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人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 。用於體外定量檢測人血清 、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並檢查試

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係尊龍凱時 。

胰島素簡介 ：

胰島素是糖代謝中最主要的激素之一 。胰腺的 ß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並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

中糖轉運到肝髒中貯存起來 。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於酶標板上 ，當同時加入標本或參考品和HRP耦

連的抗人胰島素抗體時 ，其中胰島素的不同位點會與捕獲抗體和HRP耦連的抗人胰島素抗體結合 ，形成夾心複合物 ，錨定在固相載體板上 ，

其它遊離的成分通過洗滌的過程被除去 。最後加入顯色劑 ，若樣本中存在胰島素將會形成免疫複合物 ，辣根過氧化物酶會催化無色的顯色

劑氧化成藍色物質 ，在加入終止液後呈黃色 。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 ，通過參考品繪製

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 。

人胰島素定量分析酶聯免疫檢測試劑盒組成:

組分 規格（96T/48T）

人胰島素預包被板 12條/6條

標準品稀釋液 10ml/5ml

人胰島素標準品 2支/1支(凍幹)*

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後即可 ；

B.血清標本應是自然凝固後 ，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.標本收集後請分裝 ，凍存於－20℃ ，避免反複凍融 。

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 ，高血脂或汙染的標本檢測 ，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後再配製工作液 。

3.標準品複溶加樣後 ，剩餘部分請丟棄 。

4.底物請勿接觸氧化劑和金屬 。

5.加樣時 ，請及時更換槍頭 ，避免交叉汙染 。

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 ，在加液後請輕輕叩擊邊緣以保證混勻 。

8.室溫反應 ，請嚴格控製在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並導致結果誤差較大

10.試驗中標準品和樣本檢測時建議作雙複孔 。

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後應置室溫（25～28℃）平衡20分鍾 ；每次檢測後剩餘試劑請及時於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標準品稀釋液 ，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: 按標簽複溶體積加入標準品稀釋液複溶使胰島素終濃度達到200 mIU/L ，室溫反應 ，請嚴格控製在25～28℃ ，靜置15~20分鍾後

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後依次加入檢測孔中 。（標準曲線取七個點 ，最高濃度為200 mIU/L,

標準品稀釋液直接加入作為0濃度.）

洗滌方法:

自動洗板機或人工洗板 ：每孔洗滌液為300ul ，注入與吸出間隔15-30秒 。洗板5次 。最後一次洗板完成後將板倒扣著在厚吸水紙上用力拍幹 。

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.通過計算並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 ，保存於4℃ 。

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

3.分別將標本或不同濃度標準品(25ul/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分鍾 。

4.洗板5次 ，且最後一次置厚吸水紙上拍幹 。

5.加入顯色劑TMB100ul/孔 ，避光室溫（25～28℃）孵育10分鍾 。

6.加入終止液50ul/孔,混勻後即刻測量OD450值 。

上海尊龍凱時生物科技有限公司 www.ruianjituan.com人胰島素參考標準曲線

0

0.5

1

1.5

2

2.5

0 6.25 12.5 25 50 100 200

濃度（mIU/L）

O

D值

上海尊龍凱時生物科技有限公司 www.ruianjituan.com

結果判斷:

1.複孔的值在20%的差異範圍內結果才有效 ，複孔的值平均後可作為測量值。

2.每個標準品或標本的OD值應減去本底校正孔的OD值 。

3.手工繪製標準曲線 。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值高於標準曲線上限 ，應適當稀釋後重測 ，計算濃度時應乘以稀釋倍數 。

典型數值和參考曲線

濃度（mIU/L） 典型OD值1 典型OD值2 OD平均值

0 0.0957 0.1515 0.1236

6.25 0.2963 0.3653 0.3308

12.5 0.5215 0.6337 0.5776

25 0.7894 0.8764 0.8329

50 1.0825 1.1933 1.1379

100 1.4356 1.5614 1.4985

200 1.9482 2.0006 1.9744

人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 ，特異性和重複性:

1.靈敏度 ：多次重複結果表明 ，最小檢出量為1.54mIU/L 。

2.特異性 ：與人的C肽 ，Proinsulin ，IGF-I 、IGF-II和大鼠胰島素 、小鼠胰島素不反應 ，與豬 、綿羊和牛的胰島素分別有374% 、48%和31%

的交叉反應性 。

3.重複性 ：板內 ，板間變異係數均<10%.

參考文獻:

1 、FRIER, B.M., ASHBY, J.P., NAIRIN, I.M. and BAIRS, J.D. (1981).Plasma insulin, C-peptide and glucagon concentrations in patients

with insulin-independent diabetes treated with chlorpropamide. Diab. Metab.. 7, 1, 45-49.

2 、Rudovich NN, Rochlitz HJ, Pfeiffer AF. (2004) Reduced hepatic insulin extraction in response to gastric inhibitory polypeptide

compensates for reduced insulin secretion in normal-weight and normal glucose tolerant first-degree relatives of type 2 diabetic patients.

Diabetes 53:2359-2365

3、JUDZEWITSCH, R.G., PFEIFER, M.A., BEST, J.D:, BEARD J.C., HALTER, J.B. and PORTE D. Jr. (1982). Chronic chlorpropamide

therapy of noninsulin-dependent diabetes augments basal and stimulated insulin secretion by increasing islet sensitivity to glucose. J. Clin. End. And Metab. 55, 2, 321-328.

4 、Riserus U, Vessby B, Arner P, Zethelius B. (2004) Supplementation with trans10cis12-conjugated linoleic acid induces

hyperproinsulinaemia in obese men: close association with impaired insulin sensitivity. Diabetologia 47:1016-1019

5 、Gaines-Das, R.E. and Bristow, A.F. (1988) WHO International reference reagents for human proinsulin and human C-peptide. J Biol

Stand 16:179-186

6 、 Lindstrom T, Hedman CA, Arnqvist HJ. (2002) Use of a novel double-antibody technique to describethe pharmacokinetics of

rapid-acting insulin analogs. Diabetes Care 25:1049-1054

上海尊龍凱時生物科技有限公司 www.ruianjituan.com上海尊龍凱時生物科技有限公司 www.ruianjituan.com

ELISA Kit for the Quantitative Analysis of Human Insulin

The human Insulin ELISA (enzyme-linked immunosorbent assay) kit is used for detection of human Insulin in cell culture

supernatants, human 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 ß-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 human Insulin. An anti- human Insulin monoclonal

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

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

washing. The human Insulin HRP-conjugated antibody were added and binds to human 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 human Insulin in the original specimen.

Materials provided with the kits:

reagent 96/48Test Kit

Human Insulin Antibody-Coated Wells 12 strips/6 strips

Standard Diluent 10ml/5ml

Human 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 2～8℃ 。

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 25～28℃.

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 .

上海尊龍凱時生物科技有限公司 www.ruianjituan.comHuman Insulin Standard Cruve

0

0.5

1

1.5

2

2.5

0 6.25 12.5 25 50 100 200

Concentration（mIU/L）

O

p

t

i

c

a

l

D

e

n

s

i

t

y

上海尊龍凱時生物科技有限公司 www.ruianjituan.com

4.Five times wash process were repeated..

5. Add 100ul of TMB ，Lucifugal incubation for 10 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（mIU/L） Typical data 1 Typical data 2 Average

0 0.0957 0.1515 0.1236

6.25 0.2963 0.3653 0.3308

12.5 0.5215 0.6337 0.5776

25 0.7894 0.8764 0.8329

50 1.0825 1.1933 1.1379

100 1.4356 1.5614 1.4985

200 1.9482 2.0006 1.9744

Human Insulin standard curve

Sensitivity, Specificity, Repeatability

Sensitivity: repeated assays were evalsuated and the minimum detectable dose was 1.54mIU/L.

Specificity: No cross-reactivity with human C peptide, Proinsulin, IGF-I,IGF-II and mouse insulin,rat insulin, have 374%,48% and 31%

reactivity with Porcine,sheep and bovine insulin.

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

REFERENCES:

1 、FRIER, B.M., ASHBY, J.P., NAIRIN, I.M. and BAIRS, J.D. (1981).Plasma insulin, C-peptide and glucagon concentrations in patientswith insulin-independent diabetes treated with chlorpropamide. Diab. Metab.. 7, 1, 45-49.

2 、Rudovich NN, Rochlitz HJ, Pfeiffer AF. (2004) Reduced hepatic insulin extraction in response to gastric inhibitory polypeptide

compensates for reduced insulin secretion in normal-weight and normal glucose tolerant first-degree relatives of type 2 diabetic patients.

Diabetes 53:2359-2365

3 、JUDZEWITSCH, R.G., PFEIFER, M.A., BEST, J.D:, BEARD J.C., HALTER, J.B. and PORTE D. Jr. (1982). Chronic chlorpropamide

therapy of noninsulin-dependent diabetes augments basal and stimulated insulin secretion by increasing islet sensitivity to glucose. J.

Clin. End. And Metab. 55, 2, 321-328.

4 、Riserus U, Vessby B, Arner P, Zethelius B. (2004) Supplementation with trans10cis12-conjugated linoleic acid induces

hyperproinsulinaemia in obese men: close association with impaired insulin sensitivity. Diabetologia 47:1016-1019

5 、Gaines-Das, R.E. and Bristow, A.F. (1988) WHO International reference reagents for human proinsulin and human C-peptide. J Biol

Stand 16:179-186

6 、 Lindstrom T, Hedman CA, Arnqvist HJ. (2002) Use of a novel double-antibody technique to describethe pharmacokinetics of

rapid-acting insulin analogs. Diabetes Care 25:1049-1054

上海尊龍凱時生物科技有限公司 www.ruianjituan.com