小鼠胰岛素说明书.docx

1、小鼠胰岛素说明书小鼠胰岛素说明书(1)小鼠胰岛素定量分析酶联免疫检测试剂盒本试剂盒仅供科研使用。用于体外定量检测小鼠血清、血浆或细胞培养上清液中的胰岛素浓度。使用前请仔细阅读说明书并检查试剂组分是否完整, 如有疑问请与上海巧伊生物科技有限公司联系，我们将提供力所能及的帮助。如您有其它需求，请登录上海巧伊生物科技有限公司网站或致电本公司。胰岛素简介：胰岛素是糖代谢中最主要的激素之一。胰腺的?细胞岛细胞产生胰岛素前体蛋白，前体蛋白被加工成C肽和胰岛素。它们以等摩尔浓度进入血循环中。成熟的胰岛素由A、B两条链组成。这两条链是通过两个二硫键桥接形成有功能的胰岛素分子。血浆葡萄糖浓度的变化是胰岛素产生并

2、分泌的最主要刺激因素，产生的胰岛素具有一些代谢调节作用。其最主要的作用是，将外周血中糖转运到肝脏中贮存起来。一些诸如肝糖生成障碍或在促进血糖升高的激素诸如胰高血糖素、肾上腺素、生长激素和皮质醇等作用下促进肝糖分解都可拮抗胰岛素的作用。检测原理:本试剂盒采用双抗体夹心ELISA法检测样本中胰岛素的浓度。胰岛素捕获抗体已预包被于酶标板上，当加入标本或参考品时，其中的胰岛素会与捕获抗体结合，其它游离的成分通过洗涤的过程被除去。当加入与HRP耦连的抗胰岛素抗体后，抗小鼠胰岛素抗体与胰岛素接合，形成夹心的免疫复合物，其它游离的成分通过洗涤的过程被除去。最后加入显色剂，若样本中存在胰岛素将会形成免疫复合物

3、，辣根过氧化物酶会催化无色的显色剂氧化成蓝色物质，在加入终止液后呈黄色。通过酶标仪检测，读其450nm处的OD值，胰岛素浓度与OD450值之间呈正比，通过参考品绘制标准曲线，对照未知样本中OD值，即可算出标本中胰岛素浓度。小鼠胰岛素定量分析酶联免疫检测试剂盒组成:组分小鼠胰岛素预包被板 标准品稀释液 小鼠胰岛素标准品 小鼠胰岛素抗体HRP结合物 浓缩洗涤液 20 TMB底物 终止液 封板胶纸 说明书规格（96T/48T） 12条/6条 10ml/5ml 2支/1支(冻干) 10ml/5ml 30ml/15ml 10ml/5ml 5ml/3ml 3/2张 1份标本收集:1.标本的收集请按下列流程

4、进行操作； A.细胞上清标本离心去除悬浮物后即可；B.血清标本应是自然凝固后，取上清，避免在冰箱中凝固血液； C.血浆标本，推荐用EDTA的方法收集若待测样本不能及时检测， D.标本收集后请分装，冻存于20，避免反复冻融。 2.血清标本不应添加任何防腐剂或抗凝剂；3.标本应清澈透明，检测前样本中如有悬浮物应通过离心去除。 4.请勿使用溶血，高血脂或污染的标本检测，否则结果将不准确。注意事项:1.试剂盒请保存在28。2.浓缩洗涤液因在低温下可能有结晶，请水浴加热使结晶完全溶解后再配制工作液。 3.标准品复溶加样后，剩余部分请丢弃。 4.底物请勿接触氧化剂和金属。5.加样时，请及时更换枪头，避免交

5、叉污染。 6.严禁混用不同批号的试剂盒组份。7.充分混匀对保证反应结果的准性很重要，在加液后请轻轻叩击边缘以保证混匀。 8.室温反应，请严格控制在2528。9.洗涤过程是至关重要的，洗涤不充分会使精确度下降并导致结果误差较大。 10.试验中标准品和样本检测时建议作双复孔。 11.加样过程中避免气泡的产生。12.血清和血浆标本的检测时，检测抗体的孵育时间应适当延长。检测前准备工作:1.试剂盒自冰箱中取出后应置室温（2528）平衡20分钟；每次检测后剩余试剂请及时于28保存。 2.将浓缩洗涤液用双蒸水或去离子水稀释(1份加19份水)。3.标准品:加入标准品稀释液至冻干标准品瓶中使胰岛素终浓度达到m

6、l，室温反应，请严格控制在2528，静置1015分钟后轻轻混悬（建议抽吸几次）待彻底溶解，用标准品稀释液倍比梯度稀释后依次加入检测孔中。（标准曲线取七个点，最高浓度为ml,标准品稀释液直接加入作为0浓度.）洗涤方法:自动洗板机或人工洗板：每孔洗涤液为300ul，注入与吸出间隔15-30秒。洗板5次。最后一次洗板完成后将板倒扣着在厚吸水纸上用力拍干。实验过程需自备的材料：1.不同规格的加样枪及相应的枪头； 2.酶标仪；3.自动洗板机； 4.去离子水或双蒸水；操作步骤:1.通过计算并确定一次性实验所需的板条数，取出所需板条放置在框架内，暂时用不到板条请放回铝箔袋密封，保存于4。2.建议设置本底较正

7、孔，即空白孔,设置方法为该孔只加TMB显色液和中止液。每次实验均需做标准品对照并画出标准曲线。3.分别将标本或不同浓度标准品(10ul/孔)加入相应孔中，快速加入小鼠胰岛素抗体HRP结合物(100ul/孔)。用封板胶纸封住反应孔，室温（2528）孵育120分钟。 4.洗板5次，且最后一次置厚吸水纸上拍干。5.加入显色剂TMB100ul/孔，避光室温（2528）孵育10分钟。 6.加入终止液50ul/孔,混匀后即刻测量OD450值。结果判断:1.复孔的值在20%的差异范围内结果才有效，复孔的值平均后可作为测量值。2.每个标准品或标本的OD值应减去本底校正孔的OD值。3.手工绘制标准曲线。以标准品

8、浓度作横坐标，OD值作纵坐标，以平滑线连接各标准品的坐标点。通过标本的OD值可在标准曲线上查出其浓度。4.若标本OD值高于标准曲线上限，应适当稀释后重测，计算浓度时应乘以稀释倍数。典型数值和参考曲线浓度ng/ml典型OD值1典型OD值2OD平均值 0 小鼠胰岛素参考标准曲线注意：本图仅供参考，应以同次试验标准品所绘标准曲线计算标本含量。灵敏度，特异性和重复性:1.灵敏度：多次重复结果表明，最小检出量为ml。2.特异性：不与IGF-I、IGF-II、 小鼠 C肽、大鼠 C肽反应，与猪胰岛素、绵羊胰岛素、大鼠胰岛素、牛胰岛素及人胰岛素分别有628%，256%，146%，110%和195%交叉反应。

9、 3.重复性：板内，板间变异系数均10%.参考文献:Korner J, Savontaus E, Chua SC, Jr., Leibel RL, Wardlaw SL (2001) Leptin regulation of Agrp and Npy mRNA in the mousehypothalamus. J Neuroendocrinol 13:959-966Olsson R and Carlsson PO (2005) Better vascular engraftment and function in pancreatic islets transplanted without

10、prior culture. Diabetologia 48:469-476Rydtren T and Sandler S (2002) Efficacy of 1400 W, a novel inhibitor of inducible nitric oxide synthase, in preventing interleukin-1beta-induced suppression of pancreatic islet function in vitro and multiple low-dose streptozotocin-induced diabetes in vivo. Eur

11、J Endocrinol 147:543- 551 10ELISA Kit for the Quantitative Analysis of Mouse InsulinThe mouse Insulin ELISA (enzyme-linked immunosorbent assay) kit is used for detection of mouse Insulin in cell culture supernatants,mouse serum and ELISA KIT IS FOR RESEARCH USE ONLY. Please read this instruction man

12、ual carefully and check out the material provided before use, and you can contact with our company if any questions. You can enter our website or call us for other aim.IntroductionInsulin is the principal hormone responsible for the control of glucose metabolism. It is synthesized in the ?-cells of

13、the islets of Langerhans as the precursor, proinsulin, which is processed to form C-peptide and insulin. Both are secreted in equimolar amounts into the portal circulation. The mature insulin molecule comprises two polypeptide chains, the A chain and B chain (21 and 30 amino acids respectively). The

14、 two chains are linked together by two inter-chain disulphide bridges.Secretion of insulin is mainly controlled by plasma glucose concentration, and the hormone has a number of important metabolic principal function is to control the uptake and utilisation of glucose in peripheral tissues via the gl

15、ucose transporter. This and other hypoglycaemic activities, such as the inhibition of hepatic gluconeogenesis and glycogenolysis are counteracted by the hyperglycaemic hormones including glucagon, epinephrine (adrenaline), growth hormone and cortisol.Principles of the TestThe kits is a solid sandwic

16、h enzyme-linked immunosorbent assay for detection of mouse Insulin. An anti- mouseInsulin monoclonal antibody has been absorbed onto the wells of the microtiter strips provided. Samples including specimens or standards were pipetted into wells. The mouseInsulin in specimens or standards would be cap

17、tured by the coated antibody and the free others were removed by washing. The mouse Insulin HRP-conjugated antibody were added and binds to mouse Insulin captured by the first antibody, which formed a sandwich. After this, subtrate solution would be added and catalyzed by the HRP, and a coloured pro

18、duct is formed. The intensity of the colored product is used to calculate in proportion to the amount of mouse Insulin in the original specimen.Materials provided with the kits:reagentMouse Insulin Antibody-Coated Wells Standard Diluent Mouse Insulin Standard HRP coupled Antibody Wash Buffer Concent

19、rate 20 TMB Stop Solution Plate CoversComplete Instruction Manual96/48Test Kit 12strips/6strips 10ml/5ml 2/1vial(s) 10ml/5ml 30ml/15ml 10ml/5ml 5ml/3ml 3/2 1Specimen Collection specimen as following: particulate of the cell culture supernatants should be removed before use. was obtained from clot at

20、 room temperature. collect plasma with EDTA. immediately or store samples at-20. Avoid free-thaw cycles. and anticoagulant should not appear in Serum samples. particulate should be removed from samples before use. 4. Do not use grossly hemolyzed or lipemic samples.Note: Strongly recommend that the s

21、erum and plasma samples should be diluent as doubling dilution before use.Precautions for use: storage the Kit at 28。2. Washing buffer concentrate may have crystal in low temperature, and you can melt its in water-bath before use. 3. Please discard the dissolved standard after 3 days for use. contac

22、t of substrate solution with oxidizing agents and metal. of disposable pipette tips avoid microbial contamination or cross-contamination of reagents or specimens. 6. Do not mix or substitute reagents with those from other lots or other sources.7. To ensure the adequate mixure of added reagents, plea

23、se tap gently the plate after the wells were filled with liquid. 8. Incubation temperature should be 2528. 9. Wash step was crucial for whole assay process.10. Duplicate wells of the same sample were recommended in assay process. 11. Avoid the foam while pour the liquid into wells.12. For serum or p

24、lasma samples ,the biotin-conjugated antibody should be incubate for at least 90 minutes.Reagent Preparation reagents should be warmed up to room temperature before use. The remanent reagents must reseal and put into refrigeratory again as soon as possible. 1ml of wash buffer Concentrate into 19ml d

25、eionized or distilled water to work.3. Add ml standard diluent to bottle wait15 minutes for complete dissolution. And in turn add the half concentration diluent by standard diluent .Wash step:Automated microplate washer or operating by pipette: Each well should be pour into300ul wash buffer and soak

26、 15 or 30 seconds,then be aspirated, five times process were repeated. After the last wash, remove remaining wash buffer by the plate and blot it against clean paper towels.Materials Required But Not Provided1. pipettes and pipette tips2. Microwell strip reader capable of reading at 450 nm (540 nm a

27、s optional reference wave length) 3. automated microplate washer or deionized waterAssay procedure needed strips were putted into the frame, the remains were returned into foil pouch and resealed. well were recommended, which only color reagent and stop solution be added. It is suggested that each t

28、esting with gradient density of standard for standard curve. 10ul of standard or sample then add 100ul of HRP- antibody with the Plate Covers for 120 minutes at room temperature . times wash process were repeated.5. Add 100ul of TMB，Lucifugal incubation for 10 minutes at room temperature.6. Add 50ul

29、 of stop solution to each well, determine the optical density of each well within 10 minutes.Calculation of Results should be within 20 per cent of the mean. Average absorbance values for each set of duplicate samples were used as detection results. blank absorbance values of subtract should be dedu

30、cted. a best fit curve through the points of graph. Draw the standard curve by plotting assayed OD valure (on the Y axis) vs. concentration (on the X axis). The sample concentration was obtained based on its OD value founding in the standard concentration curve. the values obtained are not within th

31、e expected range of the standard, Samples should be dilute and assay again.Typical Data and Standard Curveconcentration (ng/ml)Typical data 1Typical data 2Average0 Mouse Insulin standard curveSensitivity, Specificity, RepeatabilitySensitivity: repeated assays were evaluated and the minimum detectable dose was ml.Specificity: No significant cross-reactivity or interference with IGF-I,IGF-II, Mouse C-peptide,Rat C-peptide and has 628% cross-reactivity with porcine