Rabbit Hepatocyte Growth Factor ELISA Kit

96 Tests

Catalogue Number: E04H0208

Store all reagents at 2-8°C

Collect sample: serum or blood plasma

FOR LABORATORY RESEARCH USE ONLY. NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS!PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE BEGINNING!

INTENDED USE

This B.G HGF 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 450 nm using a spectrophotometer. In order to measure the concentration of HGF in the sample, this HGF 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 HGF concentration. The concentration of HGF in the samples is then determined by comparing the O.D. of the samples to the standard curve.

PRINCIPLE OF THE ASSAY

The coated well immunoenzymatic assay for the quantitative measurement of serum HGF utilizes a monoclonal anti-HGF and a HGF-HRP conjugate. The assay asample and buffer are incubated together with anti-HGF antibody coated plate for sixty and washed. The diluted HGF-HRP conjugate is then added to each well and incubated. After the incubation period, the wells are decanted and washed three times. The wells are then incubated with a substrate for the enzyme. The product of the enzyme-substrate reaction forms a blue colored complex. Finally, a stopping solution is added to stop the reaction, which will then turn the solution yellow.The intensity of color is measured spectrophotometrically at 450nm in a microplate reader. The intensity of the color is inversely proportional to the HGF concentration since HGF from samples and HGF-HRP conjugate compete for the anti-HGF antibody binding site. Since the number of sites is limited, as more sites are occupied by HGF from the sample, fewer sites are left to bind HGF-HRP conjugate.Standards of known HGF concentrations are run concurrently with the samples being assayed and a standard curve is plotted relating the intensity of the color （Optical Density） to the concentration of HGF. The unknown HGF

concentration in each sample is interpolated from this curve.

REAGENTS PROVIDED

All reagents provided are stored at 2-8° C. Refer to the expiration date on the label.

1.          MICROTITER PLATE  96 wells

2.          ENZYME CONJUGATE  6.0 mL  1 vial

3.          STANDARD.1  0ng/ml   1 vial

4.          STANDARD.2  1.0ng/ml   1 vial

5.          STANDARD.3  2.5ng/ml   1 vial

6.          STANDARD.4  5.0ng/ml   1 vial

7.          STANDARD.5  10ng/ml   1 vial

8.          STANDARD.6  25ng/ml   1 vial

9.          SUBSTRATE A  6.0 mL  1 vial

10.       SUBSTRATE B  6.0 mL  1 vial

11.       STOP SOLUTION  6.0 mL  1 vial

12.       WASH SOLUTION x100  10 mL  1 vial

13.       Instruction   1

SAMPLE COLLECTION AND STORAGE

Serum- Use a serum separator tube（SST） and allow samples to clot for 30minutes before centrifugation for 15minutes at approximay 1000 x g.Remove serum and assay immediay or aliquot and store samples at -20 °C or -80°C.

Plasma - Collect plasma using EDTA or heparin as an anticoagulant.Centrifuge samples for 15 minutes at 1000 x g at 2-8°C within 30minutes of collection.Store samples at -20°C or -80°C.Avoid repeated freeze-thaw cycles.

Cell culture fluid and other biological fluids - Remove particulates by centrifugation and assay immediay or aliquot and store samples at -20°C or -80°C.Avoid repeated freeze-thaw cycles.

NOTE: Serum, plasma, and cell culture fluid samples to be used within 7 days may be stored at 2-8°C, otherwise samples must stored at -20°C（≤2months） or -80°C（≤6months） to avoid loss of bioactivity and contamination. Avoid freeze-thaw cycles .When performing the assay slowly bring samples to room temperature.

DO NOT USE HEAT-TREATED SPECIMENS.

MATERIALS REQUIRED BUT NOT SUPPLIED

1.          Microplate reader capable of measuring absorbance at 450 nm.

2.          Precision pipettes to deliver 2 ml to 1 ml volumes.

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

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

5.          100 ml and 1 liter 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.

PRECAUTIONS

1.          Do not substitute reagents from one kit lot to another. Standard, conjugate and microtiter plates are matched for optimal performance. Use only the reagents supplied by manufacturer.

2.          Allow kit reagents and materials to reach room temperature （20-25°C） before use. Do not use water baths to thaw samples or reagents.

3.          Do not use kit components beyond their expiration date.

4.          Use only deionized or distilled water to dilute reagents.

5.          Do not remove microtiter plate from the storage bag until needed. Unused strips should be stored at 2-8°C in their pouch with the desiccant provided.

6.          Use fresh disposable pipette tips for each transfer to avoid contamination.

7.          Do not mix acid and sodium hypochlorite solutions.

8.          Serum and plasma should be handled as potentially hazardous and capable of transmitting disease. Disposable gloves must be worn during the assay procedure, since no known test method can offer complete assurance that products derived from human blood will not transmit infectious agents. Therefore, all blood derivatives should be considered potentially infectious and good laboratory practices should be followed.

9.          All samples should be disposed of in a manner that will inactivate viruses.

10.       Solid Waste: Autoclave 60 min. at 121°C.

11.       Liquid Waste: Add sodium hypochlorite to a final concentration of 1.0%. The waste should be allowed to stand for a minimum of 30 minutes to inactivate the viruses before disposal.

12.       Substrate Solution is easily contaminated. If bluish prior to use, do not use.

13.       Substrate B contains 20% acetone, keep this reagent away from sources of heat or flame.

14.       Remove all kit reagents from refrigerator and allow them to reach room temperature （ 20-25°C）.

ASSAY PROCEDURE

Prepare all Standards before starting assay procedure （see Preparation Reagents）. It is recommended that all Standards and Samples be added

1.          in duplicate to the Microtiter Plate.

2.          First, secure the desired number of coated wells in the holder, then add 100 μL of Standards or Samples to the appropriate well of the antibody pre-coated Microtiter Plate.

3.          Add 50 μL of Conjugate to each well. Mix well. Complete mixing in this step is important. Cover and incubate for 1 hours at 37°C.

4.          Prepare Substrate Solution no more than 15 minutes 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 container. Using a squirt bottle, fill each well compley 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 at 350 μL/well/wash （range: 350-400 μL）. 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 of 10 seconds or shaking time of 5 seconds between washes.

8.          Add 50 μL Substrate A&B to each well. Cover and incubate for 15 minutes at 20-25°C.

9.          Add 50 μL of Stop Solution to each well. Mix well.

10.       Read the Optical Density （O.D.） at 450 nm using a microtiter plate reader within 30 minutes.

B.G CALCULATION OF RESULTS

1.        Calculate the mean absorbance value A₄₅₀ for each set of reference standards and samples. 

2.        Divide the average A450 value for each standard, control and test sample by the average A450 of standard0 and multiply by 100 to obtain %B/B0 for each sample.

3.        Prepare a standard curve by plotting the average absorbance of each standard versus the corresponding concentrations of the standards on linear-log graph paper or the %B/B0 value for each standard versus the corresponding concentration of the standard on linear-log or logit-log graph paper. logit=ln（B/ B0）/（1－B/ B0）

4.        Any values obtained for diluted samples must be further converted by applying the appropriate dilution factor in the calculations.

5.        The standard density is a X, the B/ B0 is a Y, sitting to mark the paper in the logit- log up draw a standard curve.According to the B/ B0 that need to be measured the sample can from sit to mark the density value that the paper looks up the sample up.

6.        The sensitivity by this assay is 0.01ng/ml

7.        The standard curve presented here is an example of the data typically produced with this kit; however, your results will not be identical to these.

8.        Standard curve

Disposal Note Safety

1.          This kit contains materials with small quantities of sodium azide. Sodium azide reacts with lead and copper plumbing to form explosive metal azides. Upon disposal, flush drains with a large volume of water to prevent azide accumulation. Avoid ingestion and contact with eyes, skin and mucous membranes. In case of contact, rinse affected area with plenty of water. Observe all federal, state and local regulations for disposal.

2.          All blood components and biological materials should be handled as potentially hazardous. Follow universal precautions as established by the Centers for Disease Control and Prevention and by the Occupational Safety and Health Administration when handling and disposing of infectious agents.

Additional Materials Required Quality Control

1.          When not in use, kit components should be refrigerated. All reagents should be warmed to room temperature before use.

2.          Microtiter plates should be allowed to come to room temperature before opening the foil bags. Once the desired number of strips has been removed, immediay reseal the bag and store at 2 - 8°C to maintain plate integrity.

3.          Samples should be collected in pyrogen/endotoxin-free tubes.

4.          Samples should be frozen if not analyzed shortly after collection. Avoid multiple freeze-thaw cycles of frozen samples. Thaw compley and mix well prior to analysis.

5.          When possible, avoid use of badly hemolyzed or lipemic sera. If large amounts of particulate matter are present, centrifuge or filter prior to analysis.

6.          It is recommended that all standards, controls and samples be run in duplicate.

7.          When pipetting reagents, maintain a consistent order of addition from well-to-well. This ensures equal incubation times for all wells.

8.          Cover or cap all reagents when not in use.

9.          Do not mix or interchange different reagent lots from various kit lots.

10.       Do not use reagents after the kit expiration date.

11.       Read absorbances within 2 hours of assay completion.

12.       The provided controls should be run with every assay. If control values fall outside pre-established ranges,the accuracy of the assay is suspect.

13.       All residual wash liquid must be drained from the wells by efficient

aspiration or by decantation followed by tapping the plate forcefully on absorbent paper. Never insert absorbent paper directly into the wells.

14.       Because Stabilized Chromogen is light sensitive, avoid prolonged exposure to light. Also avoid contact between Stabilized Chromogen and metal, or color may develop.

15.       Incomplete washing will adversely affect the test outcome. All washing must be performed with Wash Buffer provided.

16.       Washing can be performed manually as follows: compley aspirate the liquid from all wells by gently lowering anaspiration tip （aspiration device） into the bottom of each well. Take care not to scratch the inside of the well.

17.       After aspiration, fill the wells with at least 0.4 mL of diluted wash solution. Let soak for 15 to 30 seconds, then aspirate the liquid. Repeat as directed under ASSAY METHOD. After the washing procedure, the plate is inverted and tapped dry on absorbent tissue.

18.       Alternatively, the wash solution may be put into a squirt bottle. If a squirt bottle is used, flood the plate with wash buffer, compley filling all wells. After the washing procedure, the plate is inverted and tapped dry on absorbenttissue.

19.       If using an automated washer, the operating instructions for washing equipment should be carefully followed.

20.       Assay Procedure Preliminary notes: Do not mix reagents from different lots.It is recommended that assays be performed in duplicate.Standards and samples must be assayed at the same time.Avoid exposing the substrate to direct sunlight.

兔肝细胞生长因子

标本：血清或血浆

使用说明书

产品编号：E04H0208

本试剂盒仅供科研使用，不得用于临床及诊断使用！

预 期 应 用

   ELISA法定量检测兔血清或者血浆中肝细胞生长因子的含量。

试 剂 组 成

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

样本的采集及保存

一般的生物标本包括有：

血液、体液、内脏器官、粪便、胃液、活检组织、组织提取液、天然孔分泌物及各种表达系统的表达产物等

一般为无菌操作，低温保存（-80℃、液氮）

一．如果采集的实质器官则要求：

1、   器官实质不能太小

2、   以采集实质性病灶区为佳：如淋巴结、心脏、非、肺、脾以及肠管等病毒、病菌聚集的地方为佳

3、   同一病例装入同一容器，并做好标记

4、   应在0-8℃的低温储存和运输

5、   实质性器官的处理：

5.1、取实质性器官约0.5mg左右，充分剪碎或研磨

5.2、加入约500ul的PBS/生理盐水，充分混匀

5.3、离心5000rmp x10min，去上清

5.4、-20℃保存，作为待检标本（切勿反复冻融）

二．体液（常见的包括血清、血浆、唾液以及尿液等）的处理方式

1、血液包括血浆和血清，它们的主要区别就是：血清凝血而血浆不凝血，所以它们的处理方式也就不一样

1.1、采集血清时一般不加抗凝剂（主要为枸橼酸盐和柠檬酸盐），采集后立即离心800-1000rmp x 5min分离，取上清，-20℃或4℃保存备用；

1.2、如要采集血浆，一般要加入总体积1%的抗凝剂，采集后先室温或4℃静置半小时后，800-1000rmp x 5min分离，取上清，-20℃或4℃保存备用；

2、胃液和唾液的采集方式一般现采现用，但是一般饭后半小时内不易采集，因为刚进食的唾液中富含丰富的唾液淀粉酶，的采集的时间时

空腹采集；

3、尿液的采集方式基本和唾液一样的，即现采现用，但是一般隔夜尿不采集；

4、组织提取液如肺泡灌洗液等，采集后离心分离，800-1000rmp x 5min，取上清，-20℃或4℃保存备用；

三．粪便以及天然孔排泄物：采集后一般现用PBS/生理盐水溶解，充分混匀，800-1000rmp x 5min分离，取上清，-20℃或4℃保存备用；

四．活检组织一般进行穿刺检测，zui常见的就是肝活检，像病毒性肝炎、脂肪肝、各种类型的肝硬化等进行活检简单方便、准确。

三、表达产物的检测

（一）真核表达产物

1、   细胞上清（分泌性蛋白）

1.2、贴壁细胞或半贴壁，直接将细胞培养上清吸出，10000rmp x10min， 取上清，-20℃或4℃保存，作为标本进行检测，如有需要可进行透析或纯化处理；

1.2、不贴壁细胞，将培养基和细胞转移至10ml离心管，500-800rmp x10min，吸取上清至另一10ml离心管中，10000rmp x10min， -20℃或4℃保存，作为标本进行检测，如有需要可进行透析或纯化处理；

2、   细胞裂解物（非分泌性蛋白）

2.1、贴壁细胞或半贴壁，直接将细胞培养上清吸出，然后用0.01M PBS（pH 7.4）将细胞吹下至10ml离心管，500-800rmp x10min，弃上清，沉淀转移至1.5ml离心管中， DDW重悬，置冰水浴中用超声波裂解仪进行裂解，10000rmp x10min，取上清，-20℃或4℃保存，作为标本进行检测，如有需要可进行透析或纯化处理；

2.2、不贴壁细胞，将培养基和细胞转移至10ml离心管，500-800rmp x10min，弃上清，沉淀移至另一10ml离心管中，001M PBS（pH 7.4）重悬，500-800rmp x10min，弃上清，沉淀移至1.5ml离心管中， DDW重悬，置冰水浴中用超声波裂解仪进行裂解，10000rmp x10min，取上清， -20℃或4℃保存，作为标本进行检测，如有需要可进行透析或纯化处理；

（二）原核（大肠杆菌表达系统）表达产物

1、表达上清（非融合性蛋白）

直接将培养物和上清吸出，10000rmp x10min， 取上清，-20℃或4℃保存，作为标本进行检测，如有需要可进行透析或纯化处理；

2、菌体裂解物（融合性蛋白）

直接将培养物和上清吸出，10000rmp x10min，弃上清，沉淀用PBS洗一遍，500-800rmp x10min，弃上清，DDW重悬沉淀，冰水浴中用超声波裂解仪进行裂解，10000rmp x10min，取上清，-20℃或4℃保存，作为标本进行检测，如有需要可进行透析或纯化处理；

注 意 事 项

1. 试剂准备：所有试剂都必须在使用前达到室温，使用后请立即按照说明书要求保存。实验操作中必须使用一次性吸头，避免交叉污染。

2. 加样：加样时，要控制加样速度，避免*孔与zui后一孔之见的时间间隔过大，否则将会导致不同的预孵育时间，从而影响实验的准确性以及重复性；一般加样时间控制在10分钟内，如果样本数量过多，可使用多道移液器。

3. 孵育：样品要在密闭的容器内进行孵育，严格按照说明书上规定的孵育时间和温度进行。

4. 洗涤：洗涤过程中反应孔中的残留的洗涤液应在滤纸上充分拍干，同时要消除板底残留的液体和手指痕迹，避免影响zui后的酶标仪读数。

5. 反应时间的控制：加入底物后请定时观察反应孔的颜色变化（比如，10分钟左右），如果颜色较深，请提前加入终止液终止反应。

6. 建议实验前预测样品含量，如样品浓度过高，应对样品进行稀释，计算结果时乘以相应的稀释倍数。

7. 建议使用本试剂盒时先做预实验（即先做标准曲线，试用几个标本），如果对本试剂盒有任何疑问，可和所购经销商，如果因运输过程导致试剂盒失效，可要求调换，但概不承担产品本身以外的任何损失。

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100μl的标准品溶液于空白微孔中。

3.          空白微孔中加入100μl的样品，空白对照加入100μl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*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15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.          百分结合率计算：设S0管计数为B0，各标准管或样品管计数为B，非特异管计数为NSB，则百分结合率计算公式如下：B/ B0=（B－NSB）/（ B0－NSB）×*

3.          logit计算:各标准点或样品管的logit值计算公式如下:logit=ln（B/ B0）/（1－B/ B0）

4.          将标准品的OD均值与标准品0点的OD均相除，为标准点的百分结合率，在log-logit坐标纸上绘图。

5.          Log-logit双对数标准曲线：坐标纸上横轴从左至右*个1-9表示为*个10进位，第二个1-9表示为第二个10进位。第三个1-9表示为第三个10进位。坐标纸纵轴为百分比（1-99），即各标准吸光值的百分结合率。取一条通过各点的直线。要求尽可能多的点在线上，同时剩余的点均匀分布在直线的两边。样品也同样由吸光值计算百分结合率，再从纵轴上的相应结合率找到直线上的点，此点对应的横坐标浓度即为样品的浓度，无须换算。

6.          人工处理：以标准浓度取log值为横坐标，对应的logit值为纵坐标在普通坐标纸上或以标准浓度为横坐标，对应的B/B0为纵坐标在logit-log坐标纸上画出标准曲线（理想化时是一条直线）。根据待测样品的B/B0可以从坐标纸上查出样品的浓度值。如果使用普通坐标纸，查出的数值应取反对数才是zui后的浓度值。

7.          自动处理：使用logit-log或四参数数据处理模式，由电脑自动计算得出结果。

8.          敏感度：0.01ng/ml；

9.          图例