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資料下載

人全段甲狀旁腺素（i-PTH）ELISA檢測(cè)試劑盒

點(diǎn)擊次數(shù)：857    發(fā)布時(shí)間：2017/8/3 18:15:13

· 上 傳 者： 上海極威生物科技有限公司

· 上傳時(shí)間：2017/8/3 18:15:13

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· 所屬分類：應(yīng)用方法

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 本試劑盒只能用于科學(xué)研究，不得用于醫(yī)學(xué)診斷

人全段甲狀旁腺素（i-PTH）ELISA檢測(cè)試劑盒

使用說(shuō)明書

檢測(cè)原理

試劑盒采用雙抗體一步夾心法酶聯(lián)免疫吸附試驗(yàn)（ELISA）。往預(yù)先包被人全段甲狀旁腺素（i-PTH）捕獲抗體的包被微孔中，依次加入標(biāo)本、標(biāo)準(zhǔn)品、HRP標(biāo)記的檢測(cè)抗體，經(jīng)過(guò)溫育并徹底洗滌。用底物TMB顯色，TMB在過(guò)氧化物酶的催化下轉(zhuǎn)化成藍(lán)色，并在酸的作用下轉(zhuǎn)化成*終的黃色。顏色的深淺和樣品中的人全段甲狀旁腺素（i-PTH）呈正相關(guān)。用酶標(biāo)儀在450nm 波長(zhǎng)下測(cè)定吸光度（OD值），計(jì)算樣品濃度。

樣品收集、處理及保存方法

1.  血清：使用不含熱原和內(nèi)毒素的試管，操作過(guò)程中避免任何細(xì)胞刺激，收集血液后，3000轉(zhuǎn)離心10分鐘將血清和紅細(xì)胞迅速小心地分離。

2.  血漿：EDTA、檸檬酸鹽或肝素抗凝。3000轉(zhuǎn)離心30分鐘取上清。

3.  細(xì)胞上清液：3000轉(zhuǎn)離心10分鐘去除顆粒和聚合物。

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

5.  保存：如果樣本收集后不及時(shí)檢測(cè)，請(qǐng)按一次用量分裝，凍存于-20℃，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。

自備物品

1. 酶標(biāo)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

 

操作注意事項(xiàng)

1. 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(huì)有結(jié)晶，這屬于正�，F(xiàn)象，水浴加熱使結(jié)晶完全溶解后再使用。

2. 實(shí)驗(yàn)中不用的板條應(yīng)立即放回自封袋中，密封（低溫干燥）保存。

 

3. 預(yù)處理后的樣本無(wú)需稀釋，直接取10μL加樣即可。

4. 嚴(yán)格按照說(shuō)明書中標(biāo)明的時(shí)間、加液量及順序進(jìn)行溫育操作。

5. 所有液體組分使用前充分搖勻。

試劑盒組成名稱

96孔配置

48孔配置

備注

微孔酶標(biāo)板

12孔×8條

12孔×4條

無(wú)

標(biāo)準(zhǔn)品

0.3mL

0.3mL

無(wú)

樣本稀釋液

6mL

3mL

無(wú)

檢測(cè)抗體-HRP

10mL

5mL

無(wú)

20×洗滌緩沖液

25mL

15mL

按說(shuō)明書進(jìn)行稀釋

底物A

6mL

3mL

無(wú)

底物B

6mL

3mL

無(wú)

終止液

6mL

3mL

無(wú)

封板膜

2張

2張

無(wú)

說(shuō)明書

1份

1份

無(wú)

自封袋

1個(gè)

1個(gè)

無(wú)

注：標(biāo)準(zhǔn)品濃度依次為：480、240、120、60、30、15pg/mL.

試劑的準(zhǔn)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(nèi)液體，每孔加滿洗滌液，靜置1min后甩盡孔內(nèi)液體，在吸水紙上拍干，如此洗板5次。

2. 自動(dòng)洗板機(jī)：每孔注入洗液350μL，浸泡1min，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，剩余板條用自封袋密封放回4℃。

2. 設(shè)置標(biāo)準(zhǔn)品孔和樣本孔，標(biāo)準(zhǔn)品孔各加不同濃度的標(biāo)準(zhǔn)品50μL；

3. 待測(cè)樣本孔先加待測(cè)樣本10μL，再加樣本稀釋液40μL；

4. 隨后標(biāo)準(zhǔn)品孔和樣本孔中每孔加入辣根過(guò)氧化物酶（HRP）標(biāo)記的檢測(cè)抗體100μL，用封板膜封住反應(yīng)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，靜置1min，甩去洗滌液，吸水紙上拍干，如此重復(fù)洗板5次（也可用洗板機(jī)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(nèi)，在450nm波長(zhǎng)處測(cè)定各孔的OD值。

結(jié)果判斷

繪制標(biāo)準(zhǔn)曲線：在Excel工作表中，以標(biāo)準(zhǔn)品濃度作橫坐標(biāo)，對(duì)應(yīng)OD值作縱坐標(biāo)，繪制出標(biāo)準(zhǔn)品線性回歸曲線，按曲線方程計(jì)算各樣本濃度值。

試劑盒性能

1. 準(zhǔn)確性：標(biāo)準(zhǔn)品線性回歸與預(yù)期濃度相關(guān)系數(shù)R值，大于等于0.9900。

2. 靈敏度：檢測(cè)濃度小于1.0pg/mL。

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

4. 重復(fù)性：板內(nèi)、板間變異系數(shù)均小于15%。

5. 貯藏：2-8℃，避光防潮保存。

6. 有效期：6個(gè)月

 

免責(zé)聲明

1. 試劑盒僅供研究使用，不得用于臨床實(shí)驗(yàn)或人體實(shí)驗(yàn)，否則所產(chǎn)生的一切后果，由實(shí)驗(yàn)者承擔(dān)，本公司概不負(fù)責(zé)。

2. 嚴(yán)格按照說(shuō)明書操作，實(shí)驗(yàn)者違反說(shuō)明書操作，后果由實(shí)驗(yàn)者承擔(dān)。

FOR RESEARCH USE ONLY.

NOT FOR USE IN DIAGNOSTIC PROCEDURES.

Humanintact Parathormone(i-PTH)ELISA Kit instruction

Intended use

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

Samplecollection 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 30minutes 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 suppliedName

96determinations

48determinations

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:

480、240、120、60、30、15pg/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 sample10μl Then add sample diluent40μl to testing sample well; Blank welldoesn’t add anyting.

4. Add 100μl of HRP-conjugate reagentto each well,cover with anadhesive stripand incubate for 60 minutes at 37°C.

5. Aspirate each well ａnd wash, repeating the process fourtimes for a total of five washes.Wash by filling each well with Wash Solution(400μl) using a squirt bottle, manifolddispenseror autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Solutionby aspirating ordecanting. 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. Protectfrom light.

7. Add 50μl Stop Solution to each well. The color in the wells should change from blue toyellow. 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 is1.0pg/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!

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