途游斗地主

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途游斗地主Bio-techne|HUAXUEFAGUANGFA:BUSHISUOYOUDE ELISA DOUYAOCE OD ZHIYO

途游斗地主DAJIAHAO,WOSHIXIAOCAIJIZHANGSAN,WANWANMEIXIANGDAOSHIXIONGRANGWOJIJIDULIZUO ELISA LE。ZHEIGEWODONG,PINGSHIGENZHESHIXIONGQINXUEKULIAN,PAIBANJIAYANGWOSHIYOUMOYOUYANG。

DAIWOZUOSHOUYIGE OD450 nm,YOUSHOUYIGE 540,NANI?HUAXUEFAGUANGFA ELISA SHUOMINGSHUSHANGZENMEMEIYOU OD ZHIDEYAOQIUA?「SHAONIANLANG,BUSHISUOYOUDE ELISA DOUYAOCE OD ZHIYO。」ANZHONGGUANCHAYIJIUDESHIXIONGDEYIYANGYANGDIPIAOGUO。

下面是小博士上课时间

什么是化学发光法 ELISA?

途游斗地主MEILIANMIANYIXIFUCEDINGFA(ELISA)。SHOUXIANJIANGMEIZHIJIEBIAOJIZAIKANGYUANHUOKANGTISHANG,ZAIJINGGUOKANGYUANYUKANGTIJIEHEXINGCHENGKANGYUANKANGTIMIANYIFUHEWU。TONGGUOBUTONGDIWUYUMEIFANYINGHOUCHANSHENGDETEDINGCHANWUDETEXING,DADAODINGLIANGJIANCETEDINGFENZIDEMUDE。GENJUDIWUJIXIANGYINGCHANWUDEBUTONG,ELISA KEFENWEIBISEFA(Colorimetric)YUHUAXUEFAGUANGFA(Chemiluminescent)LIANGLEI。

途游斗地主HUAXUEFAGUANGFAMEILIANMIANYIFENXIXITONG(Chemiluminescent ELISA)SHIZAIMIANYIFANYINGJIESHUHOU,JIARUMEIDEFAGUANGDIWU,CHANSHENGCHUYUJIFATAIDEZHONGJIANTI。ZHIHOUZHONGJIANTIHUIFASHEGUANGZISHIFANGNENGLIANGYIHUIDAOWENDINGDEJITAI,CIFAGUANGQIANGDUKEYOUGUANGDUJI(Luminometer)JINXINGJIANCE,BINGLIYONGBIAOZHUNQUXIANJISUANCHUBEICEWUDEHANLIANG。XIANGBIYUBISEFA,HUAXUEFAGUANGFAJUYOUGENGGAODELINGMINDUHEGENGKUANDEJIANCEFANWEI。

途游斗地主CHANGJIANDEHUAXUEFAGUANGZUHESHILUMINUO(3-aminophthal hydrazide, Luminol)DIWUYULAGENGUOYANGHUAWUMEIFANYING。LUMINUOYU 1928 NIANSHOUCIBEIHECHENGWEIHUAXUEFAGUANGDIWU,GAIDIWUDEFAGUANGFANYINGXUYAOKEKEDETIAOJIAN,FAGUANGANDANQIECHIXUSHIJIANDUAN。

途游斗地主ZHIHOUYANJIURENYUANFAXIAN,JIARUZENGQIANGJIDEZENGQIANGXINGLUMINUOFANYING(TU 1)NENGGOUZHANGSHIJIANCHANSHENGMINGLIANGQIEWENDINGDEFAGUANGXIAOGUO,CONGERSHIDEQIGUANGFANYINGYONGYUMIANYIFENXI,MUQIAN R&D Systems® DE QuantiGlo® XILIEJI Novus Biologicals® DE Chemiluminescent ELISA JUNCAIYONGCIYUANLI。

图 1:增强 luminol/peroxide 底物用于化学发光检测

QuantiGlo® ELISA Kit 的基本检测步骤

第一步:途游斗地主向包被有捕获抗体的微孔板中加入样品或标准品,孵育使分析物与包被的抗体结合,将未结合的其他分子清洗掉。

第二步:加入 HRP 偶联的抗体(检测抗体)并与一抗捕获的分析物结合。未结合的检测抗体被冲洗掉。

第三步:途游斗地主加入增强的鲁米诺底物,使之与 HRP 反应发光,发光强度与样品中存在的分析物数量成正比。利用微孔板光度计测量发光强度。

QuantiGlo® 试剂盒检测参数设置及应用举例

RU QuantiGlo® SHIJIHESHUOMINGSHUSUOLIE,GUANGDUJISHEDINGKEYISHIYONGYIXIACANSHUHUOTONGDENGCANSHU:1.0 FENZHONGZHIHOUSHIJIAN、JIANCESHIJIAN 0.5 MIAO/KONG、QIUHEMOSHI、ZIDONGZENGYIHUOTONGDENG。

XUYAOZHUYIDESHI:

途游斗地主XIANGDUIGUANGDANWEI(Relative light units,RLUs)SHUZHIYOUBUTONGGUANGDUJIJIANCEKENENGBUTONG,XUYAOANZHAOYIQIZHIZAOSHANGDEJIANYIDIAOZHENGSHEZHI。

途游斗地主XIANGDUIGUANGDANWEIDESHUZHIKENENGZAI 15 FENZHONGDEYUEDUCHUANGKOUQIJIANNEIFASHENGBIANHUA,YOUYUXUYAOCANKAOBIAOZHUNQUXIANSHUZHIHUANSUAN,SUOYIZHEIBINGBUHUIYINGXIANGJIEGUO。

 

应用举例 1:Human VEGF QuantiGlo ELISA Kit(货号 QVE00B)

途游斗地主SHIYONG MOLECULAR DEVICE PINPAIDE SpectraMax® L WEIKONGBANHUAXUEFAGUANGMEIBIAOYIJINXINGJIANCE(SpectraMax® M5/M5e HE FlexStation® 3 DUOGONGNENGMEIBIAOYIDEHUAXUEFAGUANGJIANCEMOSHIYESHIYONG)。SHUJUDESHOUJIHEJISUANTUIJIANYOU SoftMax® Pro RUANJIANWANCHENG,QIYUSHEDEMOBANJIDADIJIANHUALEXIANGYINGLIUCHENG。

途游斗地主SHIYANBUZHOUJIYIQICANSHUSHEZHI:

1. MEIKONGJIARU 150 μL SHIYANXISHIYE RD1-8;

2. ZAIWEIKONGBANZHONGAN 3 FUKONGXINGSHIJIARU 50 μL BIAOZHUNPINHUOKONGBAIDUIZHAO(Calibrator XISHIYE)。SHIWENQINGDUZHENDANG(TUIJIAN  500 ± 50 rpm)WEIKONGBAN 2 XIAOSHI;

3. FUYUJIESHUHOUSHIYONGPAIQIANGXIGANKONGNEIFANYINGYE,BINGYONG 400 μL XIDIYEQINGXI 4 BIAN。ZUIHOUXIGANSHENGYUXIDIYEBINGZAIGANJINGDEXISHUIZHISHANGPAIGAN;

4. MEIKONGJIARU 200 μL VEGF FUHEWU,TIESHANGXINDEZHANHEDAI。SHIWENQINGDUZHENDANG (TUIJIAN 500± 50 rpm) WEIKONGBAN 3 XIAOSHI;

途游斗地主5. ANZHAOBUZHOU 3 JINXINGQINGXIBUZHOU;

6. MEIKONGJIARU 100 μL Glo GONGZUOYE,SHIWENBIGUANGFUYU 5 ZHI 20 FENZHONG;

7. YONG SpectraMax® L WEIKONGBANHUAXUEFAGUANGMEIBIAOYICEDING RLU ZHI。XIANGGUANCANSHURUXIA:1 MIAOJIANCESHIJIAN,PMT ZIDONGHE 470 nm MUBIAOXIAOZHUNBOZHANG。

途游斗地主JIEGUO:XIANSHIYOU SpectraMax® L WEIKONGBANHUAXUEFAGUANGMEIBIAOYIANZHAOSHANGSHUDECANSHUSUODEDE VEGF BIAOZHUNQUXIAN, QIDONGTAIFANWEICHAOGUO 4 GESHULIANGJI,JISUANSUODEDEJIANCEXIAN,YEJIUSHIKONGBAIDUIZHAOXINHAOBIAOZHUNCHA 3 BEIZHIDEXIANGYINGLIANGWEI 1.5 pg/mL,YUGAISHIJIHEPINGJUNJIANCEXIAXIANWEI 3.3 pg/mL XIANGFU。

图 2,20,000-1.3 pg/mL 的标准品检测曲线图。实验由 SpectraMax® L 微孔板化学发光酶标仪按照 1 秒检测时间,PMT 自动和 470 nm 目标校准波长的参数进行读取。标准曲线由 SoftMax® Pro 软件绘制。

应用举例 2:Human IL-6 QuantiGlo ELISA Kit(货号 Q6000) 

途游斗地主SHIYONG DYNEX TECHNOLOGIES PINPAIDE MLX™ WEILIANGDIDINGGUANGDUJICELIANGGUANGDEQIANGDU,HUIZHIBIAOZHUNQUXIAN,BINGFENXIJIEGUO。YOUYUDIWUFAGUANGCHIXUSHIJIANZHANG,SUOYIYUEDUCHUANGKOUQIKEYISHIJIARUDIWUHOUDE 20~40 FENZHONGZHIJIAN。SHIYANBUZHOUJUNYANGEANZHAOSHIJIHESHUOMINGSHUCAOZUO。

YIQICANSHUSHEDING:

① QISHIMOSHI(Start Mode):1 FENZHONGZHIHOUSHIJIAN

② ZHENDANGJIWENDUSHEDING:GUANBI(Off)

③ JIANCEXUANXIANG:

  • 自动读取 (Auto Gain)
  • 求和模式 (Sum all Readings)
  • 检测时间 1 秒/孔

 

RUANJIANCAOZUO:

① 只含有校准稀释液(Calibrator Diluent)的标准品零值孔被定义为空白孔(Blank):
含有其他不同浓度标准品的孔被定义为 S1-S5 孔。
含有阳性对照(Control)的孔根据对应标签使用「用户可定义的类型(User Definable Types)」定义为 LL、L、M、H。
含有样本的孔需定义为测试品(Tests)。

② ZAIKONGBAIMOSHICHUANGKOUZHONG,XUANZEPINGJUN(Average)。JIANGSUOYOUKONGBAIKONG(BIAOZHUNPINLINGZHIKONG)QUPINGJUNZHI,QITAKONGZHIJIANGTONGYIJIANQUZHEIGEPINGJUNZHI。

途游斗地主③ ZAIQUXIANNIHE(curve fit)CHUANGKOUZHONG,SHURU S1-S5 DE IL-6 BIAOZHUNPINNONGDU,BINGZAIXIANGDUIGUANG(Relative Light)CHUANGKOUSHIYONG log/log Cubic Spline MOSHINIHEBINGHUIZHIQUXIAN。

④ SHUJUJUZHEN(Data Matrix)JINIHEQUXIANTUJIANGCHENGXIANZAICESHIBAOGAO(Test Report)ZHONG。

JIEGUO:

——「WO,WOMINGBAILE,SHIXIONGXUANZHEIGEHEZISHIKAOLVDAOSHIYANZUJIANYANGBENZHONGMUBIAOFENXIWUDEHANLIANGCHAYIDA,GUODUODEXISHICAOZUOYIFANGMIANFEISHIFEILI,LINGYIFANGMIANRONGYIZAOCHENGWUCHADUIBA,SHIXIONGGUORANLIHAI!」

——「QISHI,ZHUYAOHAISHIYINWEIWOLAN……」

 

以上文字和图片来源于Bio-techne

 

 

 

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