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1、本试剂盒只能用于科学研究,不得用于医学诊断人（Human ）抗角蛋白丝聚集素/集蛋白抗体（AFA ）ELISA检制试剂盒使用说明书检满原理试剂盒采用双杭一步夹心法酬/免授吸阳试除（ELISA）。flft 先包被抗丝集贸白植原包被4孔中，依次*人标本、HRP标记的检潇 杭原，势过温育并网眼洗涤。用E物TMB显色,TMB在过氧化物曲 的催化下特化应需包，并在酸的作用下转化成最终的负色。用的标 仅在450nm流长下溥定吸光度（OD值），与CUTOFF值相比较，U 而判定标本中折角蛋白丝聚集和丝集用白杭体（AFA）的存在与否。 样品收集、处理及保存方法L血清：使用不含热隙和内毒索的试管，操作过程中诩免
2、任何细脆 也和，收集血液后，3000转离心10介抻将血清和红绢的迅速小心地 分岗O2.血浆：EDTA、行校酸盐或肝素抗源。3000转离心30分钟取上消3 . 3胞上精液:3000转离心10分算去除颗粒和聚合物。4 .组织匀浆：将组班加入适量生理盐水捣碎。3000特离心10分钟 取上清。5 .保存：如果样本收集后不及M也图，请按一次用量分装，深存于 -2()七，逋免反且冻融，在室温下解沫并瑞保样品均匀地充分解漾。 自备物品1. 1标仪(450nm)2.容精度加样耨及枪头:0.5-10uL. 2-20uL、20-2()0uL. 2(X)-l()(X)uL3. 37七恒洱箱操作注意甲H1 .试剂盒保
3、存在2-8七，使用前室温平衙20分仲 M冰箱取出的浓 缩洗涤淞会有绍品，这回于正常现象，水浴加热使笫品完全落第片 再便用2 .实哈中不用的板条应立即放M自封袋中，密到(低温干燥)保存。3,预处理后的样本无筋冷择，直接取50uL加样即可。4 .严格技型说明书中标明的时间、加消量及IQR近行温育榇作。5 .丽百液体组分使用前充分摇匀。忱剂宜组成名称96孔配置48HKS备注M孔梅版收12孔x8条12 11x4 条无阳性对照0.5mL0.5mL无RlttMR0.5mL0.5 mL无m m6mL3mL无检需杭原-HRPlOinL5 mL无20x沈兴城冷瀛25mL15mL技说明书道卜称并Ktt A6inL
4、3 mL无物B6mL3mL无巽it液6mL3 mL无板底2弦2张无说明书1份1份无自封袋1个1 t无4剂的准备2()x洗涤线冲淞的柿程：二修水按1： 2()林铎，即1份的20x洗涤援冲淞加19份的蓑用水。洗板方法1 .手工洗板:甩用孔内液体，每孔加满洗涤液，静置hnin后甩尽 孔内液体，在吸樽上柏干，如此洗板5次。2 .自动洗板机：每孔注入洗液350UL,浸泡Imin,洗板5次。操作步。1 .北室沿平帽20min后的用苗装中取出所篇板条，利余板条用自时 来密封收回4七。2 .设置阴、阳性对照孔和样本孔，阳、阳性对照孔中加入阴性对照、 阳性对照各50uL;3 .待酒样本孔加侍网样本10山，样本修
5、辞液40pL;4 .脑后阴、阳性对照孔和样本孔中每孔加入源根抗氧化物朗（HRP） 标记的松河抗原100mL,用封板展封住反应孔,37七水浴福或恒洱 箱洱育60mino5 .弃去液体，吸水纸上花干，得孔ID满洗涤油，前冒Imin,用去洗 涤油，吸水纸上拍干，如此垂更洗板5次（也可用洗板机洗板）。6 .库孔加人版物A、B &50pL, 37tjg光需育15mino7 .每孔加入券止漉50uL, 15min内,在450nm波长处已定各孔的ODfg。绐果判黄1 .试盼有奴性：阳性对照孔OD值平均值N1.00;阴竹对阳孔OD值平均值S0.15。2 .有界值(Cutoff)计算:临界值=阴性对眼孔平均值+
6、0.153 .阴性判断：样品OD值临界值(CutolT),样品为阴性4 .阳性判断：样品OD值临界值(Cutoff),样品为阳性筑剂盒性能1 .由旗性：阳性对照孔OD值平均值21.00;阴性对修孔OD值平均睚0/5,说明故聆结果有效。2 .特异性：不与其匕可济性舒构类依物交叉反应。3 .用复性：板内、板向变异系数均小于15%04 . t： 2-8七，啦光防潮保存。5 .有效期：6个月 免责声明1 .试剂自n供研究使用，不得用于临床实野或大体实盼，否则所产 生的一切后果，由实验者承担，本公司拗不负责。2 .严格拉阳说明书操作，实聆者违反说明力掾作，后果由实盼者承 担。FOR RESEARCH U
7、SE ONLY.NOT FOR USE IN DIAGNOSTIC PROCEDURES.Human ?nti-filaggrin antibody(AFA)ELISA Kit instructionIntended useThis AFA ELISA kit is intended Laboratory for Research use only and is noi for use in diagnostic or therapeutic procedures.Tlie Slop Solution changes the color from blue to yellow and ihe
8、intensiiy of the color is measured at 450 mn using a spectrophotometer. In order(o measure the concentration of AFA in the sample, (his AFA ELISA Kil includes a set of calibration standards. Tlie calibration standards are assayed at the same time as the samples and allow the operator(o produce a sta
9、ndard curve of Optical Density versus AFA concentration. The concentration of AFA in the samples is then determined by comparing the O.D. of (he samples to the standard curve.Sample collection and storagesSerum - Use a serum separator tube and allow samples to clot for 30 minutes before centrifugati
10、on fbr 10 minutes at approximately 3(XX)xg. Remove serum and assay immediately or aliquot and store samples at IOC or -80C.Avoid repeated freeze-thaw cyclesPlasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 30 minutes at 3000xg at 2-8X? within 30 minutes of col
11、lection. Store samples al -20*Cor -80C. Avoid repealed freeze-thaw cycles.Cell culture supernates and other biological fluids - Remove pariiculates by centrifugalion and assay iinmediaiely or aliquot and store samples at -20*Cor 80C Avoid repealed freeze-thaw cycles.Note：The samples shoule be centri
12、fugated dequately and no hemolysis or granule was allowed.Materials required but not supplied1. Standard microplate reader450nm)2. Precision pipettes and Disposable pipetie tips.3. 37 C incubatorPrecautions1. Do not substitute reagents from one kit to another. Standaixl. conjugate and microplates ar
13、e matched for optimal pertonmnce. Use only the reagents supplied by manufacturer.2. Do not renwve microplate from the storage bag until needed. Unused strips should be stored at 2-8 in their pouch with the desiccant provided.3. Mix all reagents before using.Remove all kit reagents from refrigerator
14、and allow them to reach room temperature(20-25)Materials suppliedName96 determinations48 determinationsMicroelisa stripplateI2*8stripsI2*4stripsNegative control0.5ml0.5mlPositive control0.5ml0.5mlSample Dilution6.0ml3.0mlHRP-Conjugatc reagent10.0ml5.0ml20X Wash solution25mlI5mlChromogen Solution A6.
15、0ml3.0mlChromogen Solution B6.0ml3.0mlStop Solution6.0ml3.0mlClosure plate membrane22User manual11Sealed bags11Reagent preparation20xwash solution:Dilute with Distilled or deionized water 1:20.Assay procedure1. Prepare all reagents before starting assay procedure. It is recommended that all Standard
16、s and Samples be added in duplicate to the Microclisa Stripplatc.2. Separately add Positive control and Negative control 50m to the Positive and Negative well, add lOpl of Sample and 40Hl of Sample dilution to testing sample well.3. Add I OOpl of HRP-conjugate reagent (o each well, cover with an adh
17、esive strip and incubate fc)r 60 minutes at 37.4. Aspirate each well and wash, repealing the process four times for a total of five washes. Wash by filling each well with Wash Solution (400pl) using a squirt boule, manifold dispenser or autowasher. Complete removal of liquid al each step is essentia
18、l to good performance. After ihe Iasi wash, remove any remaining Wash Solution by aspirating or decarning. Invert the plate and blot it against clean paper towels.5. .Add chromogen solution A 50Hl and chromogen solution B 50pl to each well. Gently mix and incubate for 15 minutes al 37. Protect from
19、light.6. Add 50Hl Stop Solution to each well. The color in (he wells should change from blueyellow. If the color in the wells is gren or the color change does not appear uniibrm. gently (ap the plate to ensure thorough mixing.7. Read the Optical Density (O.D.) at 450 nin using a microtiier plate rea
20、der within 15 minutes.Determine the result1. Test validity: the average of Positive control welll.OO; the average ofNegative 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 Positive.Storage and validityStorage：2-8 C.validity： six months.FOR RESEARCH USE ONLY;NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS!PLEASE READ THROUGH ENTIRE PROCEDURE BEFOREBEGINNING!