人酶联检测剂盒使用说明书,人基质裂解素（ST3）ELISA试剂盒

本试剂仅供研究使用 

供应商：上海乔羽生物科技有限公司

目的：本试剂盒用于测定小鼠血清，血浆及相关液体样本中基质裂解素（ST3）的含量。
实验原理：
     本试剂盒应用双抗体夹心法测定标本中人基质裂解素（ST3）水平。用纯化的转化生长因子（ST3）抗体包被微孔板，制成固相抗体，往包被单抗的微孔中依次加入转化生长因子（ST3），再与HRP标记的羊抗鼠抗体结合，形成抗体-抗原-酶标抗体复合物，经过彻底洗涤后加底物TMB显色。TMB在HRP酶的催化下转化成蓝色，并在酸的作用下转化成最终的黄色。颜色的深浅和样品中的转化生长因子（ST3）呈正相关。用酶标仪在450nm波长下测定吸光度（OD值），通过标准曲线计算样品中人基质裂解素（ST3）浓度。

试剂盒内容及其配制 ：

 
试剂盒组成：
 

自备材料 ：

1. 蒸馏水。

2. 加样器：5ul、10ul 、 50ul 、 100ul 、 200ul 、500ul、1000ul。

3. 振荡器及磁力搅拌器等。

样品收集、处理及保存方法 ：

1、血清……操作过程中避免任何细胞刺激。使用不含热原和内毒素的试管。收集血液后，1000×g离心10分钟将血清和红细胞迅速小心地分离。

2、 血浆……EDTA、柠檬酸盐、肝素血浆可用于检测。1000 ×g离心30分钟去除颗粒。

3、 细胞上清液……1000 ×g离心10分钟去除颗粒和聚合物。

4、 组织匀浆……将组织加入适量生理盐水捣碎。1000 ×g离心10分钟，取上清液。

 5、 保存……如果样品不立即使用，应将其分成小部分-70℃保存，避免反复冷冻。尽可能的不要使用溶血或高血脂血。如果血清中大量颗粒，检测前先离心或过滤。不要在37℃或更高的温度加热解冻。应在室温下解冻并确保样品均匀地充分解冻。

人基质裂解素ELISA试剂盒操作注意事项：

●   试剂应按标签说明书储存，使用前恢复到室温。稀稀过后的标准品应丢弃，不可保存。

●   实验中不用的板条应立即放回包装袋中，密封保存，以免变质。

●   不用的其它试剂应包装好或盖好。不同批号的试剂不要混用。保质前使用。

●   使用一次性的吸头以免交叉污染，吸取终止液和底物A、B液时，避免使用带金属部分的加样器。

●   使用干净的塑料容器配置洗涤液。使用前充分混匀试剂盒里的各种成份及样品。

●   底物A应挥发，避免长时间打开盖子。底物B对光敏感，避免长时间暴露于光下。避免用手接触，有毒。实验完成后应立即读取OD值。

●   加入试剂的顺序应一致，以保证所有反应板孔温育的时间一样。

●   按照说明书中标明的时间、加液的量及顺序进行温育操作。

安全性 ：

1.   避免直接接触终止液和底物A、B，一旦接触到这些液体，请尽快用水冲洗。

2.   实难中不要吃喝、抽烟或使用化妆品。

3.   不要用嘴吸取试剂盒里的任何成份。

试剂的准备 ：

1.   标准品：标准品的系列稀释应在实验时准备，不能储存。稀释前将标准品振荡混匀。稀释比例按下表中进行：

2.   洗涤缓冲液（50×）的稀释：蒸馏水50倍稀释。

试剂盒性能 ：

1.   灵敏度：最小的检测浓度小于1号标准品。稀释度的线性。样品线性回归与预期浓度相关系数R值为0.990。

2.   特异性：不与其它细胞因子反应。

3.   重复性：板内、板间变异系数均小于10%。

人基质裂解素ELISA检测试剂盒 操作步骤：

1.   使用前，将所有试剂充分混匀。不要使液体产生大量的泡沫，以免加样时加入大量的气泡，产生加样上的误差。

2.   根据待测样品数量加上标准品的数量决定所需的板条数。每个标准品和空白孔建议做复孔。每个样品根据自己的数量来定，能使用复孔的尽量做复孔。

3.   加入稀释好后的标准品50ul 于反应孔、加入待测样品 50 ul 于反应孔内。立即加入 50 ul的生物素标记的抗体。盖上膜板，轻轻振荡混匀，37℃温育45分钟。

4.   甩去孔内液体，每孔加满洗涤液，振荡30秒，甩去洗涤液，用吸水纸拍干。重复此操作4次。如果用洗板机洗涤，洗涤次数增加一次。

5.   每孔加入100ul的亲和链酶素-HRP，轻轻振荡混匀，37℃温育30分钟。

6.   甩去孔内液体，每孔加满洗涤液，振荡30秒，甩去洗涤液，用吸水纸拍干。重复此操作4次。如果用洗板机洗涤，洗涤次数增加一次。

7.   每孔加入底物A、B各50ul，轻轻振荡混匀，37℃温育5分钟。避免光照。

8.   取出酶标板，迅速加入50ul终止液，加入终止液后应立即测定结果。

9.   在450nm波长处测定各孔的OD值。

结 果 判 断 与 分 析 ：

1、 仪器值：于波长450nm的酶标仪上读取各孔的OD值

2、 以吸光度OD值为纵坐标（ Y ），相应的 OT 标准品浓度为横坐标（ X ），做得相应的曲线，样品的 OT 含量可根据其OD 值由标准曲线换算出相应的浓度，再乘以稀释倍数；或用标准物的浓度与OD值计算出标准曲线的回归方程式，将样品的OD值代入方程式，计算出样品浓度，再乘以稀释倍数，即为样品的实际浓度。

3、 检测值范围： 0-100ng/ml

4、 敏感度：0.39ng/ml

Owned material:

1 distilled water.

2 pipettes: 5ul, 10ul, 50ul, 100ul, 200ul, 500ul, 1000ul.

3 oscillator and a magnetic stirrer etc..

The collection, processing and preservation methods samples:

1, serum...... To avoid any cell stimulation during operation. Pyrogen and endotoxin free tube. Blood was collected after 1000 x g centrifugation for 10 minutes in serum and red blood cells rapidly and carefully isolated.

2, plasma...... EDTA, citrate and heparin plasma can be used to detect. 1000 * g centrifuge for 30 minutes to remove particles.

3, cell supernatant...... 1000 * g centrifuge for 10 minutes to remove particles and polymer.

4, homogenate...... The organization will add saline ground. 1000 * g supernatant was centrifuged for 10 minutes.

5, save...... If the samples are not used immediately, it should be divided into small parts of -70 C preservation, to avoid repeated freezing. As far as possible do not use hemolysis or high blood lipids. If a large number of particles in the serum, testing prior to centrifugation or filtration. Don't thaw at 37 DEG C or higher heating temperature. Should be thawed at room temperature and ensure the uniform fully thawed samples.

Human tissue factor ELISA kit:

The reagent should label storage instructions, return to room temperature before use. The standard should be discarded after thinning, can not be saved.

No - Experiment strips should be immediately put back the bag, sealed to prevent spoilage.

Other reagents - not should be packaged or covered. Do not mix different batches of reagents. Shelf life before use.

] using disposable suction head to avoid cross contamination and absorb the termination of the liquid and the substrate of a and B liquid, avoid using metal parts of the sample adding device.

Plastic container configuration washing liquid - clean. All samples of ingredients and mix well before use in the kit.

The substrate A should be volatile, avoid long time to open the lid. The B substrate is sensitive to light, avoid long time exposure to light. Avoid hand contact, toxic. After completion of the experiment should be immediately read od.

The added reagents should be consistent in order to ensure that all reaction plate hole incubation time.

The temperature in accordance with the amount of liquid and the sequence of instructions indicated time, sterile operation.

Safety:

1 to avoid direct contact with the stop solution and the substrate A, B, once exposed to these fluids, wash with water as soon as possible.

2 to eat and drink, do not smoke or use cosmetics.

3 don't learn any of the ingredients of the reagent box with the mouth.

Preparation of reagent:

1: standard serial dilution standard should be prepared in the experiment, can not be stored. The dilution standard oscillating mixing. Press the dilution ratio in the table:

One hundred

Ng/ml (Standard No. 6) times the original concentration without dilution directly into 50ul

Fifty

Ng/ml (5 standard) standard product original times standard 100ul joined 100ul dilution

Twenty-five

Ng/ml (4 standard) standard 100ul Standard No. 5 product with 100ul diluent

Twelve point five

Ng/ml (3 standard) standard 100ul Standard No. 4 product with 100ul diluent

Six point two five

Ng/ml (2 standard) standard 100ul Standard No. 3 product with 100ul diluent

Three point one two

Ng/ml (1 standard) standard 100ul Standard No. 2 product with 100ul diluent

Zero

Ng/ml (blank control) the original concentration times without dilution directly into 50ul

2 washing buffer (50 *) dilution: distilled water diluted 50 times.

The performance of kit:

1 sensitivity: minimum detection concentration is less than 1 standard. Linear dilution. Sample linear regression correlation with expected concentration coefficient R value is 0.990.

2: no specific reaction with other cytokines.

3 repeatability: plate, plate between the coefficients of variation were less than 10%.

Human tissue factor ELISA kit steps:

1 before use, all reagents and mixing. Don't make the liquid to produce a large amount of foam, so as to avoid when adding a lot of bubbles, the error on the sample.

2 according to determine the number of sample number plus standard strip number. Each standard and blank should be wells. Each sample is determined by the number of them, can use complex hole to do complex hole.

3 diluted after standard 50ul in reaction hole, added to the sample 50 UL in reaction hole to be measured. Immediately joined the 50 UL antibody biotin. Cover film plate, gently shake, 37 degrees 45 minutes of incubation.

4 left hole liquid, each hole filled with washing liquid, oscillating 30 seconds off the washing liquid, pat dry with absorbent paper. This operation is repeated 4 times. If you use the washing machine washing, washing times increase a.

5 per hole adding chain affinity enzyme -HRP 100ul, gently oscillating mixing, 37 degrees 30 minutes incubation.

6 left hole liquid, each hole filled with washing liquid, oscillating 30 seconds off the washing liquid, pat dry with absorbent paper. This operation is repeated 4 times. If you use the washing machine washing, washing times increase a.

7 per hole adding substrate A, B 50ul, gently oscillating mixing, 37 degrees 5 minutes incubation. Avoid light.

8 remove ELISA plate, quickly add 50ul terminated liquid, adding the stop solution immediately after the determination results.

9 od determination of each hole at the wavelength of 450nm.

The result of judgment and analysis:

1, instrument value: Yu Bo 450nm ELISA od read the hole on the instrument

2, to the OD value as a vertical coordinate (y), corresponding ot standard concentrations as a horizontal coordinate (x), do have corresponding curve, sample ot content can be according to the OD value by standard curve conversion out corresponding concentration, multiplied by the dilution multiple; or with the standard concentration and the OD value calculated the regression equation of the standard curve, the sample OD value in the equation.

3, the detection range: 0-100ng/ml

4, sensitivity: 0.39ng/ml

人酶联检测剂盒使用说明书，人基质裂解素(ST3)ELISA试剂盒