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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
甘薯羽狀斑駁病毒(SPFMV)ELISA檢測(cè)試劑盒
使用說(shuō)明書(shū)
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)先包被甘薯羽狀斑駁病毒(SPFMV)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*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zui終的黃色。顏色的深淺和樣品中的甘薯羽狀斑駁病毒(SPFMV)呈正相關(guān)。用酶標(biāo)儀在450nm 波長(zhǎng)下測(cè)定吸光度(OD 值),判斷樣品是否含有甘薯羽狀斑駁病毒(SPFMV)。
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
操作注意事項(xiàng)
試劑盒組成
名稱(chēng) | 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) |
陰性對(duì)照 | 0.6mL | 0.6mL | 無(wú) |
陽(yáng)性對(duì)照 | 0.6mL | 0.6mL | 無(wú) |
* | 6mL | 3mL | 無(wú) |
檢測(cè)抗原-HRP | 10mL | 5mL | 無(wú) |
20×洗滌緩沖液 | 25mL | 15mL | 按說(shuō)明書(shū)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(shū) | 1份 | 1份 | 無(wú) |
自封袋 | 1個(gè) | 1個(gè) | 無(wú) |
試劑的準(zhǔn)備
20×洗滌緩沖液的稀釋?zhuān)赫麴s水按1:20稀釋?zhuān)?份的20×洗滌緩沖液加19份的蒸餾水。
洗板方法
操作步驟
結(jié)果判斷
1. 試驗(yàn)有效性:陽(yáng)性對(duì)照孔OD值平均值≥1.00;
陰性對(duì)照孔OD值平均值≤0.15。
2. 臨界值(Cut off)計(jì)算:臨界值=陰性對(duì)照孔平均值+0.15
3. 陰性判斷:樣品OD值<臨界值(Cut off),樣品為陰性
4. 陽(yáng)性判斷:樣品OD值>臨界值(Cut off),樣品為陽(yáng)性
試劑盒性能
1. 準(zhǔn)確性:陽(yáng)性對(duì)照孔OD值平均值≥1.00;陰性對(duì)照孔OD值平均值≤0.15,說(shuō)明試驗(yàn)結(jié)果有效。
2. 特異性:不與其它可溶性結(jié)構(gòu)類(lèi)似物交叉反應(yīng)。
3. 重復(fù)性:板內(nèi)、板間變異系數(shù)均小于15%。
4. 貯藏:2-8℃,避光防潮保存。
5. 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(shū)操作,實(shí)驗(yàn)者違反說(shuō)明書(shū)操作,后果由實(shí)驗(yàn)者承擔(dān)。
FOR RESEARCH USE ONLY.
NOT FOR USE IN DIAGNOSTIC PROCEDURES.
Sweet Potato Feathery Mottle Virus (SPFMV) ELISA Kit instruction
Intended use
This SPFMV 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 SPFMV in the sample, this SPFMV 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 SPFMV concentration. The concentration of SPFMV 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 approximay 3000×g. Remove serum and assay immediay 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 immediay or aliquot and store samples at -20℃or -80℃. Avoid repeated freeze-thaw cycles.
Note: The samples shoule be centrifugated dequay 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
Name | 96 determinations | 48 determinations |
Microelisa stripplate | 12*8strips | 12*4strips |
Negative control | 0.6ml | 0.6ml |
Positive control | 0.6ml | 0.6ml |
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 |
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. Separay add Positive control and Negative control 50μl to the Positive and Negative well, Add testing sample 10μl Then add sample diluent 40μl to testing sample well; Blank well doesn’t add anyting.
3. Add 100μl of HRP-conjugate reagent to each well, cover with an adhesive strip and incubate for 60 minutes at 37°C.
4. 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.
5. 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.
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.
Determine the result
1. Test validity: the average of Positive control well≥1.00; the average of Negative control well ≤0.15.
2. Calculate Critical (CUT OFF): Critical= the average of Negative control well + 0.15.
Negative Result: sample OD< Calculate Critical (CUT OFF) is Negative.
Positive Result: sample OD≥ Calculate Critical (CUT OFF) is Positive.
Storage and validity
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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