本試劑盒只能用于科學(xué)研究，不得用于醫(yī)學(xué)診斷
人（human）磷酸化tau217蛋白（p-tau217）
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)先包被磷酸化tau217蛋白（p-tau217）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(zhuǎn)化成藍(lán)色，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磷酸化tau217蛋白（p-tau217）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.濃度為0的s0號(hào)標(biāo)準(zhǔn)品即可視為陰性對(duì)照或者空白；按照說(shuō)明書操作時(shí)樣本已經(jīng)稀釋5倍，最終結(jié)果乘以5才是樣本實(shí)際濃度。
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*6管
0.3ml*6管
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（s0-s5）濃度依次為：0、125、250、500、1000、2000 pg/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.樣本孔先加待測(cè)樣本10μl，再加樣本稀釋液40μl；空白孔不加
4.除空白孔外，標(biāo)準(zhǔn)品孔和樣本孔中每孔加入辣根過氧化物酶（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：最-低檢測(cè)濃度小于10 pg/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.
humantau(p-tau217)elisa kit instruction
intended use
thisp-tau217elisa kit is intended labohumanory 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 concenthumanion oftauin the sample, thisp-tau217elisa kit includes a set of calibhumanion standards. the calibhumanion standards are assayed at the same time as the samples and allow the opehumanor to produce a standard curve of optical density versusp-tau217concenthumanion. the concenthumanion ofp-tau217in the samples is then determined by comparing the o.d. of the samples to the standard curve.
samplecollection and storages
serum- use a serum sepahumanor tube and allow samples to clot for 30 minutes before centrifugation for10 minutes at approximately3000×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 for30minutes at3000×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.donotsubstitutereagentsfromone kitto another.standard, conjugateandmicroplates arematchedfor optimal performance.useonlythereagentssuppliedbymanufacturer.
2. donotremovemicroplatefromthestorage baguntilneeded.unusedstripsshouldbestored at2-8°cintheir pouchwiththe desiccantprovided.
3.mix all reagents before using.
remove allkitreagentsfromrefrigehumanor and allowthemtoreachroomtempehumanure( 20-25°c)
materials supplied
name
96determinations
48determinations
microelisa stripplate
12*8strips
12*4strips
standard
0.3ml*6tubes
0.3ml*6tubes
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 (s0→s5) concenthumanion was followedby:0,125,250,500,1000,2000 pg/ml
reagent prepahumanion
20×wash solution:dilute withdistilled or deionized water1:20.
assay procedure
1.prepare allreagentsbeforestartingassayprocedure.itisrecommendedthatallstandardsand samplesbe addedin duplicatetothemicroelisastripplate.
2.addstandard:set standard wells,testing sample wells.add standard 50μl to standard well.
3. addsample: add testing sample10μlthen add40μlof sample diluentto testing sample well; blank welldoesn’t add anyting.
4. add100μlofhrp-conjugate reagentto each well,cover with anadhesive stripandincubatefor60 minutesat37°c.
5.aspihumane each well and wash, repeating the processfourtimes for a total of fivewashes.wash by filling each well with washsolution(400μl) using a squirt bottle, manifolddispenseror autowasher. complete removal of liquid at each step is essential to goodperformance. after the last wash, remove any remaining washsolutionby aspihumaning 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 mixandincubate for 15 minutes at37°c.protect from light.
7.add 50μlstop 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.
8readtheopticaldensity(o.d.)at450nmusingamicrotiterplatereaderwithin15minutes.
calculation of results
1.this standard curve is used to determine the amount in an unknown sample. the standard curve is genehumaned by plotting the average o.d. (450 nm) obtained for each of the six standard concenthumanions on the vertical (y) axis versus the corresponding concenthumanion 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 concenthumanion.
4.any variation in opehumanor, pipetting and washing technique, incubation time or tempehumanure, and kit age can cause variation in result. each user should obtain their own standard curve.
5.the sensitivity by this assay is10 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!
關(guān)鍵詞：洗板機(jī) 水浴鍋 酶標(biāo)儀 恒溫箱