猴子胰岛素ELISA试剂盒 北京驰明瑞

猴子胰岛素

使用说明书

产品编号：

本试剂盒仅供科研使用，不得用于临床及诊断使用！

试剂组成

名称规格数量保存

包被平底微孔板96孔1可拆卸板2-8℃密封冷藏

酶结合物6毫升1瓶2-8℃冷藏

标准品1毫升6瓶2-8℃冷藏

显色剂A6毫升1瓶2-8℃冷藏

显色剂B6毫升1瓶2-8℃避光冷藏

终止液6毫升1瓶2-8℃冷藏

浓缩洗涤液（100倍

10毫升1瓶2-8℃冷藏

稀释）

使用说明书1份

自备物品

1.酶标仪（尽量提前预热）

2.微量加液器、吸头

3.蒸馏水或去离子水以及滤纸

操作步骤

1.取出试剂盒，于室温（20-25℃）放置15-30分钟。实验过程应在室温

（20-25℃）内进行。

2.取出酶标板，按照标准品的次序分别加入50μl的标准品溶液于空白微孔

中。

3.空白微孔中加入50μl的样品，空白对照加入50μl的蒸馏水；

4.在各孔中加入50μl的酶标记溶液；（不含空白对照孔）

5.将酶标板用封口胶密封后，37℃孵育反应1小时；（在孵育箱中保持稳定

的温度与湿度）

6.充分清洗酶标板3-5次，保持各孔有充足的水压；（浓缩洗涤液以1：100

的比例与蒸馏水稀释）

7.酶标板洗涤后用吸水纸彻底拍干；

8.各孔加入显色剂A、B液各50μl；（不含空白对照孔）

9.20-25℃下避光反应10分钟；

10.各孔加入50μl终止液，终止反应；

结果判断

1.30分钟内在波长450nm的酶标仪上读取各孔的OD值；

2.以OD值为纵坐标，以标准品的浓度为横坐标，绘制曲线图；

3.根据样品的OD值查找对应的浓度范围；

4.敏感度：1.0μIU/ml.

5.图例

M o n k ey In Ins

s u l i n E L I SA Kit 96Tes Test

t s Ca Cat t alog alogu u e Nu Num m be ber r :S to tor r e all r e ag age e n ts at 2-8°C

FOR LA LAB B O R A T O RY RESE RESEA A R C H USE O NL NLY Y .N O T FOR T H ER ERA A PEUTIC O R DI DIAG AG AGN N O S TIC APPLIC APPLICA A T I O NS!NS!P P LEASE RE REA

A D T H R O U G H ENT ENTI I RE PR PRO O CE CED D U R E

B EFO EFOR

R E B E G I N N I N G !I N TEN TEND

D ED US

E This BOGOO INS ELISA kit is intended Laboratory for research use only and is not for use in diagnostic or therapeutic procedures.The Stop Solution changes the color from blue to yellow and the intensity of the color is measured at 450nm using a spectrophotometer.In order to measure the concentration of INS in the sample,this INS ELISA Kit includes a set of calibration standards.The calibration standards are assayed at the same time as the samples and allow the operator to produce a standard curve of Optical Density versus INS concentration.The concentration of INS in the samples is then determined by comparing the O.D.of the samples to the standard curve.

PRI PRIN N C I PLE O F T H E AS ASS

S AY This INS enzyme linked immunosorbent assay applies a technique called a

quantitative sandwich immunoassay.The microtiter plate provided in this kit has been pre-coated with a monoclonal antibody specific for INS.Standards or samples are then added to the microtiter plate wells and INS if present,will bind to the antibody pre-coated wells.In order to quantitatively determine the amount of INS present in the sample,a standardized preparation of horseradish peroxidase (HRP)-conjugated polyclonal antibody,specific for INS are added to each well to “sandwich”the INS immobilized on the plate.The microtiter plate undergoes incubation,and then the wells are thoroughly washed to remove all unbound components.Next,A and B substrate solution is added to each well.The enzyme (HRP)and substrate are allowed to react over a short incubation period.Only those wells that contain INS and enzyme-conjugated antibody will exhibit a change in color.The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wave length of 450nm.

RE REAG AG AGENTS ENTS PR PRO

O V I D ED All reagents provided are stored at 2-8°C.Refer to the expiration date on the label.

1.MICROTITER PLATE 96wells

2.ENZYME CONJUGATE 6.0mL 1vial

3.STANDARD.10μIU/ml 1vial

4.STANDARD.250μIU/ml 1vial

5.STANDARD.3100μIU/ml 1vial

6.STANDARD.4250μIU/ml 1vial

7.STANDARD.5500μIU/ml 1vial

8.STANDARD.61000μIU/ml 1vial

9.SUBSTRATE A 6.0mL 1vial 10.SUBSTRATE B 6.0mL 1vial 11.STOP SOLUTION 6.0mL 1vial

12.WASH SOLUTION x10010mL 1vial 13.INSTRUCTION 1

M ATE ATER R I A LS RE REQ

Q U I RED B U T N O T S UPPLIED 1.Microplate reader capable of measuring absorbance at 450nm.2.Precision pipettes to deliver 2ml to 1ml volumes.

3.Adjustable 10ml -100ml pipettes for reagent preparation.

4.Adjustable 10ml -100ml pipettes for reagent preparation.

5.100ml and 1liter graduated cylinders.

6.Calibrated adjustable precision pipettes,preferably with disposable plastic

tips.(A manifold multi-channel pipette is desirable for large assays.)7.Absorbent paper.8.37°C incubator.

9.Distilled or deionized water.

10.Data analysis and graphing software.Graph paper:linear(Cartesian),log-log

or semi-log,or log-logit as desired.

11.Tubes to prepare standard or sample dilutions.

AS ASS S AY PR PRO O CE

CED D U R E

1.Prepare all Standards before starting assay procedure(see Preparation

Reagents).It is recommended that all Standards and Samples be added in duplicate to the Microtiter Plate.

2.First,secure the desired number of coated wells in the holder,then add50μL

of S t a n d a r d s or Sa Sam m p les to the appropriate well of the antibody pre-coated Microtiter Plate.

3.Add50μL of C on onj j u g a te to each well.Mix https://www.360docs.net/doc/4c10311764.html,plete mixing in this

step is important.Cover and incubate for1hours at37°C.

4.Prepare Substrate Solution no more than15minutes before end of incubation

(see Preparation of Reagents).

5.Wash the Microtiter Plate using one of the specified methods indicated

below:

6.Manual Washing:Remove incubation mixture by aspirating contents of the

plate into a sink or proper waste https://www.360docs.net/doc/4c10311764.html,ing a squirt bottle,fill each well completely with distilled or de-ionized water,then aspirate contents of the plate into a sink or proper waste container.Repeat this procedure four more times for a total of FIVE washes.After final wash,invert plate,and blot dry by hitting plate onto absorbent paper or paper towels until no moisture appears.Note:Hold the sides of the plate frame firmly hen washing the plate to assure that all strips remain securely in frame.

7.Automated Washing:Aspirate all wells,then wash plate FIVE times using

distilled or de-ionized water.Always adjust your washer to aspirate as much liquid as possible and set fill volume at350μL/well/wash(range:350-400μL).After final wash,invert plate,and blot dry by hitting plate onto absorbent paper or paper towels until no moisture appears.It is recommended that the washer be set for a soaking time of10seconds or shaking time of5 seconds between washes.

8.Add50μL Substrate A&B to each well.Cover and incubate for10minutes at

20-25°C.

9.Add50μL of Stop Solution to each well.Mix well.

10.Read the Optical Density(O.D.)at450nm using a microtiter plate reader

within30minutes.

LATI I O N O F RESULTS

LCU U LAT

OGOO O C A LC

B OGO

1.This standard curve is used to determine the amount in an unknown sample.

The standard curve is generated by plotting the average O.D.(450nm) obtained for each of the six standard concentrations on the vertical(Y)axis versus the corresponding concentration on the horizontal(X)axis.

2.First,calculate the mean O.D.value for each standard and sample.All O.D.

values,are subtracted by the mean value of the zero standard before result interpretation.Construct the standard curve using graph paper or statistical software.

3.T o determine the amount in each sample,first locate the O.D.value on the

Y-axis and extend a horizontal line to the standard curve.At the point of intersection,draw a vertical line to the X-axis and read the corresponding concentration.

4.Any variation in operator,pipetting and washing technique,incubation time or

temperature,and kit age can cause variation in result.Each user should obtain their own standard curve.

5.The sensitivity by this assay is1.0μIU/ml

6.Standard curve