elis

Parallelism is regularly used to______________amintain a balance in a sentence

parallelismpar.al.lel.ismAHD:[p瞨“…-l祃-箊”…m] D.J.[6p#r*lel7iz*m]K.K.[6p#r*lWl7!z*m]n.（名词）The quality or condition of being parallel; a parallel relationship.平行性：处于平行的性质或状态；平行关系Likeness, correspondence, or similarity in aspect, course, or tendency.类似，一致：在观点、路线或趋势上的一致、对应或相似Grammar The use of identical or equivalent syntactic constructions in corresponding clauses.【语法】 平行关系，平行结构的运用：在相应的句子中运用相同的或可替换的句法结构Philosophy The doctrine that to every mental change there corresponds a concomitant but causally unconnected physical alteration.【哲学】 并行论：一种认为每一个精神变化都有一个随之而来但并非与之直接联系的生理变化的学说repetitionrep.e.ti.tionAHD:[r祊”

parallelism是排比，平行的修辞手法。排比句(Parallelism sentence）是修辞手法之一，这种修辞法是把两个或两个以上的结构大体相同或相似，意思相关，语气一致的短语、句子排列成串，形成一个整体。并且句子或句子之间的联系由于语义上的一致和句法关系的相关得到加强。例如：I think he exaggerates the parallelism between the two cases.我认为他夸大了两件事的相似之处。Brackets shall be spaced at proper intervals to provide rigidity and parallelism.平台之间应该有适当的空间间隔，这样有利于提高坚硬性和平行性。Parallelism与antithesis是英语中广泛使用的修辞手段。Antithesis 意味相关、相对，是把相反的词、短语或从句用平行或对称的结构排列起来，以表达相反或相对的意思。使两层意思互相对照，达到加强语气的目的。Parallelism及antithesis与汉语的排比、对偶有相似之处，也有很大的不同。关于prallism有三种常见的译法：平行式，排比，对偶(亦称对句法)。

Parallelism 排比， 平行。这种修辞法是把两个或两个以上的结构大体相同或相似，意思相关，语气一致的短语.句子排列成串，形成一个整体。英语的常用修辞手法Simi1e明喻明喻是将具有共性的不同事物作对比，这种共性存在于人们的心里，而不是事物的自然属性。标志词常用：like, as, seem, as if, as though, similar to, such as等。metaphor隐喻，暗喻隐喻是简缩了的明喻，是将某一事物的名称用于另一事物，通过比较形成。metonymy借喻，转喻

ingredient是材料relish是开胃的东西

歌曲名:Slowly歌手:Melissa Manchester专辑:Hey RickyMatt Cardle - SlowlySaw your face, turned awayNever gave a second thought, never looked at you that waySearching for, someone elseI didn"t hear the fireworks, we were dancing with ourselvesAnd then you come close and knock me out, ohSlowly, slowly, changing me slowlyDriving with your heart, kissing in the darkThe way you hold me, hold me, turn with me slowlyThis is where it starts, kissing in the darkYou"re a thorn, in my soulThat cuts at first but now I"m feeling something wonderfulMore fool me, to let you driveYou can push me all you want but I"ll fall when I decideBut then you come close and knock me out, ohSlowly, slowly, changing me slowlyDriving with your heart, kissing in the darkThe way you hold me, hold me, turn with me slowlyThis is where it starts, kissing in the darkIt makes me shiver, that I could have missed youIt makes me shiver, that I could have missed youSlowly, slowly, changing me slowlyDriving with your heart, kissing in the darkThe way you hold me, hold me, turn with me slowlyThis is where it starts, kissing in the darkhttp://music.baidu.com/song/7489370

ElisaSchrey外文名：ElisaSchrey职业：演员代表作品：《塔楼》合作人物：NikiasChryssos

change list更改名单，更改列表

WhiteList”，这个词与“BlackList（黑名单）”是相对的。

英文原文：queen elizabeth英式音标：[kwiu02d0n] [u026au02c8lu026azu0259bu0259θ] 美式音标：[kwin] [u026au02c8lu026azu0259bu0259θ]

三者其实并没有本质的区别，都是作家的意思。文章除了你说的，此外还有：article，essay，passage都可以表示。

VangelisSeitanidisVangelisSeitanidis，导演编剧制作人，主要作品有：另一半、在你化妆之后、Aioniosfoititis等外文名：VangelisSeitanidis职业：导演，编剧，制作人代表作品：《Aioniosfoititis》合作人物：GiorgosKaramihos

歌曲名:3 + 3歌手:Vangelis专辑:Spiralan unreleased song from Asylum B-SideI often wonder why I sit and wait to dieWhat have I done to justify the sentence they gave?Too many hours spent in darkness questioning"How and why?"I can"t remember many moments in the lightThis retribution has defiled me to no endAnd execution without merit looms aheadTell me why!I was a boy who had to live his own wayI never fit into the model they had designedI chose a path less takenI stood with others who had often felt the sameWe got stares from passersbyOur dark attire seemed to frighten people awayAnd when three young boys diedThey pointed fingers and entangled us in a lie!No hope in sightI sit here terrified!(terrified, terrified)Can"t someone try to bring the truth to light?(bring it to light, bring it to light)Won"t anybody open up their eyes?(open your eyes, open your eyes)Before the three of us have lost our lives!∮Why was everybody so damn afraid?They blamed the devil for the heinousness of the crimeAnd to justify their own faithWe were labeled followers of Satan for a timeThe three of us were attacked and disgracedFor many hours we were questioned and someone liedOne of us couldn"t take itHis simple manner was manipulated in timeFear made him lieNow I sit here terrified!(terrified, terrified)Can"t someone try to bring the truth to light?(bring it to light, bring it to light)Won"t anybody open up their eyes?(open your eyes, open your eyes)Before the three of us have lost our lives!Now I am terrified!(terrified, terrified, terrified)Can"t someone try to bring the truth to light?(bring it to light, bring it to light)Won"t anybody open up their eyes?(open your eyes, open your eyes)Before the three of us have lose our lives!Tell me why (tell me why, tell me why)Can"t anybody see through their disguise?!(see through their disguise, see through their disguise)Believe we didn"t take their lives!(take their lives, take their lives)And now the three of us have lost our livesI often wonder why I sit and wait to dieWhat have I done to justify the sentence they gave?Too many hours spent in darkness questioning"How and why?"I can"t remember many moments in the lightThis retribution has defiled me to no endAnd execution without merit looms aheadTell me whyyyyyyy!http://music.baidu.com/song/9080623

VangelisKaravelisVangelisKaravelis，演员，主要作品《公开信》。外文名：VangelisKaravelis职业：演员代表作品：《公开信》合作人物：YiorgosStamboulopoulos

歌曲名:Voices歌手:Vangelis专辑:VoicesMacross Plus - Voices (english) LyricsThe first word in my dreamsI could clearly seePlanet Eden high beyond the skiesBeautiful and sadis the story I`ll tellof the winged travellers eagertwice was one daythe wind guided him where to golike an eagle high above the flewWaving from down belowhe flew out of sightinto the mystical darknessNeither a smile nor a cryI gave when he leftfeeling my spleen declineAnd hopingone day we"d fly overback to the places we once sharedWhere vessels glidein silky waves and of golddeep in the gulf such planet liesSurrounded by this universeof love and hateconfusion breaks through and dwells.Cast a spell, from the old magic book.Set a path.In the black magic box, something strange will happen,it will take you so far.So try...We can fly,we have wings,we can touch floatingdreams.Call me from so far through the wind in the light.Someone came from the dark over from the stars.Protecting my heart from crying.Taken back by surprise my traveller returned.What went wrong? Why did he change?http://music.baidu.com/song/54625016

柴可夫斯基擅长以旋律优美动听打动人心，瓦格纳的曲风壮阔雄伟，范吉利斯的音乐有此二人的这些特点，才这么说的。个人意见。

英语是音乐家范吉利斯的名字范吉利斯(Vangelis)本名Evangelos Odyssey Papathanassiou，1943年3月29日出生于希腊，几乎没有受过任何音乐教育的他，4岁便能演奏钢琴，6岁举行个人作品发表会， 可谓真正的音乐天才。范吉利斯年轻时期接受摇滚音乐洗礼，学生时期自组的乐团「Forminx」不仅是1960年代希腊最受欢迎的音乐团体，范吉利斯更是将近代流行音乐带进希腊的第一人。而为了寻找更宽广的音乐空间，25岁的范吉利斯前往巴黎发展，他自组的「爱神之子合唱团」(Aphrodite"s Child)红遍整个欧洲，也成功开发了前卫摇滚乐的新方向。1970年代的范吉利斯全心投入电子音乐创作，并转往伦敦为电影制作配乐，开启他辉煌的电影配乐时代，其中包括1981年荣获奥斯卡金像奖最佳配乐的「火战车」、1982年的「银翼杀手」、1983年的「南极物语」、1992年的「1492征服天堂」等重要作品，范吉利斯开创了电子音乐与电影配乐的崭新前景，奠定了所谓未来太空音乐的发展，可谓居功厥伟。 也许正是因为范吉利斯的无师自通，他的音乐具有独特的无法复制性，他以键盘音乐为基础，巧妙融合古典、流行、电子、new age等音乐元素，发展出绵密的管弦乐法以及崭新的电子音乐语法，而他身兼作曲、编曲、制作的能力，更完整地呈现他的音乐理念，从早期古典乐器到后来的新电子音乐，在范吉利斯手中诞生出崭新的表现方式，成为一种未来感十足的新音乐。范吉利斯的成就，不仅反应在全球的唱片销售数字以及无数的得奖纪录，国际天文联盟更决定将新发现的「6354号小行星」正式定名为「Vangelis」，藉以向这位音乐大师致敬。 2001年6月28日在雅典宙斯神殿举办的21世纪第一个最伟大的音乐会，演出曲目便是范吉利斯的合唱交响曲《Mythodea》，这场由希腊文化部长亲自宣布的音乐会，是由世界知名的两大黑人女高音洁西·诺曼、凯萨琳·芭托担纲演唱、伦敦大都会管弦乐团伴奏，另外还有120人编制的希腊国家歌剧合唱团、以及20位打击乐名家共同演出，而范吉利斯则亲自担任电子键盘的主奏。这项最引人注目的音乐会，不仅透过电视实况转播传送到全世界，美国太空总署(NASA)更将这部世纪巨作带上外层空间，成为2001年火星计划的代表音乐！

歌曲名:A Song歌手:Vangelis专辑:EarthVangelisI would like to write a songThat is so vibrant and so intimateThat the earth would adopt itAs if it had sprung like the streamAs if no one had written it but life itselfAnd my song would travel alongFrom bird to wing, to treeTo breeze to heart to breath to songBecause a song belongs to everyoneLike the springhttp://music.baidu.com/song/14113217

www.BT.comBT 里头的东西很全的，你可以试试啊！百度你试了吗？

歌曲名:Come To Me歌手:Vangelis专辑:VoicesVangelis - Come To MeCome to meWhile I"m sleepingSo their wagging tonguesMight not find you, my loveAll the whileThey lie downi"ll let a so sweetFor I"ll die for my loveSo stop this night so strongThat I will rememberAnd have it ever afterCome to meWhile I"m sleepingSo their wagging tonguesMight not find you, my loveCome to meEnd my dreamingSo their wagging tonguesMight not find you, my love?round the cornerBack of handsWhether or not we?d everThey"ll still be talkingWhether we?d ever, my lovehttp://music.baidu.com/song/8489218

歌曲名:Longing歌手:Vangelis专辑:Vangelis Blade Runner - Trilogy「LONGING」作词/作曲∶奥井雅美编曲：藤田淳平歌∶JAM Project幼い顷　见上げた空飞びたくて駆け登ったビルディング手を伸ばしてつかんだ梦それは最果てよりも高く戦いという代偿さえ　Ah　忘れ去る苍さよ钢の頬、触れる风　瞳をとじて想ったI wish the end of the warいつかこの身体に巡った血潮で风を感じ君と二人　そっと頬寄せ合い次の梦を未来(あす)を観る手にしたのは…剣に映すFighting spirit几亿もの惑星(ほし)の中で美しく光放つ地球(ガイア)谁もが皆　描いた梦叶う时を信じ戦う静寂という闇の中に　Ah　引き込まれないよう钢の腕、触れられない华奢な君の手を想ったI wish for love and peaceきっとこの身体に宿った魂　还る日まで君と二人　そっと手を取り合い次の梦を未来(あす)を行く笑颜がそう…怯え消えないTruth world戦いという现実へと　With　生まれ落ちた同志(とも)よ钢の胸、脉打った命の鼓动を想うI wish the end of the warいつかその身体に宿った魂　风を感じ苍き空の果てに争いのない世界梦を未来(あす)を観る爱しき人…剣で护るFighting spirit钢で护るTruth world【 おわり 】http://music.baidu.com/song/8530855

歌曲名:Voices歌手:Vangelis专辑:The CollectionMacross Plus - Voices (english) LyricsThe first word in my dreamsI could clearly seePlanet Eden high beyond the skiesBeautiful and sadis the story I`ll tellof the winged travellers eagertwice was one daythe wind guided him where to golike an eagle high above the flewWaving from down belowhe flew out of sightinto the mystical darknessNeither a smile nor a cryI gave when he leftfeeling my spleen declineAnd hopingone day we"d fly overback to the places we once sharedWhere vessels glidein silky waves and of golddeep in the gulf such planet liesSurrounded by this universeof love and hateconfusion breaks through and dwells.Cast a spell, from the old magic book.Set a path.In the black magic box, something strange will happen,it will take you so far.So try...We can fly,we have wings,we can touch floatingdreams.Call me from so far through the wind in the light.Someone came from the dark over from the stars.Protecting my heart from crying.Taken back by surprise my traveller returned.What went wrong? Why did he change?http://music.baidu.com/song/15025637

歌曲名:Message歌手:Vangelis专辑:DirectB.Reith - MessSomebody get me out this mess I"m inSee I"ve been tryin" to do the best I canBut I keep on stumblin" over and over againI ain"t got no more excuses for yaAll that I can say is sorryWon"t you please come rescue me as quickly as you canIt"s crazy how far we goJust to put a little gas in our egoThat"s what happens when your self esteem is lowWe"ll do anything to get people to notice us I knowIt"s tough but hey, imagine us throwin" our lives awayBecause we"re listenin" to the voice inside that chantsSays we"ll never be significant if we can"tFit in so we get into all kinds of trouble tryin" to be somebody we"re notSince in the beginning we"ve been givin" into the lie that says that we gotTo keep up with the Jones"s or get left behindSo we end up in debt tryin" to catch up it"s messed up"Cause ten years from now we can"t press rewindI want to invest in what stands the test of timeI"m so sick and tired of being down and outTryin" to do what"s right but wrong keeps comin outI keep on tryin to write but songs ain"t comin outPlus now I"m getting older and time is runnin out I know,Growth"s a process but I can"t see my progressAnd I"m startin" to feel that my labor"s in vainSo I hold on to the promise instead of my problemsOtherwise this stress is gonna drive me insaneYes there"s more to life than workin" 9 to 5Buyin" things that make you feel fuzzy insideI tried it and it worked at first for five minutesBut left me in the cold to hang like icicles oh!Now I"m froze like a popsicleFeel"s like my mission"s impossibleI"m stuck up in a maze full of obstaclesKeep on ending where I"m not supposed to goI just want to be okay with who I am,not have to run awayI"m so tired of trends I can"t keep pretendingPlaying these foolish gameshttp://music.baidu.com/song/8222916

　　曾经以《火战车》电影配乐一举成名的Vangelis ，多年来便是以个人独特的作曲风格扬名於世。然而Vangelis早在七O年代初期便已经陆续发表了数张非常实验的电子乐作品，那时电子乐才刚刚萌芽，因而他的许多作品都只有流传於较为小众的圈子，而且毫无疑问地，他亦是日后许多所谓Ambient、Trance、New Age等乐派的先趋人物及界定者。而Vangelis和当时同时期的大师级人物如Brian Eno最大的不同处，在於他善用电子乐器的各种音响性格，尤其在每种声响里，你都能感受到他所赋予每个乐器角色的定位，进而让听者能够产生非常意象式的情感，而这也是为何许多导演喜爱采用他的音乐做为影像配乐的原因之一。　　进入九O年代的Vangelis，由於俨然已为一代知名的大作曲家气势，因此许多导演及制片便开始纷纷请他出马为其电影做配乐。比较著名的作品包括有90年的 「The City」、92年的「1492」电影配乐、94年的「Blade Runner 银翼杀手」电影配乐、96年的「Oceanic」以及98年的「El Greco」。还包括为数不少的剧场、芭蕾作品，展现他多方涉猎且娴熟的文化涵养，堪称是当代配乐同行的异数。　　命运悲歌--顽强的德军官兵　　“如果我们为命运女神所抛弃，如果我们从此不能回到故乡，如果子弹结束了我们的生命，如果我们在劫难逃，那至少我们忠实的坦克,会给我们一个金属的坟墓。”　　——第三帝国《装甲兵之歌》　　1944，1月。东线。切尔卡瑟。自1942年夏天以来，南线一直是苏德战场上的焦点，双方最大最强的主力集团在这里展开着激烈的厮杀。1944年初，苏联军两个方面军对德军的卡涅夫突出部形成挟击之势。为掌握主动权，苏军不顾冬春泥泞地形，决心拔除这枚钢钉。　　1月28日，以苏联近卫第5坦克集团军为首的精锐部队完成了对突出部德军的合围，史称“切尔卡瑟钢铁口袋”。德军两个军近6万人被困，其中唯一的装甲师是著名的武装SS“维京师”。　　德国元帅曼施坦因决心集结兵力，在解围的同时重创苏军，不幸的是他高估了此时手中握有的实力。自2月10日至2月15日，十余万德军与更多的苏军殊死血战。包围圈内外的德军仅距离不到10千米，但是这最后几千米的路程，对外围业已精疲力尽的帝国军人而言，是无论如何也走不完的.曼施坦因无奈地电告被围部队：救援部队力量以耗尽，你部只能自行突围。风雪交加、连夜血战中的被围德军心情异常沉重。　　被围德军最高指挥官是施特默尔曼将军，不愧为典型的德意志军官，期间他整编部队，保持士气，顽强抵抗，拒绝投降。苏军派来使者要求德军投降，将军尽可能找来好酒好菜招待了使者，并约定战场上再见。此时将军下定决心，自行突出重围。　　2月16日，暴风雪夜。被困德军丢弃了所有辎重，含泪放弃了两千余名重伤员（在东线，无论苏德都没有收容敌方重伤员的习惯）。施特默尔曼将军布置好突围步骤后，平静地对部属说：“我将与后卫部队在一起，兄弟们，包围圈外见。”　　五万五千名德军以维京师为先头，企图悄悄突围，但很快就被苏军发现，熊熊烈火中，腥风血雨，尸横遍野。骄傲的维京尖刀捅开了缺口，被围德军在2月17日中午冲到格尼洛伊提基河边，友军就在彼岸——但是，没有桥，也没有船。在放弃了所有重装备后，德军自发组织起来，维持秩序，三万五千人在浅水区成功渡河。　　尽管后卫部队殊死抵抗，但强大的苏军仍然切断了缺口，没来得及渡河的还有两万多德军。他们在冰凉的荒野中，没有任何重武器，又累又饿。苏联将军的指示是：“我们已经给过德国人不止一次投降的机会了，既然他们不珍惜，那么，骑兵小伙子们，尽情地攻击吧！”在密集的炮火攻击后，骑兵冲上来猛砍猛杀，许多投降德军举起的双手也被劈掉。　　定格瞬间：战后，苏军打扫战场时，在战况最激烈的高地上发现了施特默尔曼将军的尸体，手里紧紧握着一枝步枪。他的制服血迹斑斑，但胸前的勋章却熠熠生辉；他的身旁，横七竖八地躺着维京师的后卫营——担任阻击任务的这支小部队，以尽数战死的代价，换来了大量同胞的生还.苏联人默然良久，最后以军礼安葬了这位从容倒在后卫阵地上的德国将军。　　天空依然阴霾，依然有鸽子在飞翔，谁来证明那些，没有墓碑的爱情和生命，雪依然在下那村庄依然安祥，年轻的人们消逝在白桦林……　　......

完整歌名应该是 ucc60uc6b0ucc60uc6b0-uc544ubb34ub9ac uc560ub97c uc4f0uace0 ub9c9uc544ubcf4ub824 ud574ub3c4 ub108uc758 ubaa9uc18cub9acuac00 ub4e4ub824（Chow Chow - 无论怎么挣扎怎么阻挡还是听到你的声音）表达的是一个人时的孤独 对于恋人的想念 来自内心的呼唤chow chow 个人认为可能是某种语气词

歌曲名:Bed Time歌手:Under Melissa Blue专辑:It"s Bluer Than YouUsher & BabyfaceBedtime"I just want you so much. Can I have you baby, do you hear me baby?(verse 1)Craving your body all through the night, feels like I"m going through withdrawI want to hold you, I want you inside of these armsI"m dying to taste you, I can"t denyI can"t get it out of my mindPlease let me hold youCuz" I need your lovin" tonight(chorus)Whenever I"m near you, my love just comes down (just comes down)whenever I see you, I just want you around (oh baby, oh suga)"Cause I wanna feel you all through the day and the night (oh lady)whenever I"m near you it feels like bedtime(verse 2)Constantly thinking of things to do, places where we can make loveCuz" I wanna freak you, I"ll freak you where ever you wantPlacing my kisses all over you, caressing you so nice and slowOh how I want you, girl theres something I want you to know(chorus)Whenever I"m near you, my love just comes down (just comes down)whenever I see you, I just want you around (I just want it baby...)"Cause I wanna feel you all through the day and the night (Oh)whenever I"m near you, it feels like bedtime(bridge)Do you get lonely (ooh, I get lonely)thinking about me? (thinkin" about you)Am I the only...(Girl I dream of, I think of at all)Are you into me, outta your mind for me?Say you can"t go a day with out me...Girl I love you,and I want you,and I need you, I need you, I need you(chorus)Whenever I"m near you, my love just comes down (my love comes down)whenever I see you, I just want you around (Oh I want you, I want you)"Cause I wanna feel you all through the day and the nightwhenever I"m near you, it feels like bedtime(chorus)Whenever I"m near you, my love just comes down (my love, my love my love)whenever I see you, I just want you around (I just need you...cuz)"Cause I wanna feel you all through the day and the nightwhenever I"m near you, it feels like bedtime (It feels like bedtime)It feels like bed time...(baby)http://music.baidu.com/song/15075000

《Torture》英文歌词：You know, you knowYou know that it"s tortureThe sweetest words you whisper in my ear"Cause you know what I wanna hearThe way you hold me like it"s something real"Cause you know what I wanna feelIt takes two to lieOne to lie and one to listenEmpty promises are wearin" thinOhhh...StopWhat you"re doin" to meStopLike an arrow through meStopThis is torture to me"Cause it"s gotta be all or it"s nothing at allI"m tellin" you to stopThis is torture to meYou know that it"s tortureI lose control when you"re touchin" me"Cause you know what I really needBut I need more than physicalitySo you know how it"s gotta beIt takes two to lieOne to lie but I"m done listenin"If you"re heart"s half in it then we"re finishedOhhh...StopWhat you"re doin" to meStopLike an arrow through meStopThis is torture to me (Torture to me)"Cause it"s gotta be all or it"s nothing at allI"m tellin" you to stopThis is torture to meYou know that it"s tortureCan"t keep goin" through thisCan"t keep on leadin" me onWhy you bein" so elusive?Find More lyrics at www.sweetslyrics.comYou"re in or you"re outThere"s no middle groundOhhh... noIt"s killin" me, it"s killin" meYou know that it"s torture (This is torture)It"s killin" me, it"s killin" me (It"s killin" me)You know that it"s tortureI lose control when you"re touchin" me"Cause you know what I really needBut I need more than physicalityOhhhh...StopWhat you"re doin" to me (Stop what you"re doin" to me)StopLike an arrow through meStopThis is torture to me (This is torture to me)"Cause it"s gotta be all or it"s nothing at all (Ohhh...)I"m tellin" you to stopWhat you"re doin" to meStopLike an arrow through meStopThis is torture to me (Torture, torture, you know that it"s torture)"Cause it"s gotta be all or it"s nothing at all (Nothing at all)I"m tellin" you to stopThis is torture to me (Torture to me)You know that it"s tortureIt"s killin" me, it"s killin" meYou know that it"s torture (Know that it"s torture)It"s killin" me, it"s killin" me (it"s killin" me)You know that it"s torture (Torture)It"s killin" me, it"s killin" me(This is torture to me) You know that it"s tortureIt"s killin" me, it"s killin" me (This is torture to me)You know that it"s torture

please turn the light on

歌曲名:Lil Star歌手:Kelis featuring Cee-Lo专辑:The HitsThere is nothing special about meI am just a lil starIf it seems like I"m shining brightlyIt"s probably a reflection of something you already areI forget about myself sometimeWhen there"s so many other aroundWhen deep inside you feels darkestThat is where I can always be foundThat is where I can always be foundThat is where I can always be foundJust keep trying and tryingIt"s just a matter of timingThough the grinding is tiringDon"t let "em stop you from smilingJust keep trying and tryingSooner or later you"ll find itIt"s surprising how inspiringIt is to see you shiningCause in the dark of the night you"re all i can seeand you sure look like a star to meThere is nothing special about meI am just a lil starIf you try to reach out an touch meyou"ll see I"m not really that farI may not be the brightest no am I the last one you"ll seeBut as long as you notice, that"s just fine with meEverything"s just fine with meEverything"s just fine with meJust keep trying and tryingIt"s just a matter of timingThough the grinding is tiringDon"t let "em stop you from smilingJust keep trying and tryingSooner or later you"ll find itIt"s surprising how inspiringIt is to see you shiningCause in the dark of the night you"re all i can seeand you sure look like a star to meThere is nothing special about meI am just a lil starI"ve been running and jumping, but barelyGetting, getting over the barI plan on being much more than I, am but that"s in do timeBut until then I"m guilty, and being humans my crimeBeing human that is my crimeBeing human that is my crimeJust keep trying and tryingIt"s just a matter of timingThough the grinding is tiringDon"t let "em stop you from smilingJust keep trying and tryingSooner of later you"ll find itIt"s surprising how inspiringIt is to see you shiningCause in the dark of the night you"re all i can seeand you sure look like a star to mehttp://music.baidu.com/song/3460276

...S|side too much like a Gemini He was freaky like a Taurus the way he handled me yeah Flirtatious like and Aries Which sign is best for me? Capricorn Aquarius Pisces Aries Taurus Gemini Cancer oh! Leo Virgo Libra Scorpio Sagittarius I love all y"all Capricorn Aquarius Pisces Aries Taurus Gemini Cancer oh! Leo Virgo Libra Scorpio Sagittarius I love all y"all I wish he was a Virgo, the same sign as me And know how to show me love, cause I"ve been hurt by a Pisces I wish he was a Virgo, the same sign as me And know how to show me love, cause I"ve been hurt by a Pisces I want him sexy like a Scorpio The way he holds me close yeah Sensitive as a Cancer Every sign from coast to coast Capricorn Aquarius Pisces Aries Taurus Gemini Cancer oh! Leo Virgo Libra Scorpio Sagittarius I love all y"all Capricorn Aquarius Pisces Aries Taurus Gemini Cancer oh! Leo Virgo Libra Scorpio Sagittarius I love all y"all I can"t explain why I love him Bossy like Leo"s do Got game like an Aquarius Switch moves after he"s been with you Fun like a Libra He stays on my mind He"s so unpredictable He reminds me of every sign Capricorn Aquarius Pisces Aries Taurus Gemini Cancer oh! Leo Virgo Libra Scorpio Sagittarius I love all y"all Capricorn Aquarius Pisces Aries Taurus Gemini Cancer oh! Leo Virgo Libra Scorpio Sagittarius I love all y"all Every sign from the first to the last Everyday of the month has its good and its bad I love you all, all 12 signs

歌曲名:Bounce歌手:Calvin Harris (ft. Kelis)专辑:18 MonthsBounce (Radio Edit)--Calvin Harris (ft. Kelis)~I can"t believe that every day and every nightIt"s getting better with you out of my lifeIt"s like I flicked a switch and now I"m feeling goodNo way to stop it, now you wish that you couldWhen the weekend comes I know I"ll feel aliveYou will be the last thing on my mindNo regrets, do you know what that means?.It"s hot, it"s now, it"s whenWe bounce to this trackAnd I don"t care what anybody thinks about thatHands to the sky, and throw your head back.I can love again tonightWe bounce to this trackAnd I don"t care what anybody thinks about thatHands to the sky, and throw your head back.Bounce, bounce, bounce.It"s, it"s now, it"s whenWe bounce to this trackAnd I don"t care what anybody thinks about thatI don"t care what anybody thinks about thathttp://music.baidu.com/song/30674885

是这个??徐:要一见钟情 要一箭穿心 一瞬间就决定 才是真的爱情 刘:要想清楚 要一步一步 等缘分都停驻 才有真正的幸福 合唱:干嘛要想得很美想得很远 只要这秒有感觉 孔:吻一下 拥抱一下 都胜过誓言 徐:一恋爱我会撒娇又耍赖 刘:一恋爱我会体贴又关怀 刘/徐:看着情人小事也变得浪漫 听冷笑话都会笑到流汗 徐:一恋爱我的人会亮起来 刘:一恋爱我会活的更精采 刘/徐:热恋的人总会特可爱 微笑像有翅膀停不下来 大家来恋爱 Rap:Why you talki" that mess huh? Why you trippin"but love huh? Why can"t you just let it go Just don"t say no Give it to me now momma You must be dreamin"if you think love is so 完美 So you best stop dreamin"lemme tell you love is a game It"slike round n round spinnin here we go you be like "Is this really ture?" I say I don"t know can"t tell you right now But you can stii be my boo It"s just like somethih" about you makes me feel on so good So just don,t don"t fight the feeiing Wouidn"t stop if you could Rap:I look at love simply give me some please If you don"t want it pass it on move quickly Tese girls they need attention My time love and affection Day one all on stress Day two up in your neck Day three where did this come from Day four comes around baby girl I"m gone 还是....以下为Bossy歌词 Ay uh…. You don"t have to love me You don"t even have to like me But you will respect me You know why? ‘Cause I"ma boss! Uh uh.... watch the beat go.... Uh uh.... watch the beat go.... Uh uh.... watch the beat go.... Uh uh.... watch that beat go.... [Hook:] I"m bossy, I"m the first girl to scream on a track I switched up the beat of the drum That"s right i brought all the boys to the yard And that"s right, i"m the one that"s tattooed on his arm I"m bossy, I"m the bitch y"all love to hate I"m the chick that"s raised the stake I told young stunna he should switch debate I"m back with an 808 cause i"m bossy [Verse 1:] Ooh lemme slow it down for ya so you can catch the flow Screw it up make it go extra slow Real girls get down on the flo" (On the flo" get down, on the flo‘) Ooh i gave you a taste you want some more Touch down on it like a pro I ride the beat like a bicycle, I"m icy cold [Bridge:] Ooh from the 6-4 hefers up in crenshaw The money makin" playas up in Harlem Don"t want no problem We gon" keep it bumpin" while the 808 is jumpin" [Pre-Hook:] Diamonds on my neck Di-diamonds on my grill Diamonds on my neck Di-diamonds on my grill [Hook:] I"m bossy, I"m the first girl to scream on a track I switched up the beat of the drum That"s right i brought all the boys to the yard And that"s right, i"m the one that"s tattooed on his arm I"m bossy, I"m the bitch y"all love to hate I"m the chick that"s raised the stake I told young stunna he should switch debate I"m back with an 808 cause i"m bossy [Verse 2:] Ooh (I bet I bet I bet) I betcha neva heard it like this before My baby be cruising" them phantom doors Got the bar poppin this that you can"t afford (Can"t afford) Ooh i"m drinkin" blastin" the crowds it"s all smoking" All the while i"m all open Me and my girls we stay fly and we love to stay high [Bridge:] Ooh from the 6-4 hefers up in crenshaw The money makin" playas up in Harlem Don"t want no problem We gon" keep it bumpin" while the 808 is jumpin" [Pre-Hook:] Diamonds on my neck Di-diamonds on my grill Diamonds on my neck Di-diamonds on my grill [Hook:] I"m bossy, I"m the first girl to scream on a track I switched up the beat of the drum That"s right i brought all the boys to the yard And that"s right, i"m the one that"s tattooed on his arm I"m bossy, I"m the bitch y"all love to hate I"m the chick that"s raised the stake I told young stunna he should switch debate I"m back with an 808 cause i"m bossy [Too Short:] It"s bout time that she get with me Can"t stop starin, she"s fine and she"s pretty Damn girl, don"t hurt ‘em If niggas don"t back down, you gon work em Put your mack down, i know your background What chu want girl, you gettin mad now That"s how you do it, huh? Well i"m the coolest one The bad bitches in the back bring ‘em to the front (Biatch) Tell that man you"s a boss bitch Make some noise, raise your hand if you"s a boss bitch I don"t think he understand you"s a boss bitch Get some help if you can cause he lost it Ain"t no refunds, she spent the cash mayne In your benz with her friends in the fast lane Flossin, you say "How much it cost me?" About a million dollars playa, she"s bossy [Hook:] I"m bossy, I"m the first girl to scream on a track I switched up the beat of the drum That"s right i brought all the boys to the yard And that"s right, i"m the one that"s tattooed on his arm I"m bossy, I"m the bitch y"all love to hate I"m the chick that"s raised the stake I told young stunna he should switch debate I"m back with an 808 cause i"m bossy Uh uh.... watch the beat go.... Uh uh.... watch the beat go.... Uh uh.... watch the beat go.... Uh uh.... watch the beat go.... Uh uh.... watch the beat go.... Uh uh.... watch the beat go.... Uh uh.... watch the beat go.... Uh uh.... watch the beat go.... 翻译 瑷扛啊. 你不一定要爱我,你也不用像我这样,但你能尊重我,你知道 为什么? "事业i"ma老板! 嗯嗯…… 守望巡逻去.... 嗯嗯…… 守望巡逻去.... 嗯嗯…… 守望巡逻去.... 嗯嗯…… 守望,巡逻去.... [钩: ]我博西 我的女孩尖叫了我的轨道转起来的节拍鼓,那是我的权利 使所有男孩的小鹿,而公民的权利, 我的一点的纹身手臂我博西 我的荡妇同感爱恨我的小鸡说的提出要价,我告诉年轻stunna他应 开关辩论,我回来了808事业,我博西[一首诗: ]哇lemme缓慢下来,让雅 你可以捕捉流螺钉起来使其走额外缓慢真实女孩下床就芙萝" (关于芙萝"坐下,就 芙萝的" )哇,我给你品尝,你想多一些落地就好像一个亲我 坐击败喜欢自行车,我』 米冷冰冰[桥: ]哇,从6-4 hefers起来crenshaw钱makin " playas在哈林不想 没有问题,我们健在的不断bumpin "虽然是808的Jumpin "前钩: ]钻石在我的脖子上荻钻石对我国伽马 生病钻石对我的脖子荻钻石对我的晚餐[钩: ]我跋扈,我笔 他首先女孩尖叫了我的轨道转起来的节拍鼓那个时候我broug 羟所有男孩的小鹿,而公民的权利,我笔 他说,其中的纹身手臂我跋扈,我笔 他荡妇同感爱恨我的小鸡说的提出要价,我告诉年轻stunna他应该切换 小时辩论,我回来了808事业,我博西[韵文2 : ]哇(我猜我猜我猜)我betch 涅瓦一听到这样,我才抱着游玩的人幽灵门得到律师poppin这样,你三 模拟器负担(不起)哇,我饮酒" blastin "赛事的所有吸烟"的一切,而我全部开放,我 和我的女孩,我们留飞,我们爱留高[桥: ]哇,从6-4 hefers起来铬 enshaw钱makin " playas在哈林不想没问题,我们健在的不断bumpin " ,而我808 收盘的Jumpin "前钩: ]钻石在我的脖子上荻钻石对我的烤钻石在我的脖子上荻钻石对我的烤 [钩: ]我跋扈,我次 e首先女孩尖叫了我的轨道转起来的节拍鼓那个时候我送来 笔所有男孩的小鹿,而公民的权利,我次 五,其中的纹身手臂我跋扈,我次 电子荡妇同感爱恨我的小鸡说的提出要价,我告诉年轻stunna他应该开关 辩论中,我回到了808事业,我博西[太短: ]它的布特时候,她会不会跟我不能 starin停止,她的优秀和她妈的漂亮女孩,不伤害"电磁如果niggas不退缩,你健在 统工作,把你的麦克下来,我知道你的背景是什么麟想要女孩,你gett 在疯了,现在的您是怎么做的吧? 好我的冷淡一个坏的母犬在背部,使"电磁前线( biatch )告诉该男子 你的老板荡妇作出一些噪音, 手如果你的老板荡妇,我不觉得他明白你的老板荡妇得到一些帮助 如果你能导致他失去这不是不退款, 她用现金Mayne先生,在你的奔驰与友人在快车道flossin , 你说"多少钱"我? 约100万美元Playa的,她的跋扈[钩: ]我博西 我的女孩尖叫了我的轨道转起来的节拍鼓,那是我的权利 使所有男孩的小鹿,而公民的权利, 我的一点的纹身手臂我博西 我的荡妇同感爱恨我的小鸡说的提出要价,我告诉年轻stunna他应 开关辩论,我回来了808事业,我博西嗯嗯…… 守望巡逻去.... 嗯嗯…… 守望巡逻去.... 嗯嗯…… 守望巡逻去.... 嗯嗯…… 守望巡逻去.... 嗯嗯…… 守望巡逻去.... 嗯嗯…… 守望巡逻去.... 嗯嗯…… 守望巡逻去.... 嗯嗯…… 守望巡逻去....

歌曲名:Bounce歌手:Calvin Harris Feat. Kelis专辑:Justice Crew Party MixBounceCalvin Harris Feat. KelisI can"t believe that every day and every nightThese people better leave you out of my lifeIt"s like a fantasy setting out, now I"m feeling goodNo way to stop it, now you wish that you couldWhen the waking comes I know I feel aliveYou will be the last thing on my mindNo regrets, do you know what that meansIf how, if now, if whenWe"re down to this trackAnd I don"t care what anyobody thinks about thatHands to the sky, and throw your head backI can love again tonightWe"re down to this trackAnd I don"t care what anyobody thinks about thatHands to the sky, and throw your head backBounce, bounce, bounceIf how, if now, if whenWe"re down to this trackAnd I don"t care what anyobody thinks about thatI don"t care what anyobody thinks about thathttp://music.baidu.com/song/12487202

歌曲名:Lil Star歌手:Kelis&Cee Lo Green专辑:Kelis Was HereThere is nothing special about meI am just a lil starIf it seems like I"m shining brightlyIt"s probably a reflection of something you already areI forget about myself sometimeWhen there"s so many other aroundWhen deep inside you feels darkestThat is where I can always be foundThat is where I can always be foundThat is where I can always be foundJust keep trying and tryingIt"s just a matter of timingThough the grinding is tiringDon"t let "em stop you from smilingJust keep trying and tryingSooner or later you"ll find itIt"s surprising how inspiringIt is to see you shiningCause in the dark of the night you"re all i can seeand you sure look like a star to meThere is nothing special about meI am just a lil starIf you try to reach out an touch meyou"ll see I"m not really that farI may not be the brightest no am I the last one you"ll seeBut as long as you notice, that"s just fine with meEverything"s just fine with meEverything"s just fine with meJust keep trying and tryingIt"s just a matter of timingThough the grinding is tiringDon"t let "em stop you from smilingJust keep trying and tryingSooner or later you"ll find itIt"s surprising how inspiringIt is to see you shiningCause in the dark of the night you"re all i can seeand you sure look like a star to meThere is nothing special about meI am just a lil starI"ve been running and jumping, but barelyGetting, getting over the barI plan on being much more than I, am but that"s in do timeBut until then I"m guilty, and being humans my crimeBeing human that is my crimeBeing human that is my crimeJust keep trying and tryingIt"s just a matter of timingThough the grinding is tiringDon"t let "em stop you from smilingJust keep trying and tryingSooner of later you"ll find itIt"s surprising how inspiringIt is to see you shiningCause in the dark of the night you"re all i can seeand you sure look like a star to mehttp://music.baidu.com/song/3460506

There is nothing special about me I am just a lil star If it seems like I"m shining brightly It"s probably a reflection of something you already are I forget about myself sometime When there"s so many other around When deep inside you feels darkest That is where I can always be found That is where I can always be found That is where I can always be found 我平凡无奇 我只是一颗小星星 如果我看起来在闪烁 那也许是因为反射了你的光辉 我有时会忘记我自己 当我周围群星闪耀 但当你凝视自己内心最黑暗的角落 你总能在那里找到我 你总能在那里找到我 你总能在那里找到我 Just keep trying and trying It"s just a matter of timing Though the grinding is tiring Don"t let "em stop you from smiling Just keep trying and trying Sooner or later you"ll find it It"s surprising how inspiring It is to see you shining Cause in the dark of the night you"re all I can see and you sure look like a star to me 努力吧，努力 时间会证明一切 虽然人生路途劳顿 但不要失去你的笑容 努力吧，努力， 总有一天你会发现 这是多么令人惊喜，多么激动人心 当我看见你熠熠生辉 因为在黑夜里 你就是唯一的所在 你就是我的明星 There is nothing special about me I am just a lil star If you try to reach out an touch me you"ll see I"m not really that far I may not be the brightest nor am I the last one you?l see But as long as you notice, that"s just fine with me Everything"sjust fine with me Everything"s just fine with me 我平凡无奇 我只是一颗小星星 如果你伸出手触摸 你会发现其实我并不遥远 我并不是最明亮的那一颗 你以后还会遇到好多 但只要的的目光曾为我停留 我就很满足 我一切都好 我一切都好 Just keep trying and trying It"s just a matter of timing Though the grinding is tiring Don"t let "em stop you from smiling Just keep trying and trying Sooner or later you"ll find it It"s surprising how inspiring It is to see you shining Cause in the dark of the night you"re all I can see and you sure look like a star to me 努力吧，努力 时间会证明一切 虽然人生路途劳顿 但不要失去你的笑容 努力吧，努力， 总有一天你会发现 这是多么令人惊喜，多么激动人心 当我看见你熠熠生辉 因为在黑夜里 你就是唯一的所在 你就是我的明星 There is nothing special about me I am just a lil star I"ve been running and jumping, but barely Getting, getting over the bar I plan on being much more than I, am but that"s in due time But until then I"m guilty, and being humans my crime Being human that is my crime Being human that is my crime 我平凡无奇 我只是一颗小星星 我尽我所能 却始终不能越过那道障碍 我希望超越自我 但那时刻还没有到来 直到那时我才发现我的罪行 那就是生而为人 生而为人就是我的罪孽 生而为人就是我的罪孽 Just keep trying and trying It"s just a matter of timing Though the grinding is tiring Don"t let "em stop you from smiling Just keep trying and trying Sooner of later you"ll find it It"s surprising how inspiring It is to see you shining Cause in the dark of the night you"re all I can see and you sure look like a star to me 努力吧，努力 时间会证明一切 虽然人生路途劳顿 但不要失去你的笑容 努力吧，努力， 总有一天你会发现 这是多么令人惊喜，多么激动人心 当我看见你熠熠生辉 因为在黑夜里 你就是唯一的所在 你就是我的明星

歌曲名:Milkshake歌手:Kelis专辑:MilkshakeMilkshakeKelisMy milkshake brings all the boys to the yardAnd they"re like, It"s better than yoursDamn right, it"s better than yoursI can teach you, but I have to chargeMy milkshake brings all the boys to the yardAnd they"re like, It"s better than yoursDamn right, it"s better than yoursI can teach you, but I have to chargeI know you want it...The thing that makes meWhat the guys go crazy forThey lose their minds, the way I windI think it"s time...(La La, La La, La)Warm it up(La La, La La, La)The boys are waiting(La La, La La, La)Warm it up(La La, La La, La)The boys are waitingMy milkshake brings all the boys to the yardAnd they"re like, It"s better than yoursDamn right, it"s better than yoursI can teach you, but I have to chargeMy milkshake brings all the boys to the yardAnd they"re like,It"s better than yoursDamn right, it"s better than yoursI can teach you, but I have to chargeI can see you"re on it...You want me to teach theeTechniques that freaks these boysIt can"t be boughtJust don"t please get caughtWatch if you"re smart(La La, La La, La)Warm it up(La La, La La, La)The boys are waiting(La La, La La, La)Warm it up(La La, La La, La)The boys are waitingMy milkshake brings all the boys to the yardAnd they"re like It"s better than yoursDamn right, it"s better than yoursI can teach you, but I have to chargeMy milkshake brings all the boys to the yardAnd they"re like,It"s better than yoursDamn right, it"s better than yoursI can teach you, but I have to chargeOh, once you get involvedEveryone will look this way, soYou must maintain your charmSame time maintain your haloJust get the perfect blendPlus what you have withinThen next his eyes are squaredAnd he"s picked up your scent(La La, La La, La)Warm it up(La La, La La, La)The boys are waiting(La La, La La, La)Warm it up(La La, La La, La)The boys are waitingMy milkshake brings all the boys to the yardAnd they"re like,It"s better than yoursDamn right, it"s better than yoursI can teach you, but I have to chargeMy milkshake brings all the boys to the yardAnd they"re like, It"s better than yoursDamn right, it"s better than yoursI can teach you, but I have to chargehttp://music.baidu.com/song/3488548

歌曲名:Milkshake歌手:Kelis专辑:TastyMilkshakeKelisMy milkshake brings all the boys to the yardAnd they"re like, It"s better than yoursDamn right, it"s better than yoursI can teach you, but I have to chargeMy milkshake brings all the boys to the yardAnd they"re like, It"s better than yoursDamn right, it"s better than yoursI can teach you, but I have to chargeI know you want it...The thing that makes meWhat the guys go crazy forThey lose their minds, the way I windI think it"s time...(La La, La La, La)Warm it up(La La, La La, La)The boys are waiting(La La, La La, La)Warm it up(La La, La La, La)The boys are waitingMy milkshake brings all the boys to the yardAnd they"re like, It"s better than yoursDamn right, it"s better than yoursI can teach you, but I have to chargeMy milkshake brings all the boys to the yardAnd they"re like,It"s better than yoursDamn right, it"s better than yoursI can teach you, but I have to chargeI can see you"re on it...You want me to teach theeTechniques that freaks these boysIt can"t be boughtJust don"t please get caughtWatch if you"re smart(La La, La La, La)Warm it up(La La, La La, La)The boys are waiting(La La, La La, La)Warm it up(La La, La La, La)The boys are waitingMy milkshake brings all the boys to the yardAnd they"re like It"s better than yoursDamn right, it"s better than yoursI can teach you, but I have to chargeMy milkshake brings all the boys to the yardAnd they"re like,It"s better than yoursDamn right, it"s better than yoursI can teach you, but I have to chargeOh, once you get involvedEveryone will look this way, soYou must maintain your charmSame time maintain your haloJust get the perfect blendPlus what you have withinThen next his eyes are squaredAnd he"s picked up your scent(La La, La La, La)Warm it up(La La, La La, La)The boys are waiting(La La, La La, La)Warm it up(La La, La La, La)The boys are waitingMy milkshake brings all the boys to the yardAnd they"re like,It"s better than yoursDamn right, it"s better than yoursI can teach you, but I have to chargeMy milkshake brings all the boys to the yardAnd they"re like, It"s better than yoursDamn right, it"s better than yoursI can teach you, but I have to chargehttp://music.baidu.com/song/2622535

歌曲名:Milkshake歌手:Kelis专辑:The HitsMilkshakeKelisMy milkshake brings all the boys to the yardAnd they"re like, It"s better than yoursDamn right, it"s better than yoursI can teach you, but I have to chargeMy milkshake brings all the boys to the yardAnd they"re like, It"s better than yoursDamn right, it"s better than yoursI can teach you, but I have to chargeI know you want it...The thing that makes meWhat the guys go crazy forThey lose their minds, the way I windI think it"s time...(La La, La La, La)Warm it up(La La, La La, La)The boys are waiting(La La, La La, La)Warm it up(La La, La La, La)The boys are waitingMy milkshake brings all the boys to the yardAnd they"re like, It"s better than yoursDamn right, it"s better than yoursI can teach you, but I have to chargeMy milkshake brings all the boys to the yardAnd they"re like,It"s better than yoursDamn right, it"s better than yoursI can teach you, but I have to chargeI can see you"re on it...You want me to teach theeTechniques that freaks these boysIt can"t be boughtJust don"t please get caughtWatch if you"re smart(La La, La La, La)Warm it up(La La, La La, La)The boys are waiting(La La, La La, La)Warm it up(La La, La La, La)The boys are waitingMy milkshake brings all the boys to the yardAnd they"re like It"s better than yoursDamn right, it"s better than yoursI can teach you, but I have to chargeMy milkshake brings all the boys to the yardAnd they"re like,It"s better than yoursDamn right, it"s better than yoursI can teach you, but I have to chargeOh, once you get involvedEveryone will look this way, soYou must maintain your charmSame time maintain your haloJust get the perfect blendPlus what you have withinThen next his eyes are squaredAnd he"s picked up your scent(La La, La La, La)Warm it up(La La, La La, La)The boys are waiting(La La, La La, La)Warm it up(La La, La La, La)The boys are waitingMy milkshake brings all the boys to the yardAnd they"re like,It"s better than yoursDamn right, it"s better than yoursI can teach you, but I have to chargeMy milkshake brings all the boys to the yardAnd they"re like, It"s better than yoursDamn right, it"s better than yoursI can teach you, but I have to chargehttp://music.baidu.com/song/3460270

歌曲名:Milkshake歌手:Kelis专辑:30 Jahre Hip HopMy Milkshake brings all the boys to the yard,And their like "It"s better than yours"Damn right, It"s better than yours,I can teach you, but I have to charge (2x)My Milkshake brings all the boys to the yard,And their like "It"s better than yours"Damn right, It"s better than yours,I can teach you, but I have to chargeVrs1:I know you want it...The thing that makes me,What the guys go crazy for,They lose their minds, The way I wind,I think it"s time...Vamp:(La-La-La-La-la)Warm it up,(La-La-La-La-la)The boys are waiting,(La-La-La-La-la)Warm it up,(La-La-La-La-la)The boys are waiting,Chorus:My Milkshake brings all the boys to the yard,And their like "It"s better than yours"Damn right, It"s better than yours,I can teach you, but I have to charge (2x)My Milkshake brings all the boys to the yard,And their like "It"s better than yours"Damn right, It"s better than yours,I can teach you, but I have to chargeVrs2:I can see you"re on it...You want me to teach the,Technics that freaks these boys,It can"t be bought,Just know theives get caught,Watch if you"re smart,Vamp:(La-La-La-La-la)Warm it up,(La-La-La-La-la)The boys are waiting,(La-La-La-La-la)Warm it up,(La-La-La-La-la)The boys are waiting,Chorus:My Milkshake brings all the boys to the yard,And their like "It"s better than yours"Damn right, It"s better than yours,I can teach you, but I have to charge (2x)My Milkshake brings all the boys to the yard,And their like "It"s better than yours"Damn right, It"s better than yours,I can teach you, but I have to chargeBridge:Oh, Once you get involved,Everyone will look this way so,You must maintain your charm,Sametime maintain your halo,Just get the perfect blend,Plus what you have within,Spoken:Then next his eyes are squint,Then he"s picked up your scent,Vamp:(La-La-La-La-la)Warm it up,(La-La-La-La-la)The boys are waiting,(La-La-La-La-la)Warm it up,(La-La-La-La-la)The boys are waiting,Chorus:My Milkshake brings all the boys to the yard,And their like "It"s better than yours"Damn right, It"s better than yours,I can teach you, but I have to charge (2x)My Milkshake brings all the boys to the yard,And their like "It"s better than yours"Damn right, It"s better than yours,I can teach you, but I have to chargehttp://music.baidu.com/song/59341562

杀虫剂。kelisi主要成分是氯氰菊酯。它可以用于防治果树上的害虫，如瓢虫、蚜虫、苹果蚜等。Kelisi杀虫剂的使用方法和剂量需要根据具体情况进行调整，建议在使用前仔细阅读产品说明书，并遵循相关的安全使用指南。

歌曲名:Flashback歌手:Kelis专辑:TastyFlashback作词：buzzG作曲：buzzG编曲：buzzG呗：初音ミク翻译：cyatakuby：CHHKKEけやき通りの细い街并を 歩く梦を见る /做了个漫步在狭窄的榉树步道上的梦一人では歩き出せない /一个人便无法迈出脚步私のことよく知ってるでしょう？ /这样的我你也是很清楚的吧？幸せな甘い季节が过ぎて /幸福而甜蜜的季节已经过去长いトンネルへ /消逝在漫长隧道的彼方変わる世界 重ならぬ未来 /变化的世界 不曾交汇的未来こんなはずじゃなかったの /原本不应该是这样的吧左手が退屈そうにして空を切る /我的左手寂寞地在空气中划过君の右手を求めてる /寻求著你的右手もう枯れるまで泣いて /已经哭到泪腺乾涸逃げることも许されないのかな？ /可就连逃避也是不被容许的吗？今 手のひらに降り注ぐ几千の星の光 /此刻 徐徐注入手心的数千繁星的光芒そういう景色や 想い出が /这样美丽的景色与回忆破壊されるのは嫌だよ /我不想看到它被摧毁啊数え切れないほど夜を越えた /即便已经跨越无数夜晚今でも梦を见るよ /如今也仍在做著梦君の毎秒変わる表情が /你那每秒变幻的表情フラッシュバックする梦を /在脑海中不断闪回的梦『会いたかったからすぐきたんだ。』 /『因为想见你所以立刻就动身来了。』って映画の见すぎ！ /说什麼呢看多电影了吧！『いきなりなんて超迷惑。』 /『这麼突然感觉超麻烦的。』本当は嬉しかったよ /其实心里很高兴的喔木枯らしが穷屈そうに二人の间を /寒风从似乎很是拘束的俩人间すり抜ける日曜の午後 /穿过的那个星期天的下午もう记忆から覚めて溢れ出した /已经从记忆中醒来挤满了内心想いの行き场所探してよ /去找找思念该去往的地方吧昔见た君の仕草を /理所当然般贪图著当たり前に欲しがる日々 /曾经所见的你的身姿的时光世界には追いつけない /在这世界里已是无法追寻追いつかなくていい /也不必去追寻了呢私だけ知らなかったの？ /只有我曾不明白吗？一人分の伞も无いのに /明明连一份爱意也不剩了君にすがって寄り添うだけの /仅仅一味抓著你依靠向你的时代遅れのアイロニー /错过时机的反话---music---昔见た君の仕草を /理所当然般贪图著当たり前に欲しがる日々 /曾经所见的你的身姿的时光世界には追いつけない /在这世界里已是无法追寻追いつかなくていい /也不必去追寻了呢今 手のひらに降り注ぐ几千の星の光 /此刻 徐徐注入手心的数千繁星的光芒そういう景色や 想い出が /这样美丽的景色与回忆破壊されるのは嫌だよ /我不想看到它被摧毁啊数え切れないほど夜を越えた /即便已经跨越无数夜晚今でも梦を见るよ /如今也仍在做著梦君の毎秒変わる表情が /你那每秒变幻的表情フラッシュバックする梦を /在脑海中不断闪回的梦けやき通りの细い街并を 歩く梦を见る /做了个漫步在狭窄的榉树步道上的梦一人では歩き出せない /一个人便无法迈出脚步私のこと见てる金木犀 /金木犀正看著这样的我-END-http://music.baidu.com/song/2622552

歌曲名:Acapella歌手:Kelis专辑:One More LoveAcapellaKelisI was walkin was livingMy melody was acapellaTheres a beat I was missingNo tune or a scale I could playThe sound in the distanceNo orchestra playing togetherLike a boat out to seaThe silence was too deafeningSo come and revive meI cant feel my heartbeatJust me surviving aloneBefore youMy whole life was acapellaNow a symphonysThe only song to singBefore youMy whole life was acapellaNow a symphonysThe only song to singEverything was the sameOne color was just like the othersAn assembly routineMy memories were all black and whiteTill I stopped over-thinkingDecided to draw back the curtainsAnd I cleared all the cobwebsAnd began to let in the lightSo come and revive meI cant feel my heartbeatJust me surviving aloneBefore youMy whole life was acapellaNow a symphonysThe only song to singBefore youMy whole life was acapellaNow a symphonysThe only song to singYou are the drum in my heart beatBass and guitar leadStuck on the notes you playMy heart that you play onRed like a crayonI cant walk awayYou are the drum in my heart beatBass and guitar leadStuck on the notes you playMy heart that you play onRed like a crayonI cant walk awayYou are the drum in my heart beatBass and guitar leadStuck on the notes you playMy heart that you play onRed like a crayonI cant walk awayBefore youMy whole life was acapellaNow a symphonysThe only song to singBefore youMy whole life was acapellaNow a symphonysThe only song to singBefore youMy whole life was acapellaNow a symphonysThe only song to singBefore youMy whole life was acapellaNow a symphonysThe only song to singhttp://music.baidu.com/song/2588222

歌曲名:Stick Up歌手:Kelis专辑:TastyJackie Boyz - Stick UpMaxRNB - Your first R&B/Hiphop sourceFuegoOoh whoa whoa ohh ohyeah yeahI saw you girlon the floorLookin` like I wanna (approah) youI see you dancin" with your girlfriendsI hope they got a lotbecause I"mma (breakin)Ooh whoaDon"t be a (afraid) of my heartYou just got arrows I got somethin" to tear you apartYou want the down downI got the down downI got your head spinnin" round round roundI know you want somethin", so don"t try to runThe first shots are fun, come kiss the gunI like hearing lowThere"s nowhere to go, so don"t try to run"Cause this is stick up girlPut your hands in the air(x3)"Cause this is stick up girlPut your hands in the air(x3)Cause this is stick up girlYou in the backJust like i thoughtOoh your body softOoh girl ooh ya body softYou at the right placeAt the right timeGirl what we doin"Ooh it should be a crimeOoh whoaDon"t be a (afraid) of my heartYou just got arrows I got somethin" to tear you apartYou want the down downI got the down downI got your head spinnin" round round roundI know you want somethin", so don"t try to runThe first shots are fun, come kiss the gunI like hearing lowThere"s nowhere to go, so Don"t try to run"Cause this is stick up girlPut your hands in the air(x3)"Cause this is stick up girlPut your hands in the air(x3)Cause this is stick up girlA shot through the heart is all I need today and tonight(Today and tonight)My luck got your hostage baby at least for a nightDown, down, down(Stick up girl, stick up girl, stick up girl, stick up girl)Ohh yeah yeah yeah ooh whoa(stick up girl, stick up girl, stick up girl, stick up girl)I know you want somethin", so don"t try to runThe first shots are fun, come kiss the gunI like hearing lowYou know what I want, so don"t try to run"Cause this is stick up girlPut your hands in the air(x3)"Cause this is stick up girlPut your hands in the air(x3)cause this is stick up girlPut your hands in the air(x6)MaxRNB - Your first R&B/Hiphop sourcehttp://music.baidu.com/song/2622487

歌曲名:Lil Star歌手:Kelis专辑:Kelis Was HereThere is nothing special about meI am just a lil starIf it seems like I"m shining brightlyIt"s probably a reflection of something you already areI forget about myself sometimeWhen there"s so many other aroundWhen deep inside you feels darkestThat is where I can always be foundThat is where I can always be foundThat is where I can always be foundJust keep trying and tryingIt"s just a matter of timingThough the grinding is tiringDon"t let "em stop you from smilingJust keep trying and tryingSooner or later you"ll find itIt"s surprising how inspiringIt is to see you shiningCause in the dark of the night you"re all i can seeand you sure look like a star to meThere is nothing special about meI am just a lil starIf you try to reach out an touch meyou"ll see I"m not really that farI may not be the brightest no am I the last one you"ll seeBut as long as you notice, that"s just fine with meEverything"s just fine with meEverything"s just fine with meJust keep trying and tryingIt"s just a matter of timingThough the grinding is tiringDon"t let "em stop you from smilingJust keep trying and tryingSooner or later you"ll find itIt"s surprising how inspiringIt is to see you shiningCause in the dark of the night you"re all i can seeand you sure look like a star to meThere is nothing special about meI am just a lil starI"ve been running and jumping, but barelyGetting, getting over the barI plan on being much more than I, am but that"s in do timeBut until then I"m guilty, and being humans my crimeBeing human that is my crimeBeing human that is my crimeJust keep trying and tryingIt"s just a matter of timingThough the grinding is tiringDon"t let "em stop you from smilingJust keep trying and tryingSooner of later you"ll find itIt"s surprising how inspiringIt is to see you shiningCause in the dark of the night you"re all i can seeand you sure look like a star to mehttp://music.baidu.com/song/54054109

歌曲名:Dancing歌手:Elisa专辑:Then Comes The SunDancingby ElisaleonwikiTime is gonna take my mindand carry it far away where I can flyThe depth of life will dim my temptation to live for youIf I were to be alone silence would rock my tears"cause it"s all about love and I know betterHow life is a waving featherSo I put my arms around you around youAnd I know that I"ll be living soonMy eyes are on you they"re on youAnd you see that I can"t stop shakingNo, I won"t step back but I"ll look down to hide from your eyes"cause what I feel is so sweet and I"m scared that even my own breathOh could burst it if it were a bubbleAnd I"d better dream if I have to struggleSo I put my arms around you around youAnd I hope that I will do no wrongMy eyes are on you they"re on youAnd I hope that you won"t hurt meI"m dancing in the room as if I was in the woods with youNo need for anything but musicMusic"s the reason why I know time still existsTime still existsTime still existsSo I put my arms around you around youAnd I hope that I will do no wrongMy eyes are on you they"re on youAnd I hope that you won"t hurt meSo I put my arms around you around youAnd I hope that I will do no wrongMy eyes are on you they"re on youAnd I hope that you won"t hurt mehttp://music.baidu.com/song/8297339

歌曲名:Dancing歌手:Elisa专辑:ElisaDancingby ElisaleonwikiTime is gonna take my mindand carry it far away where I can flyThe depth of life will dim my temptation to live for youIf I were to be alone silence would rock my tears"cause it"s all about love and I know betterHow life is a waving featherSo I put my arms around you around youAnd I know that I"ll be living soonMy eyes are on you they"re on youAnd you see that I can"t stop shakingNo, I won"t step back but I"ll look down to hide from your eyes"cause what I feel is so sweet and I"m scared that even my own breathOh could burst it if it were a bubbleAnd I"d better dream if I have to struggleSo I put my arms around you around youAnd I hope that I will do no wrongMy eyes are on you they"re on youAnd I hope that you won"t hurt meI"m dancing in the room as if I was in the woods with youNo need for anything but musicMusic"s the reason why I know time still existsTime still existsTime still existsSo I put my arms around you around youAnd I hope that I will do no wrongMy eyes are on you they"re on youAnd I hope that you won"t hurt meSo I put my arms around you around youAnd I hope that I will do no wrongMy eyes are on you they"re on youAnd I hope that you won"t hurt mehttp://music.baidu.com/song/53668746

歌曲名:Dancing歌手:Elisa专辑:Soundtrack "96-"06Dancingby ElisaleonwikiTime is gonna take my mindand carry it far away where I can flyThe depth of life will dim my temptation to live for youIf I were to be alone silence would rock my tears"cause it"s all about love and I know betterHow life is a waving featherSo I put my arms around you around youAnd I know that I"ll be living soonMy eyes are on you they"re on youAnd you see that I can"t stop shakingNo, I won"t step back but I"ll look down to hide from your eyes"cause what I feel is so sweet and I"m scared that even my own breathOh could burst it if it were a bubbleAnd I"d better dream if I have to struggleSo I put my arms around you around youAnd I hope that I will do no wrongMy eyes are on you they"re on youAnd I hope that you won"t hurt meI"m dancing in the room as if I was in the woods with youNo need for anything but musicMusic"s the reason why I know time still existsTime still existsTime still existsSo I put my arms around you around youAnd I hope that I will do no wrongMy eyes are on you they"re on youAnd I hope that you won"t hurt meSo I put my arms around you around youAnd I hope that I will do no wrongMy eyes are on you they"re on youAnd I hope that you won"t hurt mehttp://music.baidu.com/song/53652162

歌曲名:Dancing歌手:Elisa专辑:Caterpillar - Itunes VersionDancingby ElisaleonwikiTime is gonna take my mindand carry it far away where I can flyThe depth of life will dim my temptation to live for youIf I were to be alone silence would rock my tears"cause it"s all about love and I know betterHow life is a waving featherSo I put my arms around you around youAnd I know that I"ll be living soonMy eyes are on you they"re on youAnd you see that I can"t stop shakingNo, I won"t step back but I"ll look down to hide from your eyes"cause what I feel is so sweet and I"m scared that even my own breathOh could burst it if it were a bubbleAnd I"d better dream if I have to struggleSo I put my arms around you around youAnd I hope that I will do no wrongMy eyes are on you they"re on youAnd I hope that you won"t hurt meI"m dancing in the room as if I was in the woods with youNo need for anything but musicMusic"s the reason why I know time still existsTime still existsTime still existsSo I put my arms around you around youAnd I hope that I will do no wrongMy eyes are on you they"re on youAnd I hope that you won"t hurt meSo I put my arms around you around youAnd I hope that I will do no wrongMy eyes are on you they"re on youAnd I hope that you won"t hurt mehttp://music.baidu.com/song/8272179

大量样本，同时检测某种蛋白（指标）的多少，意义，一组样品中 每个个体 指标多少？有什么变化趋势。一目了然。

elisa实验原理是在于抗原抗体之间的特异结合反应，所以任何优质的诊断试剂离不开优质的原料，如抗原抗体，酶等。抗原和抗体跟酶交连以后仍然能够保持活性和酶的催化性。交连后的酶根底物可以进行反应，催化分解，使其产生有色物质。根据有色物质的多少和颜色深浅通过酶测曲线得出结论。elisa是用血清来检测的，由数值来判断是阴性还是阳性的结果。特点：一、高效、灵敏、特异的抗体；二、稳定的重复性和可靠性；三、吸附性能好，空白值低，孔底透明度高的固相载体；四、适用血清、血浆、组织匀浆液、细胞培养上清液、尿液等等多种标本类型；五、节省实验经费。以上内容参考：百度百科--elisa试剂盒

ELISA的类型ELISA可用于测定抗原，也可用于测定抗体。在这种测定方法中有三个必要的试剂：（1）固相的抗菌素原或抗体，即"免疫吸附剂"（immunosorbent）；（2）酶标记的抗原或抗体，称为"结合物"（conjugate）；（3）酶反应的底物。根据试剂的来源和标本的情况以及检测的具体条件，可设计出各种不同类型的检测方法。用于临床检验的ELISA主要有以下几种类型：2.2.1 双抗体夹心法测抗原双抗体夹心法是检测抗原最常用的方法，操作步骤如下：1） 将特异性抗体与固相载体联结，形成固相抗体。洗涤除去未结合的抗体及杂质。2） 加受检标本，保温反应。标本中的抗原与固相抗体结合，形成固相抗原抗体复合物。洗涤除去其他未结合物质。3） 加酶标抗体，保温反应。固相免疫复合物上的抗原与酶标抗体结合。彻底洗涤未结合的酶标抗体。此时固相载体上带有的酶量与标本中受检抗原的量相关。4） 加底物显色。固相上的酶催化底物成为有色产物。通过比色，测知标本中抗原的量。在临床检验中，此法适用于检验各种蛋白质等大分子抗原，例如HBsAg、HBeAg、AFP、hCG等。只要获得针对受检抗原的异性抗体，就可用于包被固相载体和制备酶结合物而建立此法。如抗体的来源为抗血清，包被和酶标用的抗体最好分别取自不同种属的动物。如应用单克隆抗体，一般选择两个针对抗原上不同决定簇的单抗，分别用于包被固相载体和制备酶结合物。这种双位点夹心法具有很高的特异性，而且可以将受检标本和酶标抗体一起保温反应，作一步检测。在一步法测定中，当标本中受检抗原的含量很高时，过量抗原分别和固相抗体及酶标抗体结合，而不再形成"夹心复合物"。类同于沉淀反应中抗原过剩的后带现象，此时反应后显色的吸光值（位于抗原过剩带上）与标准曲线（位于抗体过剩带上）某一抗原浓度的吸光值相同（参见1.3.2，图1-4），如按常法测读，所得结果将低于实际的含量，这种现象被称为钩状效应（hook effect），因为标准曲线到达高峰后呈钩状弯落。钩状效应严重时，反应甚至可不显色而出现假阴性结果。因此在使用一步法试剂测定标本中含量可异常增高的物质（例如血清中HBsAg、AFP和尿液hCG等）时，应注意可测范围的最高值。用高亲和力的单克隆抗体制备此类试剂可削弱钩状效应。假使在被测分子的不同位点上含有多个相同的决定簇，例如HBsAg的a决定簇，也可用针对此决定的同一单抗分别包被固相和制备酶结合物。但在HBsAg的检测中应注意亚型问题，HBsAg有adr、adw、ayr、ayw4个亚型，虽然每种亚型均有相同的a决定簇的反应性，这也是用单抗作夹心法应注意的问题。双抗体夹心法测抗原的另一注意点是类风湿因子（RF）的干扰。RF是一种自身抗体，多为IgM型，能和多种动物IgG的Fc段结合。用作双抗体夹心法检测的血清标本中如含有RF，它可充当抗原成份，同时与固相抗体和酶标抗体结合，表现出假阳性反应。采用F（ab"）或Fab片段作酶结合物的试剂，由于去除了Fc段，从而消除RF的干扰。双抗体夹心法ELISA试剂是否受RF的影响，已被列为这类试剂的一项考核指标（参见6.2）。双抗体夹心法适用于测定二价或二价以上的大分子抗原，但不适用于测定半抗原及小分子单价抗原，因其不能形成两位点夹心。2.2.2 双抗原夹心法测抗体反应模式与双抗体夹心法类似。用特异性抗原进行包被和制备酶结合物，以检测相应的抗体。与间接法测抗体的不同之处为以酶标抗原代替酶标抗抗体。此法中受检标本不需稀释，可直接用于测定，因此其敏感度相对高于间接法。乙肝标志物中抗HBs的检测常采用本法。本法关键在于酶标抗原的制备，应根据抗原结构的不同，寻找合适的标记方法。2.2.3 间接法测抗体间接法是检测抗体常用的方法。其原理为利用酶标记的抗抗体（抗人免疫球蛋白抗体）以检测与固相抗原结合的受检抗体，故称为间接法（见图2-3）。操作步骤如下：1）将特异性抗原与固相载体联结，形成固相抗原。洗涤除去未结合的抗原及杂质。2）加稀释的受检血清，保温反应。血清中的特异抗体与固相抗原结合，形成固相抗原抗体复合物。经洗涤后，固相载体上只留下特异性抗体，血清中的其他成份在洗涤过程中被洗去。3）加酶标抗抗体。可用酶标抗人Ig以检测总抗体，但一般多用酶标抗人IgG检测IgG抗体。固相免疫复合物中的抗体与酶标抗体抗体结合，从而间接地标记上酶。洗涤后，固相载体上的酶量与标本中受检抗体的量正相关。4）加底物显色本法主要用于对病原体抗体的检测而进行传染病的诊断。间接法的优点是只要变换包被抗原就可利用同一酶标抗抗体建立检测相应抗体的方法。间接法成功的关键在于抗原的纯度。虽然有时用粗提抗原包被也能取得实际有效的结果，但应尽可能予以纯化，以提高试验的特异性。特别应注意除去能与一般健康人血清发生反应的杂质，例如以E.Coli为工程酶的重组抗原，如其中含有E.Coli成份，很可能与受过E.Coli感染者血甭中的抗E.Coli抗体发生反应。抗原中也不能含有与酶标抗人Ig反应的物质，例如来自人血浆或人体组织的抗原，如不将其中的Ig去除，试验中也发生假阳性反应。另外如抗原中含有无关蛋白，也会因竟争吸附而影响包被效果。间接法中另一种干扰因素为正常血清中所含的高浓度的非特异性。病人血清中受检的特异性IgG只占总IgG中的一小部分。IgG的吸附性很强，非特异IgG可直接吸附到固相载体上，有时也可吸附到包被抗原的表面。因此在间接法中，抗原包被后一般用无关蛋白质（例如牛血清蛋白）再包被一次，以封闭（blocking）固相上的空余间隙。另外，在检测过程中标本须先行稀释（1:40~1:200），以避免过高的阴性本底影响结果的判断。2.2.4 竞争法测抗体当抗原材料中的干扰物质不易除去，或不易得到足够的纯化抗原时，可用此法检测特异性抗体。其原理为标本中的抗体和一定量的酶标抗体竞争与固相抗原结合。标本中抗体量越多，结合在固相上的酶标抗体愈少，因此阳性反应呈色浅于阴性反应。如抗原为高纯度的，可直接包被固相。如抗原中会有干扰物质，直接包被不易成功，可采用捕获包被法，即先包被与固相抗原相应的抗体，然后加入抗原，形成固相抗原。洗涤除去抗原中的杂质，然后再加标本和酶标抗体进行竞争结合反应。竞争法测抗体有多种模式，可将标本和酶标抗体与固相抗原竞争结合，抗HBc ELISA一般采用此法。另一种模式为将标本与抗原一起加入到固相抗体中进行竞争结合，洗涤后再加入酶标抗体，与结合在固相上的抗原反应。抗HBe的检测一般采用此法。2.2.5 竞争法测抗原小分子抗原或半抗原因缺乏可作夹心法的两个以上的位点，因此不能用双抗体夹心法进行测定，可以采用竞争法模式。其原理是标本中的抗原和一定量的酶标抗原竞争与固相抗体结合。标本中抗原量含量愈多，结合在固相上的酶标抗原愈少，最后的显色也愈浅。小分子激素、药物等ELISA测定多用此法。2.2.6 捕获包被法测抗体IgM抗体的检测用于传染病的早期诊断(early diagnosis)中。间接法ELISA一般仅适用于检测总抗体或IgG抗体。如用抗原包被的间接法直接测定IgM抗体，因标本中一般同时存在较高浓度的IgG抗体，后者将竞争结合固相抗原而使一部份IgM抗体不能结合到固相上。因此如用抗人IgM作为二抗，间接测定IgM抗体，必须先将标本用A蛋白或抗IgG抗体处理，以除去IgG的干扰。在临床检验中测定抗体IgM时多采用捕获包被法。先用抗人IgM抗体包被固相，以捕获血清标本中的IgM（其中包括针对抗原的特异性IgM抗体和非特异性的IgM）。然后加入抗原，此抗原仅与特异性IgM相结合。继而加酶标记针对抗原的特异性抗体。再与底物作用，呈色即与标本中的IgM成正相关。此法常用于病毒性感染的早期诊断(early diagnosis)。甲型肝炎病毒（HAV）抗体的检测模式见图2-7。类风湿因子（RF）同样能干扰捕获包被法测定IgM抗体，导致假阳性反应。因此中和IgG的间接法近来颇受青睐，用这类试剂检测抗CMV IgGM和抗弓形虫IgM抗体已获成功。2.2.7 ABS-ELISA法ABS为亲和素(avidin)生物素(biotin)系统(system)的略语。亲和素是一种糖蛋白，分子量60000，每个分子由4个能和生物素结合的亚基组成。生物素为小分子化合物，分子量244。用化学方法制成的衍生物素-羟基琥珀酰亚胺酯可与蛋白质和糖等多种类型的大小分子形成生物素标记产物，标记方法颇为简便。生物素与亲和素的结合具有很强的特异性，其亲和力较抗原抗体反应大得多，两者一经结合就极为稳定。由于一个亲和素可与4个生物素分子结合，因此如把ABS与ELISA法可分为酶标记亲和素-生物素（LAB）法和桥联亲和素-生物素（ABC）法两种类型。两者均以生物素标记的抗体（或抗原）代替原ELISA系统中的酶标抗体（抗原）。在LAB中，固相生物素先与不标记的亲和素反应，然后再加酶标记的生物素以进一步提高敏感度。在早期，亲和素从蛋清中提取，这种卵亲和素为碱性糖蛋白，与聚苯乙烯载体的吸附性很强，用于ELISA中可使本底增高。从链霉菌中提取的链霉亲和素则无此缺点，在ELISA应用中有替代前者的趋势。由于ABS-ELISA较普通ELISA多用了两种试剂，增加了操作步骤，在临床检验中ABS-ELISA应用不多。

ELISA试验以灵敏度较高、特异性较好的特点在临床上得到了广泛的应用，但操作中的各个环节对试验的检测效果影响较大，如不注意，有可能导致显色不全、花板等结果。我将操作中各个环节常出现问题的原因及解决办法总结于下，以期给同行带来一些启发，提高试验质量。选择试剂选择质量优良的检测试剂，严格按照试剂说明书进行操作，操作前将试剂在室温下平衡30-60分钟。加 样1.血清或血浆标本分离不好即进行加样； 2.手工操作中，加样板过多造成加样后放入孵箱前等待时间过长(特别是室内温度较高时)； 3.加完标本再加酶试剂时酶溅出孔外。1.标本为血清：最好将血液先自然存放1-2小时后，再用3000rmp离心15分钟；标本为血浆：必须使用含抗凝剂的血液标本收集管，采血后必须立即颠倒采血管混合5-10次，放置一段时间后，3000rpm离心15分钟；若在几天内检测，可放在2-8℃冰箱中，若要贮存，则置于-20℃的低温冰箱内。 2.加样后及时放入孵箱。 3.加酶试剂后用吸水纸在酶标板表面轻拭吸干。 4.如果采用AT或其他全自动加样，最好选择FAME或其他后处理仪器加酶试剂。 5.标本较多时，请分批操作。孵 育1.孵育时未贴封片或加盖，使标本或稀释液蒸发，吸附于孔壁，难于清洗彻底； 2.孵育时间人为延长，导致非特异性结合紧附于反应孔周围，难以清洗彻底。1.贴封片或加盖； 2.按说明步骤严格控制操作时间。洗 板1.采用手工洗板,孔与孔之间液体交叉。 2.采用半自动洗板机洗板时，洗液量不足，导致洗板不彻底；洗板针堵塞，抽吸不完全；洗板不畅，导致洗板效果差。 3.反应板过多造成洗板等待时间长。1.保证洗液注满各孔，洗板针畅通，洗完板后最好在吸水纸（选择干净、无或少尘的吸水材料）上轻轻拍干； 2.合理安排，或多用几台洗板机。显 色1. 显色剂配制后放置时间过长或使用过期显色剂； 2. 加显色剂时溅出孔外造成液体回流。1. 显色剂尽量在临用前配制，坚持不用过期显色剂，肉眼可见浅蓝色的TMB显色剂不用； 2. 加样时保持显色剂不外流； 3.A、B液应避免接触金属器械。终 止加终止液时产生较多气泡，导致假阳性增加。加终止液时应避免产生气泡。读 板读板时板底不清洁。应保证酶标板清洁。全过程1.整个操作过程中保证酶标板不接触次氯酸; 2.实现ELISA检测标准自动化，有效提高检测质量。在实际操作中，除了选择优良试剂外，必须严格按照操作步骤进行操作，同时作好室内质控、室间质评，以严谨的工作作风检测每一份标本，才能保证检测质量。现在国内已有相当数量的单位拥有全自动酶标仪，这对于实现ELISA标准化检测、提高检测质量起到了重要作用。

ELISA的全称enzyme-linked immuno sorbent assay，即酶联免疫吸附试验，是用来检测抗原或抗体的，因为原理是抗原抗体特异性结合，所以敏感性非常高，可以检测极微量的待测物，在医学上的应用很多，只要是蛋白质基本都可以检测（只要能合成对应的抗体就行了），而且是半定量的，可以显示滴度，比如检测血清里的HIV抗体、破伤风抗毒素、流感病毒的抗原等等，不限于病种

送楼主一个传送门：http://baike.baidu.com/view/3626382.htm?fromtitle=ELISA&fromid=5474779&type=syn#viewPageContent百科资料都有的希望对楼主有帮助！

1.储存.收到试剂盒后应尽快放入冷藏室内保存,保存温度为4℃-8℃.2.回温.试剂盒在使用前应于室温（22-27℃）下充分回温,回温时间为40-60分钟.回温过程中勿将酶标板的包装袋开封,回温结束后再将其从包装袋中取出使用.3.样品稀释.样品稀释应严格按照说明书要求的稀释倍数进行稀释.如稀释倍数过大,可采用多步法进行样品稀释.例如：试剂盒要求样品需进行500倍稀释,可吸取5μl样品加入245μl样品稀释液中,充分混匀后,再吸取10μl混合液加入90μl样品稀释液中即可.4.加样.稀释好的样品在加入酶标板进行孵育的过程中,尽可能缩短第一次加样和最后一次加样的时间间隔,样品加好后马上开始计时.5.洗板.样品孵育时间结束后,马上甩去孔内液体.如多块酶标板需同时洗涤,可先将板内液体甩去,倒扣于吸水纸上,再依次进行洗涤.使用洗板机洗涤时,建议洗涤次数增加一次.未用完的洗涤液可在4℃-8℃下密封保存1月.6.显色.底物药片可在显色前3-5分钟加入底物缓冲液中,混合直到完全溶解.本试剂最好是现用现配,未用完的底物溶液可在4℃-8℃下避光保存1周.7.终止及读数.提前打开酶标仪,加入终止液后立即读数.8.同批号试剂盒中的试剂,除酶标二抗及阳性对照外,其余均可通用.9.移液器的正确使用、保存、清洁、校对.10.实验室管理桌面保持整洁,插枪头戴手套等.

1971年瑞典学者Engvail和Perlmann,荷兰学者VanWeerman和Schuurs分别报道将免疫技术发展为检测体液中微量物质的固相免疫测定方法，即酶联免疫吸附测定法(enzyme-linkedimmunosorbentassay,ELISA)。ELISA已成为分析化学领域中的前沿课题，它是一种特殊的试剂分析方法，是在免疫酶技术(immunoenzymatictechniques)的基础上发展起来的一种新型的免疫测定技术。

一、做ELISA试剂盒标准曲线样品检测时有几个问题需要注意1、样品的浓度等指标是根据标准曲线计算出来的，所以首先要把做标准曲线看作是比做正式实验还要重要的一件事，否则后面的实验结果无从谈起。2、设置标准曲线样品的标准浓度范围要有一个比较大的跨度，并且要能涵盖你所要检测实验样品的浓度，即样品的浓度要在标准曲线浓度范围之内，包括上限和下限。而对于呈S型的标准曲线，尽量要使实验样品的浓度在中间坡度最陡段，即曲线几乎成直线的范围内。3、最好采用倍比稀释法配制标准曲线中的标准样品浓度，ELISA试剂盒这样就能够保证标准样品的浓度不会出现较大的偏离。4、检测标准样品时，应按浓度递增顺序进行，以减少高浓度对低浓度的影响，提高准确性。5、标准曲线的样品数一般为7个点，但至少要保证有5个点。6、做出的标准曲线相关系数因实验要求不同而有所变动，但一般来说，相关系数R至少要大于0.98，对于有些实验，至少要0.99甚至是0.999.二、选择什么方程去拟合在S曲线的低浓度部门可以用乘幂方程很好的拟合，中低浓度部门可以用直线方程，中间部门可用对数方程，而中后段可用四参数。免疫检测时尺度点（可以是倍比稀释的，也可以不是）浓度假如和相应的吸光度（OD）值假如能够呈现直线关系当然是最理想的了，这时可以通过EXELL等利便的获得拟合曲线，进而推算出样品的浓度值。 关于尺度曲线的拟合方式，直线、二次曲线、三次曲线、指数、对数等虽都可用于ELISA及其它生物学反应中的曲线拟合，但都只合用于曲线的一部门，有的合用于前半段，有的合用于后半段，有的合用于中间一段，而Logistic曲线则对曲线的全部都有较好的合用性。在一个长的区间内，Logistic应该都能拟合得较为理想。假如用来定量，S形曲线的中间段（较陡处）是比较好的，而两真个平坦部门计算误差会大，有时甚至会很大。用于免疫检测的所谓“尺度曲线"实在称为拟合曲线比较合适。目前国际上最流行的免疫检测拟合方式是“四参数逻辑拟合"，用这种拟合方式往往能够比较精确的反映浓度和吸光度的曲线关系，从而进一步比较准确的获得样品中待测物质的浓度值。但我们做免疫检测很少有时候能够有这么理想的情况，标品浓度和相应OD值往往是"S"型的曲线关系，这时就不能用直线拟合的方式了，而对拟合方法进行选择就是必要的了。枢纽在于你的尺度曲线做到了S形的哪一部门，你想检测的样品浓度在曲线的哪一部门。当然，假如用于定量，仍是在中间一段较好。但建模型是一回事，ELISA试剂盒而用它来定量是另一回事。这些方法固然没有一种方法可以通用，但也都可以用。但并不是说它就是万能的。事实上不仅是ELISA，其它良多生物学反应都是S形曲线，也都可以用Logistic曲线拟合。

夹心法夹心法常用于检测大分子抗原，一般之操作步骤为:将具有专一性之抗体固著（coating）于塑胶孔盘上，完成后洗去多余抗体加入待测检体，检体中若含有待测之抗原，则其会与塑胶孔盘上的抗体进行专一性键结洗去多余待测检体，加入另一种对抗原专一之一次抗体，与待测抗原进行键结洗去多余未键结一次抗体，加入带有酵素之二次抗体，与一次抗体键结洗去多余未键结二次抗体，加入酵素受质使酵素呈色，以肉眼或仪器读取呈色结果间接法间接法常用于检测抗体，一般之操作步骤为：将已知之抗原固著于塑胶孔盘上，完成后洗去多余之抗原。加入待测检体，检体中若含有待测之一次抗体，则其会与塑胶孔盘上的抗原进行专一性键结。洗去多余待测检体，加入带有酵素之二次抗体，与待测之一次抗体键结。洗去多余未键结二次抗体，加入酵素受质使酵素呈色，藉仪器（ELISA reader）测定塑胶盘中的吸光值（OD值），以评估有色终产物的含量即可测量待测抗体的含量。竞争法竞争法是一种较少用到的ELISA检测机制，一般用于检测小分子抗原，其操作步骤为：将具有专一性之抗体固著于塑胶孔盘上，完成后洗去多余抗体加入待测检体，使检体中的待测抗原与塑胶孔盘上的抗体进行专一性键结加入带有酵素之抗原，此抗原也可与塑胶孔盘上的抗体进行专一性键结，由于塑胶孔盘上固著的抗体数量有限，因此当检体中抗原的量越多，则带有酵素之抗原可键结的固著抗体就越少，亦即，两种抗原皆竞相与塑胶孔盘上抗体键结，即所谓竞争法之由来。洗去检体与带有酵素之抗原，加入酵素受质使酵素呈色，当检体中抗原量越多，代表塑胶孔盘内留下之带有酵素的抗原越少，显色也就越浅。当需要侦测无法获得两种以上单一性抗体的抗原，或是不易得到足够的纯化抗体以固著于孔盘上时，一般会考虑使用竞争法ELISA。

EILSA主要是基于抗原或抗体能吸附至固相载体的表面并保持其免疫活性，抗原或抗体与酶形成的酶结合物仍保持其免疫活性和酶催化活性的基本原理。在测定时，把受检标本（测定其中的抗体或抗原）和酶标抗原或抗体按不同的步骤与固相载体表面的抗原或抗体起反应，用洗涤的方法使固相载体上形成的抗原抗体复合物与其它物质分开，最后结合在固相载体上的酶量与标本中受检物质的量有一定的比例，加入酶反应的底物后，底物被酶催化变为有色产物，产物的量与标本中受检物质的量直接相关，根据颜色反应的深浅进行定性或定量分析。

酶联免疫反应吸附试验：是酶免疫测定技术中应用最广的技术。方法1三明治法（sandwich）方法2间接法（indirect）方法3竞争法（Competitive）

两者其实就是叫法不同而已。酶联免疫法(eia) 的翻译结果:enzyme linked immunosorbent assay (eia)缩写的另一种形式就是ELISA了

　ELISA的原理　　ELISA的基础是抗原或抗体的固相化及抗原或抗体的酶标记。结合在固相载体表面的抗原或抗体仍保持其免疫学活性，酶标记的抗原或抗体既保留其免疫学活性，又保留酶的活性。在测定时，受检标本（测定其中的抗体或抗原）与固相载体表面的抗原或抗体起反应。用洗涤的方法使固相载体上形成的抗原抗体复合物与液体中的其他物质分开。再加入酶标记的抗原或抗体，也通过反应而结合在固相载体上。此时固相上的酶量与标本中受检物质的量呈一定的比例。加入酶反应的底物后，底物被酶催化成为有色产物，产物的量与标本中受检物质的量直接相关，故可根据呈色的深浅进行定性或定量分析。由于酶的催化效率很高，间接地放大了免疫反应的结果，使测定方法达到很高的敏感度。　　2.　ELISA的类型　　ELISA可用于测定抗原，也可用于测定抗体。在这种测定方法中有三个必要的试剂：（1）固相的抗菌素原或抗体，即"免疫吸附剂"（immunosorbent）；（2）酶标记的抗原或抗体，称为"结合物"（conjugate）；（3）酶反应的底物。根据试剂的来源和标本的情况以及检测的具体条件，可设计出各种不同类型的检测方法。用于临床检验的ELISA主要有以下几种类型：①双抗体夹心法②双抗原夹心法③间接法

会使检测的特异性降低，即信噪比降低。

在ELISA实验结束后，我们得到的是经酶标仪得出的标准品以及样本的OD值。其中，标准品的浓度已知，我们可以利用标准品的OD值及其相对应的浓度，以OD值为横坐标，浓度为纵坐标，做一条标准曲线，并用公式表示出来。这样，我们把样本的OD值以X值代入，便可求出Y值，即样本浓度。 下面我们就一步步来实现这个目标： 1、首先，将数据整理好输入Excel，分别为经酶标仪读出标准品的OD值及其对应的浓度值。 2、拖动鼠标选取完成的数据区，并点击图表向导，在图表类型中选“XY散点图”，并选择子图表类型的“散点图”（第一个没有连线的） 3、点击“下一步”，出现如下图界面。如是输入是如本例横向列表的就不用更改，如果是纵向列表就改选“列”。 在做ELISA标准曲线，并通过此曲线求样本浓度时，因样本OD值是已知的，因此我们需要将OD值设为X。根据上图我们可以看到，横坐标X值为OD值，纵坐标Y为标准品浓度，这正是我们想要的。 有时需要我们手动选择X值，这时可以点击“系列”来更改 如果要对横坐标和纵坐标进行调换，我们可以就点击X值和Y值文本框右边的小图标 拖动鼠标选取正确的数据区域以调整X或Y。 5、调好之后，然后点击“下一步”出现图标选项界面， 6、点击“下一步”，在弹出的窗口再点击“完成”，现在一张带标准值的完整散点图就已经完成，如下图。 7、到了这一步，散点图便完成了。我们可能会问，曲线在哪里啊？其实很简单，先点击图上的标准值点（记住一定要点选上），然后单击右键，点击“添加趋势线”。弹出趋势线类型选择对话框，有线性，对数，多项等选项 8、在选择之前，我们先对上图做一下肉眼观察，如果那些点能连成一条直线，或接近直线，我们就选择“线性”，意思就是出来的趋势线是一条直线，相应的该直线的方程就是y=ax+b的形式。不过一般的ELISA都不会是所有的标准点呈完全的线性关系的，也就是说不是一条完全的直线，这时我们可以选择“多项式”，多项式后面还有“阶数”的选择，也就是说求出来的方程是几次方，我们一般选“2阶”，即出来的方程为y=ax2+bx+c. 选择完毕后，弹出以下窗口。 9、趋势线也就是标准曲线在此便显示出来了。接下来我们需要将公式显示出来，以根据样本的OD值计算其浓度值。在鼠标点选该线（一定要选中，选中后出现下图的样子即线由很多店标识出来），单击鼠标右键，选择趋势线格式。我们在弹出的窗口中选择“选项”选项卡，出现下图，我们选择显示公式，显示R方，即相关系数，这时公式与R方值便都显示出来了。 我们将样本的OD值作为X代入上述方程，求出来的便为浓度值了。 一般ELISA的标准曲线的相关系数要达到0.99以上，本例中为0.9999，非常不错的曲线，这样就保证了由该曲线及其方程求出的样本浓度具有很强的可信性。

　ELISA的原理　　ELISA的基础是抗原或抗体的固相化及抗原或抗体的酶标记。结合在固相载体表面的抗原或抗体仍保持其免疫学活性，酶标记的抗原或抗体既保留其免疫学活性，又保留酶的活性。在测定时，受检标本（测定其中的抗体或抗原）与固相载体表面的抗原或抗体起反应。用洗涤的方法使固相载体上形成的抗原抗体复合物与液体中的其他物质分开。再加入酶标记的抗原或抗体，也通过反应而结合在固相载体上。此时固相上的酶量与标本中受检物质的量呈一定的比例。加入酶反应的底物后，底物被酶催化成为有色产物，产物的量与标本中受检物质的量直接相关，故可根据呈色的深浅进行定性或定量分析。由于酶的催化效率很高，间接地放大了免疫反应的结果，使测定方法达到很高的敏感度。　　2.　ELISA的类型　　ELISA可用于测定抗原，也可用于测定抗体。在这种测定方法中有三个必要的试剂：（1）固相的抗菌素原或抗体，即"免疫吸附剂"（immunosorbent）；（2）酶标记的抗原或抗体，称为"结合物"（conjugate）；（3）酶反应的底物。根据试剂的来源和标本的情况以及检测的具体条件，可设计出各种不同类型的检测方法。用于临床检验的ELISA主要有以下几种类型：①双抗体夹心法②双抗原夹心法③间接法

ELISA的类型 ELISA可用于测定抗原,也可用于测定抗体.在这种测定方法中有三个必要的试剂：（1）固相的抗菌素原或抗体,即"免疫吸附剂"（immunosorbent）；（2）酶标记的抗原或抗体,称为"结合物"（conjugate）；（3）酶反应的底物.根据试剂的来源和标本的情况以及检测的具体条件,可设计出各种不同类型的检测方法.用于临床检验的ELISA主要有以下几种类型： 1.双抗体夹心法测抗原　　双抗体夹心法是检测抗原最常用的方法,操作步骤如下： 1） 将特异性抗体与固相载体联结,形成固相抗体.洗涤除去未结合的抗体及杂质. 2） 加受检标本,保温反应.标本中的抗原与固相抗体结合,形成固相抗原抗体复合物.洗涤除去其他未结合物质. 3） 加酶标抗体,保温反应.固相免疫复合物上的抗原与酶标抗体结合.彻底洗涤未结合的酶标抗体.此时固相载体上带有的酶量与标本中受检抗原的量相关. 4） 加底物显色.固相上的酶催化底物成为有色产物.通过比色,测知标本中抗原的量.在临床检验中,此法适用于检验各种蛋白质等大分子抗原,例如HBsAg、HBeAg、AFP、hCG等.只要获得针对受检抗原的异性抗体,就可用于包被固相载体和制备酶结合物而建立此法.如抗体的来源为抗血清,包被和酶标用的抗体最好分别取自不同种属的动物.如应用单克隆抗体,一般选择两个针对抗原上不同决定簇的单抗,分别用于包被固相载体和制备酶结合物.这种双位点夹心法具有很高的特异性,而且可以将受检标本和酶标抗体一起保温反应,作一步检测. 在一步法测定中,当标本中受检抗原的含量很高时,过量抗原分别和固相抗体及酶标抗体结合,而不再形成"夹心复合物".类同于沉淀反应中抗原过剩的后带现象,此时反应后显色的吸光值（位于抗原过剩带上）与标准曲线（位于抗体过剩带上）某一抗原浓度的吸光值相同,如按常法测读,所得结果将低于实际的含量,这种现象被称为钩状效应（hook effect）,因为标准曲线到达高峰后呈钩状弯落.钩状效应严重时,反应甚至可不显色而出现假阴性结果.因此在使用一步法试剂测定标本中含量可异常增高的物质（例如血清中HBsAg、AFP和尿液hCG等）时,应注意可测范围的最高值.用高亲和力的单克隆抗体制备此类试剂可削弱钩状效应. 假使在被测分子的不同位点上含有多个相同的决定簇,例如HBsAg的a决定簇,也可用针对此决定的同一单抗分别包被固相和制备酶结合物.但在HBsAg的检测中应注意亚型问题,HBsAg有adr、adw、ayr、ayw4个亚型,虽然每种亚型均有相同的a决定簇的反应性,这也是用单抗作夹心法应注意的问题. 双抗体夹心法测抗原的另一注意点是类风湿因子（RF）的干扰.RF是一种自身抗体,多为IgM型,能和多种动物IgG的Fc段结合.用作双抗体夹心法检测的血清标本中如含有RF,它可充当抗原成份,同时与固相抗体和酶标抗体结合,表现出假阳性反应.采用F（ab"）或Fab片段作酶结合物的试剂,由于去除了Fc段,从而消除RF的干扰.双抗体夹心法ELISA试剂是否受RF的影响,已被列为这类试剂的一项考核指标. 双抗体夹心法适用于测定二价或二价以上的大分子抗原,但不适用于测定半抗原及小分子单价抗原,因其不能形成两位点夹心. 2.双抗原夹心法测抗体反应模式与双抗体夹心法类似.用特异性抗原进行包被和制备酶结合物,以检测相应的抗体.与间接法测抗体的不同之处为以酶标抗原代替酶标抗抗体.此法中受检标本不需稀释,可直接用于测定,因此其敏感度相对高于间接法.乙肝标志物中抗HBs的检测常采用本法.本法关键在于酶标抗原的制备,应根据抗原结构的不同,寻找合适的标记方法. 3.间接法测抗体　　间接法是检测抗体常用的方法.其原理为利用酶标记的抗抗体（抗人免疫球蛋白抗体）以检测与固相抗原结合的受检抗体,故称为间接法.操作步骤如下： 1）将特异性抗原与固相载体联结,形成固相抗原.洗涤除去未结合的抗原及杂质. 2）加稀释的受检血清,保温反应.血清中的特异抗体与固相抗原结合,形成固相抗原抗体复合物.经洗涤后,固相载体上只留下特异性抗体,血清中的其他成份在洗涤过程中被洗去. 3）加酶标抗抗体.可用酶标抗人Ig以检测总抗体,但一般多用酶标抗人IgG检测IgG抗体.固相免疫复合物中的抗体与酶标抗体抗体结合,从而间接地标记上酶.洗涤后,固相载体上的酶量与标本中受检抗体的量正相关. 4）加底物显色本法主要用于对病原体抗体的检测而进行传染病的诊断.间接法的优点是只要变换包被抗原就可利用同一酶标抗抗体建立检测相应抗体的方法. 间接法成功的关键在于抗原的纯度.虽然有时用粗提抗原包被也能取得实际有效的结果,但应尽可能予以纯化,以提高试验的特异性.特别应注意除去能与一般健康人血清发生反应的杂质,例如以E.Coli为工程酶的重组抗原,如其中含有E.Coli成份,很可能与受过E.Coli感染者血甭中的抗E.Coli抗体发生反应.抗原中也不能含有与酶标抗人Ig反应的物质,例如来自人血浆或人体组织的抗原,如不将其中的Ig去除,试验中也发生假阳性反应.另外如抗原中含有无关蛋白,也会因竟争吸附而影响包被效果. 间接法中另一种干扰因素为正常血清中所含的高浓度的非特异性.病人血清中受检的特异性IgG只占总IgG中的一小部分.IgG的吸附性很强,非特异IgG可直接吸附到固相载体上,有时也可吸附到包被抗原的表面.因此在间接法中,抗原包被后一般用无关蛋白质（例如牛血清蛋白）再包被一次,以封闭（blocking）固相上的空余间隙.另外,在检测过程中标本须先行稀释（1:40~1:200）,以避免过高的阴性本底影响结果的判断. 4.竞争法测抗体当抗原材料中的干扰物质不易除去,或不易得到足够的纯化抗原时,可用此法检测特异性抗体.其原理为标本中的抗体和一定量的酶标抗体竞争与固相抗原结合.标本中抗体量越多,结合在固相上的酶标抗体愈少,因此阳性反应呈色浅于阴性反应.如抗原为高纯度的,可直接包被固相.如抗原中会有干扰物质,直接包被不易成功,可采用捕获包被法,即先包被与固相抗原相应的抗体,然后加入抗原,形成固相抗原.洗涤除去抗原中的杂质,然后再加标本和酶标抗体进行竞争结合反应.竞争法测抗体有多种模式,可将标本和酶标抗体与固相抗原竞争结合,抗HBc 　　ELISA一般采用此法.另一种模式为将标本与抗原一起加入到固相抗体中进行竞争结合,洗涤后再加入酶标抗体,与结合在固相上的抗原反应.抗HBe的检测一般采用此法. 5.竞争法测抗原小分子抗原或半抗原因缺乏可作夹心法的两个以上的位点,因此不能用双抗体夹心法进行测定,可以采用竞争法模式.其原理是标本中的抗原和一定量的酶标抗原竞争与固相抗体结合.标本中抗原量含量愈多,结合在固相上的酶标抗原愈少,最后的显色也愈浅.小分子激素、药物等ELISA测定多用此法. 6.捕获包被法测抗体 IgM抗体的检测用于传染病的早期诊断中.间接法ELISA一般仅适用于检测总抗体或IgG抗体.如用抗原包被的间接法直接测定IgM抗体,因标本中一般同时存在较高浓度的IgG抗体,后者将竞争结合固相抗原而使一部份IgM抗体不能结合到固相上.因此如用抗人IgM作为二抗,间接测定IgM抗体,必须先将标本用A蛋白或抗IgG抗体处理,以除去IgG的干扰.在临床检验中测定抗体IgM时多采用捕获包被法.先用抗人IgM抗体包被固相,以捕获血清标本中的IgM（其中包括针对抗原的特异性IgM抗体和非特异性的IgM）.然后加入抗原,此抗原仅与特异性IgM相结合.继而加酶标记针对抗原的特异性抗体.再与底物作用,呈色即与标本中的IgM成正相关.此法常用于病毒性感染的早期诊断.甲型肝炎病毒（HAV）抗体的检测模式. 类风湿因子（RF）同样能干扰捕获包被法测定IgM抗体,导致假阳性反应.因此中和IgG的间接法近来颇受青睐,用这类试剂检测抗CMV IgGM和抗弓形虫IgM抗体已获成功. 7.ABS-ELISA法 ABS为亲和素(avidin)生物素(biotin)系统(system)的略语.亲和素是一种糖蛋白,分子量60000,每个分子由4个能和生物素结合的亚基组成.生物素为小分子化合物,分子量244.用化学方法制成的衍生物素-羟基琥珀酰亚胺酯可与蛋白质和糖等多种类型的大小分子形成生物素标记产物,标记方法颇为简便.生物素与亲和素的结合具有很强的特异性,其亲和力较抗原抗体反应大得多,两者一经结合就极为稳定.由于一个亲和素可与4个生物素分子结合,因此如把ABS与ELISA法可分为酶标记亲和素-生物素（LAB）法和桥联亲和素-生物素（ABC）法两种类型.两者均以生物素标记的抗体（或抗原）代替原ELISA系统中的酶标抗体（抗原）.在LAB中,固相生物素先与不标记的亲和素反应,然后再加酶标记的生物素以进一步提高敏感度.在早期,亲和素从蛋清中提取,这种卵亲和素为碱性糖蛋白,与聚苯乙烯载体的吸附性很强,用于ELISA中可使本底增高.从链霉菌中提取的链霉亲和素则无此缺点,在ELISA应用中有替代前者的趋势.由于ABS-ELISA较普通ELISA多用了两种试剂,增加了操作步骤,在临床检验中ABS-ELISA应用不多.

ELISA 英[u026au02c8lau026azu0259, -su0259] 美[u026au02c8lau026azu0259, -su0259] http://fanyi.baidu.com/?aldtype=85#en/zh/ELISA 楼主自己听听看

ELISA实验原理是酶分子与抗体或抗抗体分子共价结合，此种结合不会改变抗体的免疫学特性，也不影响酶的生物学活性。酶标记抗体可与吸附在固相载体上的抗原或抗体发生特异性结合。滴加底物溶液后，底物可在酶作用下使其所含的供氢体由无色的还原型变成有色氧化型，出现颜色反应。因此，可通过底物的颜色反应来判定有无相应的免疫反应，颜色反应的深浅与标本中相应抗体或抗原的量呈正比。此种显色反应可通过ELISA检测仪进行定量测定，这样就将酶化学反应的敏感性和抗原抗体反应的特异性结合起来，使ELISA方法成为一种既特异又敏感的检测方法。ELISA实验步骤及注意事项:1、固相载体选择聚苯乙烯：具有较强的吸附蛋白质的性能，抗体或抗原吸附其上后仍保留原来的免疫学活性。聚氯乙烯：聚氯乙烯对蛋白质的吸附性能比聚苯乙烯高，但空白值也略高。良好的ELISA板应该是吸附性能好，空白值低，孔底透明度高，各板之间、同一板各孔之间性能相近。2、包被①板孔的选择：ELISA级别的微孔板。②抗体选择：选择支持ELISA实验的抗体，根据推荐浓度稀释或摸索最适浓度。③包被缓冲液：pH9.6碳酸盐缓冲液或pH7.2磷酸盐缓冲液。④包被温度：2-8 ℃过夜包被或室温（37℃）包被2h。⑤包被浓度：包被浓度随载体和包被物的性质可有很大的变化。一般包被浓度为10ug/ml-20ug/ml。3、封闭①包被之后一定要立即封闭（封闭非特异性抗体）。②选择效果较好的封闭剂（细胞培养级BSA、Tween等）。4、加样及孵育①加样时应将所加物加在ELISA板孔的底部，避免加在孔壁上部，并注意不可溅出，不可产生气泡。②孵育的时间及温度参考试剂盒说明书。如有条件，建议加样孵育时使用shaker震荡仪，推荐轨道直径3mm，转速在500±50rpm。③检测抗体、酶标物的稀释比例、孵育温度、孵育时间严格根据说明书提示。④洗板对于ELISA来说，是极其关键的一步，保证ELISA的特异性。⑤封板膜一定要一次一换，切勿二次甚至多次使用，以防交叉污染。5、显色和终止①使用前需检查，底物在加入96孔板之前应为无色透明。②显色底物需现配现用，避免污染。③孵育时间，说明书上为参考范围，具体需要根据实验条件自行摸索。可参考标曲浓度最大孔的颜色，变为蓝色较明显时即可。

法还是双抗原夹

酶链免疫吸附化验是平板化验法用于探测和确定某些物质的数量，比如肽，蛋白质，抗体，和荷尔蒙。另有名称，比如 酶免疫化验（EIA）指的是同一种试验。 扩展资料 　　酶联免疫检测法（PSTVd除外）全过程包括病毒抗原的分离与纯化、病毒抗血清的制备和抗血清反应3个环节。抗血清反应实验步骤包括：抗原吸附在固相载体上，加待测抗体，再加相应酶标记抗体，生成抗原一待测抗体一酶标记抗体的复合物，再与该酶的底物反应生成有色产物。借助分光光度计的光吸收计算抗体的量。待测抗体的"量与有色产物成正比。同理也可包被抗体，测定抗原含量。ELISA常用的有直接法测定抗原、竞争法测定抗原、双抗体夹心法测定抗原等方法。 　　双抗体夹心酶联免疫检测方法是将抗体吸附于固相表面，加抗原，形成抗原一抗体复合物，再加酶标抗体，最后加底物，根据底物的降解量确定抗原有无及数量。

请参考以下链接：http://baike.baidu.com/view/3626382.htm

检测原理: 本实验采用双抗体夹心ELISA法。抗人IL-4单抗包被于酶标板上，操作步骤: 1．从已平衡至室温的密封袋中取出试验所需板条，未用的板条和

二抗广义上是指专门和一抗进行特异性反应和结合的抗体，在免疫学反应中，经常需要针对试验选择不同的二抗，艾美捷能为您的科研工作提供最适合和最全面的二抗产品。检测任何目的靶蛋白都有不止一种抗体可供选择，同时在后继试验中也会有不同的检测方案，因此在选择二抗的时候要综合考虑一抗的类型及后继检测方案的要求，一般来说，选择合适的二抗需要从下面几个方面考虑：【一抗的种属来源】二抗应选用与使用的一抗相同的物种来源，例如：如果你的一抗是小鼠源的单克隆抗体，二抗则选抗小鼠的二抗（山羊抗小鼠或者兔抗小鼠等均可）；如果一抗是从兔血清里制备的兔源多克隆抗体，则相应的二抗需要选择抗兔的二抗。即根据一抗的物种来源选择相应的抗该物种的二抗。艾美捷能为您提供最全的抗不同种属的原装进口二抗，包括抗小鼠、大鼠、兔、山羊、绵羊、人、豚鼠、猪、马、牛、鸡、鸭等二抗。【一抗的类别亚型】二抗需与一抗的类别或亚类相匹配。这通常是针对单克隆抗体而言。多克隆抗体主要是IgG类免疫球蛋白，因此相应的二抗就是抗IgG抗体。其中单克隆抗体的类别及亚类通常会在产品说明书中都会有描述，如果你的一抗是小鼠IgM，那么相应的二抗就应当是抗小鼠IgM。如果单克隆一抗是小鼠IgG的某一亚类（IgG1，IgG2a，IgG2b，IgG3），那么几乎所有的抗小鼠IgG都可以与之结合，或者你也可以选择专门针对这一亚类的二抗，例如，如果你的一抗是小鼠IgG1，那么你可以选择抗IgG1的二抗，此种抗体在双标记实验中尤其适合。在不清楚一抗为何种类/亚类的情况下，可以选用抗相应物种IgG。艾美捷能为您提供通用的IgG（H+L）二抗，或者特异性只结合IgM的Anti IgM （mu） Antibody、IgA的Anti IgA （alpha-Antibody、IgE的Anti IgE （epsilon） Antibody等。【二抗的种属来源】一般来说，不同的种属来源与二抗的质量没有必然的联系，来源于山羊的二抗与来源于驴的二抗在一般的实验里没有太多的差别。然而在一些特殊的实验里， 如双标实验里，如果其中一个一抗是山羊来源的，一个是小鼠来源的，则相应的二抗分别要抗山羊和抗小鼠的二抗，这时候，二抗就不能选择山羊或者小鼠来源的。 艾美捷有相应的驴来源的二抗，非常适合做类似双标的免疫实验。【二抗的耦联标记】一般来讲，耦联到二抗上的探针主要有酶（辣根过氧化酶HRP和碱性磷酸酶AP或其衍生物APAAP，PAP），荧光基团（FITC、 Rhodamine、Texas Red、PE、Rhodamine、Dylight等）、生物素、金颗粒。选用哪种探针的二抗主要取决于具体的实验。对于Western Blot和ELISA，最常用的二抗是酶标二抗；而细胞或组织标记实验（细胞免疫化学，组织免疫化学，流式细胞术）中通常使用荧光基团标记的二抗，免疫组化中也可以使用辣根过氧化酶或碱性磷酸酶标记的二抗。如果想要更大程度的放大检测信号，可以使用Biotin/Avidin检测系统。在一些荧光检测方案中，则需要选择不同的荧光标记；而金颗粒标记的二抗则更多的应用于免疫电镜中。艾美捷能为您提供上述所有不同类型标记的二抗。【是否经过血清吸附过的二抗】为减少二抗与样本的非特异性结合，艾美捷还能为您提供不同血清吸附的原装进口二抗。如您的检测样本是人源的蛋白，一抗是小鼠源的单克隆抗体，二抗就需要选择抗小鼠源的二抗，如果您对实验的特异性要求很高，那我们向您推荐经过人血清吸附过的抗小鼠源的二抗，这种二抗由于经过人血清吸附而排除了与人源组织（如IgG等）可能发生非特异性反应的IgG，因此将非特异性结合降低到最低。【Anti-IgG还是Anti-IgG, F（ab"）2 fragment】在一些如胸腺、脾脏、血液的组织以及造血细胞、淋巴细胞、B细胞等细胞内，通常含有较多的Fc受体，这时候选择二抗时最好选择Anti-IgG, F（ab"）2 fragment这样的特异性二抗，这样就消除了由于Fc部分与IgG的非特异性结合。常规的Anti-IgG （H+L）二抗与IgG的重链和轻链都有结合反应，即与IgG的Fc的F（ab"）2片段都能结合；由于IgG、IgM、IgA都有保守的轻链结构域，因此Anti-IgG （H+L）与它们都有交叉反应。而Anti-IgG, F（ab"）2 fragment经过了IgG Fc片段的吸附，因此只与IgG的F（ab"）2片段结合，然而IgG、IgM、IgA都有保守的轻链结构域，因此与它们也有交叉反应。【Anti-IgG（H+L）、Anti-IgG （gamma）、Anti-IgM （mu）、Anti-IgA （alpha）】在一些特殊的实验里，只需要检测样本里的IgG或者IgM，常规的Anti-IgG （H+L）由于与IgG的重链和轻链都有结合反应，而IgG、IgM、IgA都有保守的轻链结构域，因此不能特异性的检测某种类型的IgG或者IgM。这时候就需要针对特殊类型Ig分子的二抗。艾美捷备有全面的二抗现货产品并能提供最完整的二抗产品线。

WB-Re是Western Blot (Recombinant protein)WB-Ce是Western Blot (Cell lysate)IHC是Immunohistochemistry

酶联免疫吸附实验(ELISA) 即将已知的抗原或抗体吸附在固相载体表面，使酶标记的抗原抗体反应在固相表面进行的技术。该技术可用于检测大分子抗原和特异性抗体等，具有快速、灵敏、简便、载体易于标准化等优点。基本原理它采用抗原与抗体的特异反应将待测物与酶连接，然后通过酶与底物产生颜色反应，用于定量测定。测定的对象可以是抗体也可以是抗原。在这种测定方法中有3种必要的试剂：①固相的抗原或抗体（免疫吸附剂） ②酶标记的抗原或抗体（标记物）③酶作用的底物（显色剂）测量时，抗原（抗体）先结合在固相载体上，但仍保留其免疫活性，然后加一种抗体（抗原）与酶结合成的偶联物（标记物），此偶联物仍保留其原免疫活性与酶活性，当偶联物与固相载体上的抗原（抗体）反应结合后，再加上酶的相应底物，即起催化水解或氧化还原反应而呈颜色。其所生成的颜色深浅与欲测的抗原（抗体）含量成正比。 这种有色产物可用肉眼、光学显微镜、电子显微镜观察，也可以用分光光度计（酶标仪）加以测定。其方法简单，方便迅速，特异性强。elisa的临床意义，要根据具体的检测对象来看，主要是检测样本的含量以及定性，仅供参考

ELISA实验原理是酶分子与抗体或抗抗体分子共价结合，此种结合不会改变抗体的免疫学特性，也不影响酶的生物学活性。酶标记抗体可与吸附在固相载体上的抗原或抗体发生特异性结合。滴加底物溶液后，底物可在酶作用下使其所含的供氢体由无色的还原型变成有色氧化型，出现颜色反应。因此，可通过底物的颜色反应来判定有无相应的免疫反应，颜色反应的深浅与标本中相应抗体或抗原的量呈正比。此种显色反应可通过ELISA检测仪进行定量测定，这样就将酶化学反应的敏感性和抗原抗体反应的特异性结合起来，使ELISA方法成为一种既特异又敏感的检测方法。ELISA实验步骤及注意事项:1、固相载体选择聚苯乙烯：具有较强的吸附蛋白质的性能，抗体或抗原吸附其上后仍保留原来的免疫学活性。聚氯乙烯：聚氯乙烯对蛋白质的吸附性能比聚苯乙烯高，但空白值也略高。良好的ELISA板应该是吸附性能好，空白值低，孔底透明度高，各板之间、同一板各孔之间性能相近。2、包被①板孔的选择：ELISA级别的微孔板。②抗体选择：选择支持ELISA实验的抗体，根据推荐浓度稀释或摸索最适浓度。③包被缓冲液：pH9.6碳酸盐缓冲液或pH7.2磷酸盐缓冲液。④包被温度：2-8 ℃过夜包被或室温（37℃）包被2h。⑤包被浓度：包被浓度随载体和包被物的性质可有很大的变化。一般包被浓度为10ug/ml-20ug/ml。3、封闭①包被之后一定要立即封闭（封闭非特异性抗体）。②选择效果较好的封闭剂（细胞培养级BSA、Tween等）。4、加样及孵育①加样时应将所加物加在ELISA板孔的底部，避免加在孔壁上部，并注意不可溅出，不可产生气泡。②孵育的时间及温度参考试剂盒说明书。如有条件，建议加样孵育时使用shaker震荡仪，推荐轨道直径3mm，转速在500±50rpm。③检测抗体、酶标物的稀释比例、孵育温度、孵育时间严格根据说明书提示。④洗板对于ELISA来说，是极其关键的一步，保证ELISA的特异性。⑤封板膜一定要一次一换，切勿二次甚至多次使用，以防交叉污染。5、显色和终止①使用前需检查，底物在加入96孔板之前应为无色透明。②显色底物需现配现用，避免污染。③孵育时间，说明书上为参考范围，具体需要根据实验条件自行摸索。可参考标曲浓度最大孔的颜色，变为蓝色较明显时即可。

直接竞争ELISA和间接竞争ELISA区别　　1、直接竞争，标记抗原，与检测样品中的抗原竞争抗体。　　2、间接竞争，标记抗体，固相抗原与液相抗原（样品）竞争标记抗体。　　3、定义　　间接竞争法的模型：包被抗原，用HRP-抗体与样本一起加入。样本中的Ag与Solid-Ag竞争HRP-Ab，固相吸附的HRP-Ab与样本中的Ag浓度成反比。　　直接竞争法的模型：包被抗体，用HRP-抗原与样本一起加入。样本中的Ag与HRP-Ag竞争Solid-Ab，固相吸附的HRP-Ag与样本中的Ag浓度成反比。　　4、竞争法的理论基础：是限量抗体。只有在限量抗体基础上，两种抗原才会形成竞争关系。　　5、、间接竞争法具备较高灵敏度原因。　　直接竞争法里，标记抗原与待测抗原均是液相，与抗体的结合机会是一样的，例如有1份标记抗原与1份待测抗原竞争1份抗体，那么有50%的标记抗原能与抗体结合，所以标记抗原的相对结合率为50%。间接法里，固相抗原与抗体的接触面积较小，固相抗原与待测抗原的结合抗体机会是不平等的，接近顺序饱和法，即只有与待测抗原结合剩余的抗体才会与固相抗原结合，同样有1份固相抗原与1份待测抗原竞争1份抗体时，基本上抗体会被待测抗原中和掉，与固相抗原结合的抗体非常少。固相抗原的相对结合率为0%。因此，间接法的抑制曲线斜率会大于竞争法。　　因为抑制率越大则斜率越大，从而灵敏度越大。（假设零管变异5%，以两倍SD为灵敏度限，则为90%相对结合率，则间接法可以在较低的待测抗原浓度达到这一相对结合率，因此灵敏度要高。）　　在进行系统放大时，间接法一般可以使用酶标二抗。因为二抗可以针对抗体的多个部位，所以存在放大效应，从而能提高间接法的灵敏度。直接法一般难以进行放大，常用的有生物 素化抗原与酶标亲和素，但模式上似乎不存在放大效应。　　6、间接法的高灵敏度难以实现的原因：　　双抗体 夹心的免放（IRMA）模式刚出现时，也被模型证明灵敏度优于竞争法的放免（RIA），原因也是较大的斜率，但是IRMA的高灵敏度一直到单抗发展后才得以实现。

　　在国内生物研究蓬勃发展的现在，假冒无视科学研究的严谨性和严肃性，让广大的科研工作者被动造假，严重破坏了科研氛围，扰乱了市场秩序，联科生物整理了一些鉴别这些假冒伪劣ELISA试剂盒的方法，供广大的科研工作者和用户参考，让希望对广大科研工作者有所帮助。　　购买前　　【方法一】向厂家索要或从其网站下载说明书，查找ELISA的性能指标：准确度(加标回收率、稀释线性)、精密度(板内差、板间差)、灵敏度、样本值、特异性、校准。一般来说，除了校准不一定每份说明书都有外，其余的指标一般都需要罗列(部分样本需要高倍稀释的，可能没有准确度这个指标)。如果无法提供这些性能指标，则说明该试剂盒可能是假货，或者试剂盒开发过程不够科学严谨，有可能无法检测出真实的样本值。　　【方法二】对于夸大ELISA涵盖的指标的范围，尤其是不常见的种属，可先去国际知名ELISA试剂盒供应商(如eBioscience、R&D)网站上搜索，如果搜索不到该种属的ELISA试剂盒，则有可能是假货，需要小心谨慎。　　【方法三】将“厂家名”、“ELISA”和“假货”等关键词在网上搜索，有的网站或论坛如丁香园等会有打假曝光台。　　购买后　　【方法一】检查说明书、包装盒的质量，通常假冒伪劣产品为了降低成本，印刷的质量会比较差。其次仔细阅读说明书，如上所示查找ELISA的性能指标。　　【方法二】　　Ⅰ.利用干扰实验即可鉴别ELISA试剂盒是否检测目的抗原，方法如下：　　(1)将针对试剂盒特异性的重组蛋白抗原和试剂盒中的检测抗体配置成antibody:antigen=1:2的混合孵育液　　(2)37℃孵育15分钟后，代替原试剂盒中的检测抗体　　(3)后续操作按照试剂盒说明书进行，加检测抗体、酶复合物和底物　　(4)实验完毕后，检测其标准曲线，如果标准曲线良好则说明加入的目的抗原和试剂盒抗体不匹配，试剂盒检测抗体针对的是非目的抗原，该试剂盒为伪劣假造ELISA试剂盒。　　(5)如果标准曲线无线性，则说明试剂盒检测抗体已被目的抗原中和或干扰，影响了其实际试剂盒抗体的检测作用，则该试剂盒检测抗体针对的是目的抗原。　　Ⅱ.如果购买了两盒同一公司的ELISA试剂盒A和B，利用交叉实验即可鉴别ELISA试剂盒是否造假，方法如下：　　(1)取出购买的试剂盒A的包被板两条。　　(2)一条包被板加试剂盒A的标准品做标准曲线。　　(3)另一条包被板加试剂盒B的标准品做标准曲线。　　(4)后续操作按照试剂盒说明书进行，加检测抗体、酶复合物和底物。　　(5)实验完毕后，检测两条标准曲线，如果两条标准曲线一致则说明两个ELISA试剂盒检测的同一个指标而非A和B，即该试剂盒为伪劣假造ELISA试剂盒。　　(6)如果两条标准曲线不一致则说明两个ELISA试剂盒检测的不同指标，试剂盒A只能检测A，不能检测B，即该试剂盒为正常的ELISA试剂盒。

　　ELISA实验通用规则　　1、要保证移液枪的准确性,误差不能超过2%.可用水和电子天平进行确定.但最好有专业人员进行矫正.　　2、要配备20ul、50ul、100ul、1000ul和排枪各一支.吸取不同的液体后,要更换枪头.即使是吸取标准品时.　　3、要在实验前1小时将试剂盒从冰箱中取出,使各种试剂都恢复到室温,以使结果更稳定.　　4、实验时,要使底物避光保存.　　5、用枪吸取液体时速度不能太快,以免产生气泡而使吸取量不准确.　　6、吸取液体时,要用量程和需要量接近的枪去吸,减少误差.　　7、将液体加到酶标孔中时,避免枪头和孔内液体接触,可使枪头上的液滴和孔壁接触,液滴会自然流下去.　　8、液体全部加完后,可将酶标板在桌子上平行轻轻晃动30秒,混匀液体.也可以用酶标仪的晃动功能.　　9、温浴时,要用不干胶或胶带纸封好酶标板,防止水分的蒸发.　　10、洗板时,每次洗液加入后,应静置1分钟,使清洗更加彻底.没有洗板机时,倒去液体后,要将酶标板在报纸或毛纸上用力拍干.　　11、洗液不够时,可用蒸馏水自行配制PH7.4,0.02M的磷酸缓冲液,加入0.1%的吐温20作为洗液.加入1/1000的叠氮钠后可长期保存.　　12、底物是光敏感的,要在临用前现配.　　13、检测前,要打开酶标仪,使之稳定10分钟以上.　　14、底物有一定的毒性,终止液对皮肤有腐蚀性,应尽量避免接触.　　15、待检样品要澄清,否则会影响结果.　　16、温浴时间应遵守试剂盒规定.　　17、应尽量做双孔实验,这样才能保证数据的准确性.　　18、对结果有疑问的样品要用其它方法进行确证.　　二、下面所提到的是针对我公司的ELISA产品会出现的问题及解决办法　　1.加入反应终止液后,没有吸光值或吸光值偏低.　　a.原因：未加入酶标记物.　　解决办法：请按照操作步骤进行.　　b.原因：试剂盒内容物未能充分回.　　解决办法：确定试剂盒内容物回温后再进行操作.　　c.原因：室温太低.　　解决办法：若室温太低(25℃).　　解决办法：若无法降低室内温度,请适当缩短步骤4的温育时间.　　b.原因：底物溶液受到污染.　　解决办法：若发现底物溶液已呈现淡蓝色,请不要再使用.　　c.原因：反应时间超过太久.　　解决办法：请控制反应时间.　　d.原因：酶标记物污染了盒内其它试剂.　　解决办法：使用过的吸头应立即丢弃,可避免试剂间相互污染,凡是试剂(特别是酶标记物与底物溶液)接触过的容器应立即清洗干净.(先以漂白水浸泡,再以清水冲洗干净,可确保无残留)　　4.阴性标准品的吸光值低于阳性标准品的吸光值.　　a.原因：微孔中的试剂未能充分混合.　　解决办法：试剂加完后,需轻敲盘四周使其充分混合均匀.　　b.原因：洗板过程发生问题(同2-f.).　　解决办法：请参考2-f.　　c.原因：添加样品或酶标记物的量不一致.　　解决办法：请参考2-d.