檢測原理
試劑盒采用雙抗體夾心法酶聯(lián)免疫吸附試驗（elisa）。往預(yù)先包被人神經(jīng)顆粒素（nrgn）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(jīng)過溫育并*洗滌。用底物tmb顯色，tmb在過氧化物酶的催化下轉(zhuǎn)化成藍色，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人神經(jīng)顆粒素（nrgn）呈正相關(guān)。用酶標儀在450nm 波長下測定吸光度（od 值），計算樣品濃度。
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胞刺激，收集血液后，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、檸檬酸鹽或肝素抗凝。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。
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檢測，請按一次用量分裝，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
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有結(jié)晶，這屬于正常現(xiàn)象，水浴加熱使結(jié)晶*溶解后再使用。
2.實驗中不用的板條應(yīng)立即放回自封袋中，密封（低溫干燥）保存。
3.預(yù)處理后的樣本無需稀釋，直接取10μl加樣即可。
4.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。
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
微孔酶標板
12孔×8條
12孔×4條
無
標準品
0.3ml
0.3ml
無
樣本稀釋液
6ml
3ml
無
檢測抗體-hrp
10ml
5ml
無
20×洗滌緩沖液
25ml
15ml
按說明書進行稀釋
底物a
6ml
3ml
無
底物b
6ml
3ml
無
終止液
6ml
3ml
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
注：標準品濃度依次為：5000、2500、1250、625、312.5、0 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(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.自動洗板機：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)置標準品孔和樣本孔，標準品孔各加不同濃度的標準品50μl；
3.待測樣本孔先加待測樣本10μl，再加樣本稀釋液40μl；
4.隨后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(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波長處測定各孔的od值。
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，對應(yīng)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
1.準確性：標準品線性回歸與預(yù)期濃度相關(guān)系數(shù)r值，大于等于0.9900。
2.靈敏度：最di檢測濃度小于10pg/ml。
3.檢測范圍：156.25 pg/ml-5000 pg/ml
4.特異性：不與其它可溶性結(jié)構(gòu)類似物交叉反應(yīng)。
5.重復(fù)性：板內(nèi)變異系數(shù)小于10%、板間變異系數(shù)小于15%。
6.貯藏：2-8℃，避光防潮保存。
7.有效期：6個月
免責聲明
1.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(chǎn)生的一切后果，由實驗者承擔，本公司概不負責。
2.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.
humanneurogranin(nrgn) elisa kit instruction
intended use
this nrgnelisa 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 nrgnin the sample, this nrgnelisa 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 nrgnconcentration. the concentration of nrgnin 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 shouldbe centrifugated adequately 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 are matchedfor optimal performance.useonly thereagentssuppliedby manufacturer.
2. donotremovemicroplatefromthe storage baguntilneeded.unusedstripsshouldbe stored at2-8°cin their pouchwiththe desiccantprovided.
3. mix all reagents before using.
remove allkitreagentsfromrefrigerator and allowthemto reachroomtemperature( 20-25°c)
materials supplied
name
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:
5000、2500、1250、625、312.5、0 pg/ml.
reagent preparation
20×wash solution:dilute with distilled or deionized water1:20.
assay procedure
1. prepare allreagentsbeforestartingassayprocedure.itisrecommendedthatallstandardsand samplesbe addedin duplicateto the microelisastripplate.
2. add standard: set standard wells, testing sample wells. add standard 50μl to standard well.
3. add sample: add testing sample10μl then add sample diluent 40μl to testing sample well; blank welldoesn’t add anyting.
4. add100μlofhrp-conjugate reagentto each well,cover with anadhesive stripandincubatefor60 minutesat37°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. protect from 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 notappear uniform,gently tap the plate to ensure thorough mixing.
8. readtheopticaldensity(o.d.)at450nmusinga microtiterplatereaderwithin15minutes.
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 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：酶標儀 恒溫箱 洗板機 水浴鍋