兔胰岛素(Insulin)ELISA试剂盒使用说明书
本试剂盒仅供研究使用
产品名称: 兔子胰岛素(INS)ELISA Kit
英文名称: rabbit Insulin,INS ELISA Kit
货号: CSB-E06992Rb
规格: 96T
种属: Rabbit
待测物名称: insulin
缩写: INS
蛋白功能1: Biosynthesis/Metabolism
蛋白功能2: glyconeogenesis and glycometabolism
蛋白功能3: Carbohydrate metabolism
检测范围: 8 μIU/ml-140 μIU/ml
灵敏度: 5 μIU/ml
反应时间: 1-5h
所需样本体积: 50-100ul
检测波长: 450 nm
预期应用
ELISA法定量测定兔血清、血浆、细胞培养物上清或其它相关液体中Insulin含量。
概述
Insulin—胰岛素。胰岛素前体经剪切后形成A和B链,由两个二硫键结合成胰岛素,结合受体后刺激细胞摄取葡萄糖。
实验原理
本试剂盒应用双抗体夹心酶标免疫分析法测定标本中胰岛素水平。用纯化的抗体包被微孔板,制成固相抗体,往包被单抗的微孔中依次加入胰岛素抗原、生物素化的抗兔胰岛素抗体、HRP标记的亲和素,经过彻底洗涤后用底物TMB显色。TMB在过氧化物酶的催化下转化成蓝色,并在酸的作用下转化成最终的黄色。颜色的深浅和样品中的胰岛素呈正相关。用酶标仪在450nm波长下测定吸光度(OD值),计算样品浓度。
试剂盒组成及试剂配制
1. 酶联板:一块(96孔)
2. 标准品(冻干品):2瓶,每瓶临用前以样品稀释液稀释至1ml,盖好后静置10分钟以上,然后反复颠倒/搓动以助溶解,其浓度为100 U/L,做系列倍比稀释后,分别稀释成100 U/L,50 U/L ,25 U/L,12.5 U/L,6.25 U/L,3.12 U/L,1.56 U/L,其原液直接作为最高标准浓度,样品稀释液直接作为标准浓度0 U/L,临用前15分钟内配制。
如配制50 U/L标准品:取0.5ml 100 U/L的上述标准品加入含有0.5ml样品稀释液的Eppendorf管中,混匀即可,其余浓度以此类推。
3. 样品稀释液:1×20ml/瓶。
4. 检测稀释液A:1×10ml/瓶。
5. 检测稀释液B:1×10ml/瓶。
6. 检测溶液A:1×120ul/瓶(1:100)临用前以检测稀释液A 1:100稀释,稀释前根据预先计算好的每次实验所需的总量配制(每孔100ul),实际配制时应多配制0.1-0.2ml。如1ul检测溶液A加99ul检测稀释液A的比例配制,轻轻混匀,在使用前一小时内配制。
7. 检测溶液B:1×120ul/瓶(1:100)临用前以检测稀释液B1:100稀释。稀释方法同检测溶液A。
8. 底物溶液:1×10ml/瓶。
9. 浓洗涤液:1×30ml/瓶,使用时每瓶用蒸馏水稀释25倍。
10. 终止液:1×10ml/瓶(2N H2SO4)。
标本的采集及保存
1.细胞培养物上清:请离心后收集上清,并将标本保存于-20℃,且应避免反复冻融。
2.血清:标本请于室温放置2小时或4℃过夜后于1000 x g离心20分钟,取上清即可检测,或将标本放于-20℃保存,但应避免反复冻融。
3.血浆:可用肝素作为抗凝剂,标本采集后30分钟内于2 - 8° C 1000 x g离心15分钟,或将标本放于-20℃保存,但应避免反复冻融。
注:标本溶血会影响最后检测结果,因此溶血标本不宜进行此项检测。
操作步骤
各试剂在使用前平衡至室温。
1. 加样:分别设空白孔、标准孔、待测样品孔。除空白孔外,余孔分别加标准溶液或待测样品100ul,注意不要有气泡,轻轻混匀,酶标板加上盖,37℃反应120分钟。
2. 弃去液体,甩干,不用洗涤。
3. 每孔加检测溶液A工作液 100ul,37℃,60分钟。洗板3次,350ul/每孔,甩干。
4. 每孔加检测溶液B工作液 100ul,37℃,60分钟,洗板5次,甩干。
5. 依序每孔加底物溶液90ul,37℃避光显色30分钟(此时肉眼可见标准品的前3-4孔有明显的梯度兰色,后3-4孔梯度不明显)。
6. 依序每孔加终止溶液50ul,终止反应(此时兰色立转黄色)。用酶联仪在450nm波长依序测量各孔的光密度(OD值)。
注:
1. 每次实验留一孔作为空白调零孔,该孔不加任何试剂,只是最后加底物溶液及2NH2SO4。测量时先用此孔调OD值至零。
2.为防止样品蒸发,试验时将反应板放于铺有湿布的密闭盒内。
洗板方法
手工洗板方法:吸去(不可触及板壁)或甩掉酶标板内的液体;在实验台上铺垫几层
吸水纸,酶标板朝下用力拍几次;将推荐的洗涤缓冲液至少0.4ml注入孔内,浸泡1-2分钟,根据需要,重复此过程数次。
自动洗板:如果有自动洗板机,应在熟练使用后再用到正式实验过程中。
特异性
本试剂盒可同时检测重组或天然的兔胰岛素,且与其它相关蛋白无交叉反应。
计算
以标准物的浓度为横坐标(对数坐标),OD值为纵坐标(普通坐标),在半对数坐标纸上绘出标准曲线,根据样品的OD值由标准曲线查出相应的浓度;再乘以稀释倍数;或用标准物的浓度与OD值计算出标准曲线的直线回归方程式,将样品的OD值代入方程式,计算出样品浓度,再乘以稀释倍数,即为样品的实际浓度。
注意事项
1. 洗涤过程非常重要,不充分的洗涤易造成假阳性。
2. 一次加样时间最好控制在5分钟内,如标本数量多,推荐使用排枪加样。
3. 请每次测定的同时做标准曲线,最好做复孔。
4. 如标本中胰岛素含量过高,请先稀释后再测定,计算时请最后乘以稀释倍数。
5.在配制标准品、检测溶液工作液时,请以相应的稀释液配制,不能混淆。
6.底物请避光保存。
检测范围: 8 μIU/ml-140 μIU/ml
灵敏度: 5 μIU/ml
说明
1. 在储存及孵育过程中避免将试剂暴露在强光中。所有试剂瓶盖须盖紧以防止蒸发和污染,试剂避免受到微生物的污染,因为蛋白水解酶的干扰将导致出现错误的结果。
2. 小心吸取试剂并严格遵守给定的孵育时间和温度。请注意在吸取标本 / 标准品,酶结合物或底物时,第一个孔与最后一个孔加样之间的时间间隔如果太大,将会导致不同的 “预孵育”时间,从而明显地影响到测量值的准确性及重复性。而且,洗涤不充分将影响试验结果。
3. 试剂盒保存:部分试剂保存于-20℃,部分试剂保存于2-8℃,具体以标签上的标示为准。
4. 浓洗涤液会有盐析出,稀释时可在水浴中加温助溶。
5. 刚开启的酶联板孔中可能会含有少许水样物质,此为正常现象,不会对实验结果造成任何影响。
6. 中、英文说明书可能会有不一致之处,请以英文说明书为准。
7. 所有的样品都应管理好,按照规定的程序处理样品和检测装置。
8. 有效期:6个月
精密度: Intra-assay Precision (Precision within an assay): CV%<8%
Three samples of known concentration were tested twenty times on one plate to assess.
Inter-assay Precision (Precision between assays): CV%<10%
Three samples of known concentration were tested in twenty assays to assess.
样本搜集及储存: Serum: Use a serum separator tube (SST) and allow samples to clot for two hours at room temperature or overnight at 4°C before centrifugation for 15 minutes at 1000 ×g. Remove serum and assay immediately or aliquot and store samples at -20°C or -80°C. Avoid repeated freeze-thaw cycles.
Plasma: Collect plasma using EDTA, or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 ×g at 2-8°C within 30 minutes of collection. Assay immediately or aliquot and store samples at -20°C or -80°C. Avoid repeated freeze-thaw cycles.
检测步骤: Bring all reagents and samples to room temperature before use. Centrifuge the sample again after thawing before the assay. It is recommended that all samples and standards be assayed in duplicate.
1. Prepare all reagents, working standards, and samples as directed in the previous sections.
2. Refer to the Assay Layout Sheet to determine the number of wells to be used and put any remaining wells and the desiccant back into the pouch and seal the ziploc, store unused wells at 4°C.
3. Add 100μl of standard and sample per well. Cover with the adhesive strip provided. Incubate for 2 hours at 37°C. A plate layout is provided to record standards and samples assayed.
4. Remove the liquid of each well, don't wash.
5. Add 100μl of Biotin-antibody (1x) to each well. Cover with a new adhesive strip. Incubate for 1 hour at 37°C. (Biotin-antibody (1x) may appear cloudy. Warm up to room temperature and mix gently until solution appears uniform.)
6. Aspirate each well and wash, repeating the process two times for a total of three washes. Wash by filling each well with Wash Buffer (200μl) using a squirt bottle, multi-channel pipette, manifold dispenser, or autowasher, and let it stand for 2 minutes, complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining wash Buffer by aspirating ordecanting. Invert the plate and blot it against clean paper towels.
7. Add 100μl of HRP-avidin (1x) to each well. Cover the microtiter plate with a new adhesive strip. Incubate for 1 hour at 37°C.
8. Repeat the aspiration/wash process for five times as in step 6.
9. Add 90μl of TMB Substrate to each well. Incubate for 15-30 minutes at 37°C. Protect from light.
10. Add 50μl of Stop Solution to each well, gently tap the plate to ensure thorough mixing.
11. Determine the optical density of each well within 5 minutes, using a microplate reader set to 450 nm. If wavelength correction is available, set to 540 nm or 570 nm. Subtract readings at 540 nm or 570 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate. Readings made directly at 450 nm without correction may be higher and less accurate.
结果计算: Using the professional soft "Curve Expert 1.3" to make a standard curve is recommended, which can be downloaded from our web.
Average the duplicate readings for each standard and sample and subtract the average zero standard optical density.
Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the x-axis against the concentration on the y-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the INS concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data.
If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.
相关产品:
产品名称 英文名称 货号 规格
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抗体
产品名称 货号 规格
Rabbit anti-human Insulin(INS) polyclonal Antibody CSB-PA011742GA01HU 150ul
蛋白
产品名称 货号 规格 蛋白来源
Recombinant Myxine glutinosa Insulin CSB-EP011742MWW 1mg E.coli
Recombinant Myxine glutinosa Insulin CSB-YP011742MWW 1mg Yeast
Recombinant Cyprinus carpio Insulin CSB-EP011742EQE 1mg E.coli
Recombinant Cyprinus carpio Insulin CSB-YP011742EQE 1mg Yeast
Recombinant Proechimys guairae Insulin CSB-EP011742PSE 1mg E.coli
Recombinant Proechimys guairae Insulin CSB-YP011742PSE 1mg Yeast
Recombinant Chinchilla chinchilla Insulin CSB-EP011742CIK 1mg E.coli
Recombinant Chinchilla chinchilla Insulin CSB-YP011742CIK 1mg Yeast
Recombinant Acomys cahirinus Insulin CSB-EP011742ACC 1mg E.coli
Recombinant Acomys cahirinus Insulin CSB-YP011742ACC 1mg Yeast
Recombinant Platichthys flesus Insulin CSB-EP011742EWX 1mg E.coli
Recombinant Platichthys flesus Insulin CSB-YP011742EWX 1mg Yeast
Recombinant Oncorhynchus keta Insulin CSB-EP011742OBG 1mg E.coli
Recombinant Oncorhynchus keta Insulin CSB-YP011742OBG 1mg Yeast
Recombinant Katsuwonus pelamis Insulin CSB-EP011742KAB 1mg E.coli
Recombinant Katsuwonus pelamis Insulin CSB-YP011742KAB 1mg Yeast
Recombinant Crotalus atrox Insulin CSB-EP011742DYC 1mg E.coli
Recombinant Crotalus atrox Insulin CSB-YP011742DYC 1mg Yeast
Recombinant Myocastor coypus Insulin CSB-EP011742MMD 1mg E.coli
Recombinant Myocastor coypus Insulin CSB-YP011742MMD 1mg Yeast
Recombinant Camelus dromedarius Insulin CSB-EP011742CYV 1mg E.coli
Recombinant Camelus dromedarius Insulin CSB-YP011742CYV 1mg Yeast
Recombinant Balaenoptera borealis Insulin CSB-EP011742BEA 1mg E.coli
Recombinant Balaenoptera borealis Insulin CSB-YP011742BEA 1mg Yeast
Recombinant Atractosteus spatula Insulin CSB-EP011742AQQ 1mg E.coli
Recombinant Atractosteus spatula Insulin CSB-YP011742AQQ 1mg Yeast
Recombinant Myoxocephalus scorpius Insulin CSB-EP011742MMH 1mg E.coli
Recombinant Myoxocephalus scorpius Insulin CSB-YP011742MMH 1mg Yeast
Recombinant Thunnus thynnus Insulin CSB-EP011742TEK 1mg E.coli
Recombinant Thunnus thynnus Insulin CSB-YP011742TEK 1mg Yeast
Recombinant Gadus morhua subsp. callarias Insulin CSB-EP011742GAA 1mg E.coli
Recombinant Gadus morhua subsp. callarias Insulin CSB-YP011742GAA 1mg Yeast
Recombinant Anas platyrhynchos Insulin CSB-EP011742BZD 1mg E.coli
Recombinant Anas platyrhynchos Insulin CSB-YP011742BZD 1mg Yeast
Recombinant Hystrix cristata Insulin CSB-EP011742HSC 1mg E.coli
Recombinant Hystrix cristata Insulin CSB-YP011742HSC 1mg Yeast
Recombinant Elephas maximus Insulin CSB-EP011742EKA 1mg E.coli
Recombinant Elephas maximus Insulin CSB-YP011742EKA 1mg Yeast
Recombinant Cricetulus longicaudatus Insulin CSB-EP011742DXV 1mg E.coli
Recombinant Cricetulus longicaudatus Insulin CSB-YP011742DXV 1mg Yeast
Recombinant Selasphorus rufus Insulin CSB-EP011742SER 1mg E.coli
Recombinant Selasphorus rufus Insulin CSB-YP011742SER 1mg Yeast
Recombinant Anguilla anguilla Insulin CSB-EP011742BZF 1mg E.coli
Recombinant Anguilla anguilla Insulin CSB-YP011742BZF 1mg Yeast
Recombinant Chlorocebus aethiops Insulin CSB-EP011742DSU 1mg E.coli
Recombinant Chlorocebus aethiops Insulin CSB-YP011742DSU 1mg Yeast
Recombinant Macaca fascicularis Insulin CSB-EP011742MOV 1mg E.coli
Recombinant Macaca fascicularis Insulin CSB-YP011742MOV 1mg Yeast
Recombinant Anguilla rostrata Insulin CSB-EP011742AKK 1mg E.coli
Recombinant Anguilla rostrata Insulin CSB-YP011742AKK 1mg Yeast
Recombinant Pan troglodytes Insulin CSB-EP011742EQV 1mg E.coli
Recombinant Pan troglodytes Insulin CSB-YP011742EQV 1mg Yeast
Recombinant Amia calva Insulin CSB-EP011742AIW 1mg E.coli
Recombinant Amia calva Insulin CSB-YP011742AIW 1mg Yeast
Recombinant Octodon degus Insulin CSB-EP011742ODV 1mg E.coli
Recombinant Octodon degus Insulin CSB-YP011742ODV 1mg Yeast
Recombinant Rodentia sp. Insulin CSB-EP011742RFD 1mg E.coli
Recombinant Rodentia sp. Insulin CSB-YP011742RFD 1mg Yeast
Recombinant Didelphis marsupialis virginiana Insulin CSB-EP011742DKL 1mg E.coli
Recombinant Didelphis marsupialis virginiana Insulin CSB-YP011742DKL 1mg Yeast
Recombinant Zaocys dhumnades Insulin CSB-EP011742ZAE 1mg E.coli
Recombinant Zaocys dhumnades Insulin CSB-YP011742ZAE 1mg Yeast
Recombinant Torpedo marmorata Insulin CSB-EP011742TGV 1mg E.coli
Recombinant Torpedo marmorata Insulin CSB-YP011742TGV 1mg Yeast
Recombinant Alligator mississippiensis Insulin CSB-EP011742AZO 1mg E.coli
Recombinant Alligator mississippiensis Insulin CSB-YP011742AZO 1mg Yeast
Recombinant Squalus acanthias Insulin CSB-EP011742FKW 1mg E.coli
Recombinant Squalus acanthias Insulin CSB-YP011742FKW 1mg Yeast
Recombinant Callorhynchus milii Insulin CSB-EP011742CBF 1mg E.coli
Recombinant Callorhynchus milii Insulin CSB-YP011742CBF 1mg Yeast
Recombinant Polypterus senegalus Insulin CSB-EP011742PYW 1mg E.coli
Recombinant Polypterus senegalus Insulin CSB-YP011742PYW 1mg Yeast
Recombinant Piaractus mesopotamicus Insulin CSB-EP011742PJS 1mg E.coli
Recombinant Piaractus mesopotamicus Insulin CSB-YP011742PJS 1mg Yeast
Recombinant Acipenser guldenstadti Insulin CSB-EP011742ACA 1mg E.coli
Recombinant Acipenser guldenstadti Insulin CSB-YP011742ACA 1mg Yeast
Recombinant Oreochromis niloticus Insulin CSB-EP011742OET 1mg E.coli
Recombinant Oreochromis niloticus Insulin CSB-YP011742OET 1mg Yeast
Recombinant Petromyzon marinus Insulin CSB-EP011742EUC 1mg E.coli
Recombinant Petromyzon marinus Insulin CSB-YP011742EUC 1mg Yeast
Recombinant Cairina moschata Insulin CSB-EP011742CAG 1mg E.coli
Recombinant Cairina moschata Insulin CSB-YP011742CAG 1mg Yeast
Recombinant Struthio camelus Insulin CSB-EP011742FOU 1mg E.coli
Recombinant Struthio camelus Insulin CSB-YP011742FOU 1mg Yeast
Recombinant Lampetra fluviatilis Insulin CSB-EP011742LNM 1mg E.coli
Recombinant Lampetra fluviatilis Insulin CSB-YP011742LNM 1mg Yeast
Recombinant Saimiri sciureus Insulin CSB-EP011742SWD 1mg E.coli
Recombinant Saimiri sciureus Insulin CSB-YP011742SWD 1mg Yeast
Recombinant Meleagris gallopavo Insulin CSB-EP011742MQV 1mg E.coli
Recombinant Meleagris gallopavo Insulin CSB-YP011742MQV 1mg Yeast
Recombinant Trachemys dorbigni Insulin CSB-EP011742THB 1mg E.coli
Recombinant Trachemys dorbigni Insulin CSB-YP011742THB 1mg Yeast
Recombinant Oncorhynchus gorbuscha Insulin CSB-EP011742OBF 1mg E.coli
Recombinant Oncorhynchus gorbuscha Insulin CSB-YP011742OBF 1mg Yeast
Recombinant Balaenoptera physalus Insulin CSB-EP011742BED 1mg E.coli
Recombinant Balaenoptera physalus Insulin CSB-YP011742BED 1mg Yeast
Recombinant Chimaera monstrosa Insulin CSB-EP011742CII 1mg E.coli
Recombinant Chimaera monstrosa Insulin CSB-YP011742CII 1mg Yeast
Recombinant Trachemys scripta Insulin CSB-EP011742THC 1mg E.coli
Recombinant Trachemys scripta Insulin CSB-YP011742THC 1mg Yeast
Recombinant Physeter macrocephalus Insulin CSB-EP011742PJG 1mg E.coli
Recombinant Physeter macrocephalus Insulin CSB-YP011742PJG 1mg Yeast
Recombinant Hydrolagus colliei Insulin CSB-EP011742HQY 1mg E.coli
Recombinant Hydrolagus colliei Insulin CSB-YP011742HQY 1mg Yeast
Recombinant Anser anser anser Insulin CSB-EP011742AKX 1mg E.coli
Recombinant Anser anser anser Insulin CSB-YP011742AKX 1mg Yeast
Recombinant Lophius piscatorius Insulin CSB-EP011742LIG 1mg E.coli
Recombinant Lophius piscatorius Insulin CSB-YP011742LIG 1mg Yeast
Recombinant Oncorhynchus kisutch Insulin CSB-EP011742OBH 1mg E.coli
Recombinant Oncorhynchus kisutch Insulin CSB-YP011742OBH 1mg Yeast
Recombinant Aotus trivirgatus Insulin CSB-EP011742DNI 1mg E.coli
Recombinant Aotus trivirgatus Insulin CSB-YP011742DNI 1mg Yeast
Recombinant Danio rerio Insulin CSB-EP011742DIL 1mg E.coli
Recombinant Danio rerio Insulin CSB-YP011742DIL 1mg Yeast
Recombinant Dog Insulin(INS) CSB-EP011742DO 1mg E.coli
Recombinant Dog Insulin(INS) CSB-YP011742DO 1mg Yeast
Recombinant Bovine Insulin(INS) CSB-EP011742BO 1mg E.coli
Recombinant Bovine Insulin(INS) CSB-YP011742BO 1mg Yeast
Recombinant Pig Insulin(INS) CSB-EP011742PI 1mg E.coli
Recombinant Pig Insulin(INS) CSB-YP011742PI 1mg Yeast
Recombinant Rabbit Insulin(INS) CSB-EP011742RB 1mg E.coli
Recombinant Rabbit Insulin(INS) CSB-YP011742RB 1mg Yeast
Recombinant Sheep Insulin(INS) CSB-EP011742SH 1mg E.coli
Recombinant Sheep Insulin(INS) CSB-YP011742SH 1mg Yeast
Recombinant Horse Insulin(INS) CSB-EP011742HO 1mg E.coli
Recombinant Horse Insulin(INS) CSB-YP011742HO 1mg Yeast
Recombinant Cat Insulin(INS) CSB-EP011742CA 1mg E.coli
Recombinant Cat Insulin(INS) CSB-YP011742CA 1mg Yeast
Recombinant Guinea pig Insulin(INS) CSB-EP011742GU 1mg E.coli
Recombinant Guinea pig Insulin(INS) CSB-YP011742GU 1mg Yeast
Recombinant Goat Insulin(INS) CSB-EP011742GO 1mg E.coli
Recombinant Goat Insulin(INS) CSB-YP011742GO 1mg Yeast
Recombinant Human Insulin(INS) CSB-EP011742HU 1mg E.coli
Recombinant Human Insulin(INS) CSB-YP011742HU 1mg Yeast
Recombinant Chicken Insulin(INS) CSB-EP011742CH 1mg E.coli
Recombinant Chicken Insulin(INS) CSB-YP011742CH 1mg Yeast