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    大鼠胰島素定量分析酶聯免疫檢測試劑盒
    貨號:DSE006
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    • 產品信息
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    • 常見問題

    上海傳秋生物科技有限公司 www.antidotetoaging.com

    大鼠胰島素定量分析酶聯免疫檢測試劑盒

    本試劑盒僅供科研使用。用于體外定量檢測大鼠血清、血漿或細胞培養上清液中的胰島素濃度。使用前請仔細閱讀說明書并檢查試劑

    組分是否完整,如有產品包裝破損或質量投拆,請在收到貨一個月之內聯系我們。

    胰島素簡介:

    胰島素是糖代謝中最主要的激素之一。胰腺的?細胞島細胞產生胰島素前體蛋白,前體蛋白被加工成C肽和胰島素。它們以等摩爾濃度

    進入血循環中。成熟的胰島素由A、B兩條鏈組成。這兩條鏈是通過兩個二硫鍵橋接形成有功能的胰島素分子。

    血漿葡萄糖濃度的變化是胰島素產生并分泌的最主要刺激因素,產生的胰島素具有一些代謝調節作用。其最主要的作用是,將外周血

    中糖轉運到肝臟中貯存起來。一些諸如肝糖生成障礙或在促進血糖升高的激素諸如胰高血糖素、腎上腺素、生長激素和皮質醇等作用下促

    進肝糖分解都可拮抗胰島素的作用。

    檢測原理:

    本試劑盒采用雙抗體夾心ELISA法檢測樣本中胰島素的濃度。胰島素捕獲抗體已預包被于酶標板上,當同時加入標本或參考品和HRP耦

    連的抗大鼠胰島素抗體時,其中胰島素的不同位點會與捕獲抗體和HRP耦連的抗大鼠胰島素抗體結合,形成夾心復合物,錨定在固相載體板

    上,其它游離的成分通過洗滌的過程被除去。最后加入顯色劑,若樣本中存在胰島素將會形成免疫復合物,辣根過氧化物酶會催化無色的

    顯色劑氧化成藍色物質,在加入終止液后呈黃色。通過酶標儀檢測,讀其450nm處的OD值,胰島素濃度與OD450值之間呈正比,通過參考品

    繪制標準曲線,對照未知樣本中OD值,即可算出標本中胰島素濃度。

    大鼠胰島素定量分析酶聯免疫檢測試劑盒組成:

    組分 規格(96T/48T)

    大鼠胰島素預包被板 12條/6條

    標準品稀釋液 10ml/5ml

    大鼠胰島素標準品 2支/1支(凍干)*

    HRP連接的抗體結合物 10ml/5ml

    濃縮洗滌液 20× 30ml/15ml

    TMB底物 10ml/5ml

    終止液 5ml/3ml

    封板膠紙 3/2張

    說明書 1份

    標本收集:

    1.標本的收集請按下列流程進行操作;

    A.細胞上清標本離心去除懸浮物后即可;

    B.血清標本應是自然凝固后,取上清,避免在冰箱中凝固血液;

    C.血漿標本,推薦用EDTA的方法收集若待測樣本不能及時檢測,

    D.標本收集后請分裝,凍存于-20℃,避免反復凍融。

    2.血清標本不應添加任何防腐劑或抗凝劑;

    3.標本應清澈透明,檢測前樣本中如有懸浮物應通過離心去除。

    4.請勿使用溶血,高血脂或污染的標本檢測,否則結果將不準確。

    注意事項:

    1.試劑盒請保存在2~8℃。2.濃縮洗滌液因在低溫下可能有結晶,請水浴加熱使結晶完全溶解后再配制工作液。

    3.標準品復溶加樣后,剩余部分請丟棄。

    4.底物請勿接觸氧化劑和金屬。

    5.加樣時,請及時更換槍頭,避免交叉污染。

    6.嚴禁混用不同批號的試劑盒組份。

    7.充分混勻對保證反應結果的準性很重要,在加液后請輕輕叩擊邊緣以保證混勻。

    8.室溫反應,請嚴格控制在25~28℃。

    9.洗滌過程是至關重要的,洗滌不充分會使精確度下降并導致結果誤差較大

    10.試驗中標準品和樣本檢測時建議作雙復孔。

    11.加樣過程中避免氣泡的產生。

    12.血清和血漿標本的檢測時,檢測抗體的孵育時間應適當延長。

    檢測前準備工作:

    1.試劑盒自冰箱中取出后應置室溫(25~28℃)平衡20分鐘;每次檢測后剩余試劑請及時于2~8℃保存。

    2.將濃縮洗滌液用雙蒸水或去離子水稀釋(1份加19份水)。

    3.如有5X準品稀釋液,請按所需量用雙蒸水或去離子水稀釋(1份加4水)。

    4.標準品: 按標簽復溶體積加入標準品稀釋液復溶使胰島素終濃度達到5.5ng/ml,室溫反應,請嚴格控制在25~28℃,靜置10~15分鐘后輕

    輕混懸(建議抽吸幾次)待徹底溶解,用標準品稀釋液倍比梯度稀釋后依次加入檢測孔中。(標準曲線取七個點,最高濃度為5.5ng/ml,

    標準品稀釋液直接加入作為0濃度.)

    洗滌方法:

    自動洗板機或大鼠工洗板:每孔洗滌液為300ul,注入與吸出間隔15-30秒。洗板5次。最后一次洗板完成后將板倒扣著在厚吸水紙上用力拍

    干。

    實驗過程需自備的材料:

    1.不同規格的加樣槍及相應的槍頭;

    2.酶標儀;

    3.自動洗板機;

    4.去離子水或雙蒸水;

    操作步驟:

    1.通過計算并確定一次性實驗所需的板條數,取出所需板條放置在框架內,暫時用不到板條請放回鋁箔袋密封,保存于4℃。

    2.建議設置本底較正孔,即空白孔,設置方法為該孔只加TMB顯色液和中止液。每次實驗均需做標準品對照并畫出標準曲線。

    3.分別將標本或不同濃度標準品(10ul/孔)加入相應孔中,快速加入HRP連接抗體工作液(100ul/孔)。用封板膠紙封住反應孔,室溫(25~

    28℃)孵育120分鐘。

    4.洗板5次,且最后一次置厚吸水紙上拍干。

    5.加入顯色劑TMB100ul/孔,避光室溫(25~28℃)孵育20分鐘。

    6.加入終止液50ul/孔,混勻后即刻測量OD450值。

    結果判斷:

    1.復孔的值在20%的差異范圍內結果才有效,復孔的值平均后可作為測量值。

    上海傳秋生物科技有限公司 www.antidotetoaging.com大鼠胰島素標準曲線

    0

    0.5

    1

    1.5

    2

    2.5

    3

    3.5

    0 0.171875 0.34375 0.6875 1.375 2.75 5.5

    大鼠胰島素濃度(ng/ml)

    O

    D值

    上海傳秋生物科技有限公司 www.antidotetoaging.com

    2.每個標準品或標本的OD值應減去本底校正孔的OD值。

    3.手工繪制標準曲線。以標準品濃度作橫坐標,OD值作縱坐標,以平滑線連接各標準品的坐標點。通過標本的OD值可在標準曲線上查出其

    濃度。

    4.若標本OD值高于標準曲線上限,應適當稀釋后重測,計算濃度時應乘以稀釋倍數。

    典型數值和參考曲線

    濃度ng/ml 典型OD1 典型OD2 OD平均值

    0 0.1666 0.1282 0.1474

    0.171875 0.3019 0.2871 0.2945

    0.34375 0.4281 0.3529 0.3905

    0.6875 0.6073 0.5693 0.5883

    1.375 1.0635 0.9803 1.0219

    2.75 1.8406 1.7362 1.7884

    5.5 2.9785 2.8219 2.9002

    大鼠胰島素參考標準曲線

    注意:本圖僅供參考,應以同次試驗標準品所繪標準曲線計算標本含量。

    靈敏度,特異性和重復性:

    1.靈敏度:多次重復結果表明,最小檢出量為0.1ng/ml。

    2.特異性:不與IGF-I、IGF-II、 小鼠 C肽、大鼠 C肽反應,與豬胰島素、綿羊胰島素、小鼠胰島素、牛胰島素及人胰島素分別有628%,

    256%,75%,110%和195%交叉反應。

    3.重復性:板內,板間變異系數均<10%.

    參考文獻:

    Korner J, Savontaus E, Chua SC, Jr., Leibel RL, Wardlaw SL (2001) Leptin regulation of Agrp and Npy mRNA in the rat hypothalamus. J

    Neuroendocrinol 13:959-966

    Olsson R and Carlsson PO (2005) Better vascular engraftment and function in pancreatic islets transplanted without prior culture.

    Diabetologia 48:469-476

    Rydtren 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 J Endocrinol 147:543- 551 10

    ELISA Kit for the Quantitative Analysis of Rat Insulin

    The rat Insulin ELISA (enzyme-linked immunosorbent assay) kit is used for detection of rat Insulin in cell culture supernatants, rat上海傳秋生物科技有限公司 www.antidotetoaging.com

    serum and plasma.THE ELISA KIT IS FOR RESEARCH USE ONLY. Please read this instruction manual 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.

    Introduction

    Insulin is the principal hormone responsible for the control of glucose metabolism. It is synthesized in the ?-cells of 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 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 actions. Its principal function is to control the uptake and

    utilisation of glucose in peripheral tissues via the glucose 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 Test

    The kits is a solid sandwich enzyme-linked immunosorbent assay for detection of rat Insulin. An anti- rat Insulin monoclonal antibody

    has been absorbed onto the wells of the microtiter strips provided. Samples including specimens or standards were pipetted into wells.

    The rat Insulin in specimens or standards would be captured by the coated antibody and the free others were removed by washing. The

    rat Insulin HRP-conjugated antibody were added and binds to rat 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 product is formed. The intensity of the colored product

    is used to calculate in proportion to the amount of rat Insulin in the original specimen.

    Materials provided with the kits:

    reagent 96/48Test Kit

    Rat Insulin Antibody-Coated Wells 12 strips/6 strips

    Standard Diluent 10ml/5ml

    Rat Insulin Standard 2/1vial(s)

    HRP coupled Antibody 10ml/5ml

    Wash Buffer Concentrate 20× 30ml/15ml

    TMB 10ml/5ml

    Stop Solution 5ml/3ml

    Plate Covers 3/2

    Complete Instruction Manual 1

    Specimen Collection

    1.Collecting specimen as following:

    A.The particulate of the cell culture supernatants should be removed before use.

    B.Serum was obtained from clot at room temperature.

    C.Please collect plasma with EDTA.

    D.Assay immediately or store samples at 20. Avoid free-thaw cycles.

    2.Antiseptic and anticoagulant should not appear in Serum samples.

    3.Any particulate should be removed from samples before use.

    4. Do not use grossly hemolyzed or lipemic samples.

    Note: Strongly recommend that the serum and plasma samples should be diluent as doubling dilution before use.Precautions for use:

    1.Please 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.

    4.Avoid contact of substrate solution with oxidizing agents and metal.

    5.Usage 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, please 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 plasma samples ,the biotin-conjugated antibody should be incubate for at least 90 minutes.

    Reagent Preparation

    1.The reagents should be warmed up to room temperature before use. The remanent reagents must reseal and put into refrigeratory

    again as soon as possible.

    2. Dilute 1ml of wash buffer Concentrate into 19ml deionized or distilled water to work.

    3. If you have a 5x standard diluent, please dilute it with double steaming water or deionized water.

    4. Add the standard dilution solution to the bottle according to the volume of the label and 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 15 or 30 seconds,then

    be aspirated, five times process were repeated. After the last wash, remove remaining wash buffer byaspirating.Invert the plate and blot it

    against clean paper towels.

    Materials Required But Not Provided

    1. pipettes and pipette tips

    2. Microwell strip reader capable of reading at 450 nm (540 nm as optional reference wave length)

    3. automated microplate washer

    4.Glass-distilled or deionized water

    Assay procedure

    1.The needed strips were putted into the frame, the remains were returned into foil pouch and resealed.

    2.Blank well were recommended, which only color reagent and stop solution be added. It is suggested that each testing with gradient

    density of standard for standard curve.

    3.Add 10ul of standard or sample then add 100ul of HRP- antibody immediatly.Cover with the Plate Covers provided.Incubate for 120

    minutes at room temperature .

    4.Five times wash process were repeated..

    5. Add 100ul of TMB,Lucifugal incubation for 20 minutes at room temperature.

    6. Add 50ul of stop solution to each well, determine the optical density of each well within 10 minutes.

    Calculation of Results

    1.Duplicates should be within 20 per cent of the mean. Average absorbance values for each set of duplicate samples were used as

    detection results.

    2.The blank absorbance values of subtract should be deducted

    3.Drawing 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.

    4.If the values obtained are not within the expected range of the standard, Samples should be dilute and assay again.

    Typical Data and Standard Curve

    concentration (ng/ml) Typical data 1 Typical data 2 Average

    0 0.1666 0.1282 0.1474

    0.171875 0.3019 0.2871 0.2945

    0.34375 0.4281 0.3529 0.3905

    0.6875 0.6073 0.5693 0.5883

    1.375 1.0635 0.9803 1.0219

    2.75 1.8406 1.7362 1.7884

    5.5 2.9785 2.8219 2.9002

    Rat Insulin standard curve

    Sensitivity, Specificity, Repeatability

    Sensitivity: repeated assays were evaluated and the minimum detectable dose was 0.1ng/ml.

    Specificity: No significant cross-reactivity or interference with IGF-I,IGF-II, Mouse C-peptide,Rat C-peptide and has 476%

    cross-reactivity with porcine insulin, 179% with ovine insulin,75% with mouse insulin ,78% with bovine insulin and 167% with Human

    insulin.

    Repeatability: The coefficient of variation between wells or plates is less than 10 per cent.

    REFERENCES:

    Korner J, Savontaus E, Chua SC, Jr., Leibel RL, Wardlaw SL (2001) Leptin regulation of Agrp and Npy mRNA in the rat

    hypothalamus. J Neuroendocrinol 13:959-966

    Olsson R and Carlsson PO (2005) Better vascular engraftment and function in pancreatic islets transplanted without prior

    culture. Diabetologia 48:469-476

    Rydtren 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 J Endocrinol 147:543- 551 10

    上海傳秋生物科技有限公司 www.antidotetoaging.com

     


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