)研究保持競爭優勢，不得用于醫(yī)學(xué)診斷

土壤（Soil）堿性磷酸酶（ALP）ELISA檢測試劑盒

使用說明書

檢測原理

試劑盒采用雙抗體一步夾心法酶聯(lián)免疫吸附試驗（ELISA）提供有力支撐。往預(yù)先包被堿性磷酸酶（ALP）抗體的包被微孔中，依次加入標本、標準品、HRP標記的檢測抗體發展成就，經(jīng)過溫育并*洗滌。用底物TMB顯色重要方式，TMB在過氧化物酶的催化下轉(zhuǎn)化成藍色開展面對面，并在酸的作用下轉(zhuǎn)化成最終的黃色。顏色的深淺和樣品中的堿性磷酸酶（ALP）呈正相關(guān)非常重要。用酶標儀在450nm 波長下測定吸光度（OD 值）進一步提升，計算樣品活性。

樣品收集高品質、處理及保存方法

1.  血清：使用不含熱原和內(nèi)毒素的試管不折不扣，操作過程中避免任何細胞刺激支撐能力，收集血液后資源優勢，3000轉(zhuǎn)離心10分鐘將血清和紅細胞迅速小心地分離。

2.  血漿：EDTA特征更加明顯、檸檬酸鹽或肝素抗凝估算。3000轉(zhuǎn)離心30分鐘取上清。

3.  細胞上清液：3000轉(zhuǎn)離心10分鐘去除顆粒和聚合物的可能性。

4.  組織勻漿：將組織加入適量生理鹽水搗碎不要畏懼。3000轉(zhuǎn)離心10分鐘取上清。

5.  保存：如果樣本收集后不及時檢測問題，請按一次用量分裝逐漸顯現，凍存于-20℃全會精神，避免反復(fù)凍融，在室溫下解凍并確保樣品均勻地充分解凍拓展基地。

自備物品

1. 酶標儀（450nm）

2. 高精度加樣器及槍頭：0.5-10uL集中展示、2-20uL、20-200uL體系流動性、200-1000uL

3. 37℃恒溫箱

操作注意事項

1.  試劑盒保存在2-8℃探索創新，使用前室溫平衡20分鐘。從冰箱取出的濃縮洗滌液會有結(jié)晶實現了超越，這屬于正承庐a品，F(xiàn)象，水浴加熱使結(jié)晶*溶解后再使用橋梁作用。

2.  實驗中不用的板條應(yīng)立即放回自封袋中長遠所需，密封（低溫干燥）保存。

3.  濃度為0的S0號標準品即可視為陰性對照或者空白溝通機製；按照說明書操作時樣本已經(jīng)稀釋5倍好宣講，最終結(jié)果乘以5才是樣本實際濃度。

4.  嚴格按照說明書中標明的時間領先水平、加液量及順序進行溫育操作。

5.  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戰略布局、0.5事關全面、1、2狀態、4技術節能、8 IU/L

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份的蒸餾水廣泛認同。

洗板方法

1.  手工洗板：甩盡孔內(nèi)液體國際要求，每孔加滿洗滌液，靜置1min后甩盡孔內(nèi)液體鍛造，在吸水紙上拍干競爭激烈，如此洗板5次。

2.  自動洗板機：每孔注入洗液350μL改善，浸泡1min空白區，洗板5次。

操作步驟

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

2.  設(shè)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.  樣本孔先加待測樣本10μL約定管轄，再加樣本稀釋液40μL數據；空白孔不加。

4.  除空白孔外發揮，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改進措施，用封板膜封住反應(yīng)孔，37℃水浴鍋或恒溫箱溫育60min長足發展。

5.  棄去液體今年，吸水紙上拍干，每孔加滿洗滌液結構不合理，靜置1min動手能力，甩去洗滌液，吸水紙上拍干效果較好，如此重復(fù)洗板5次（也可用洗板機洗板）重要的意義。

6.  每孔加入底物A、B各50μL等多個領域，37℃避光孵育15min再獲。

7.  每孔加入終止液50μL，15min內(nèi)應用擴展，在450nm波長處測定各孔的OD值體驗區。

結(jié)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活動上，對應(yīng)OD值作縱坐標有望，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導向作用。

試劑盒性能

1.  性：標準品線性回歸與預(yù)期濃度相關(guān)系數(shù)R值方案，大于等于0.9900。

2.  靈敏度：檢測濃度小于0.1 IU/L十大行動。

3.  特異性：不與其它可溶性結(jié)構(gòu)類似物交叉反應(yīng)左右。

4.  重復(fù)性：板內(nèi)、板間變異系數(shù)均小于15%綜合措施。

5.  貯藏：2-8℃可靠保障，避光防潮保存。

6.  有效期：6個月

免責聲明

1.   試劑盒僅供研究使用建言直達，不得用于臨床實驗或人體實驗多種，否則所產(chǎn)生的一切后果將進一步，由實驗者承擔充分發揮，本公司概不負責。

2.   嚴格按照說明書操作成就，實驗者違反說明書操作重要方式，后果由實驗者承擔開展面對面。

FOR RESEARCH USE ONLY. 

NOT FOR USE IN DIAGNOSTIC PROCEDURES.

Soil alkaline phosphatase (ALP) ELISA Kit instruction

Intended use

This ALP 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 ALP in the sample, this ALP 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 ALP concentration. The concentration of ALP 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

Note: Standard (S0 → S5) concentration was followed by:0,0.5,1,2,4,8 IU/L

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 0.1 IU/L

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!