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大鼠中性粒细胞明胶酶相关脂质运载蛋白定量分析酶联免疫检测试剂盒

本试剂盒仅供科研使用。用于体外定量检测大鼠血清、血浆或细胞培养上清液中的 NGAL 浓度.使用前请仔细阅读说明书并检查试剂组

分是否完整,如有产品包装破损或质量投拆，请在收到货一个月之内联系我们。

NGAL 简介：

中性粒细胞明胶酶相关脂质运载蛋白(NGAL)又名 Lipocalin2，属于 lipocalin 结合蛋白家族，也是中性粒细胞的中性粒颗粒的组成部

分， NGAL 由 178 个氨基酸残基组成的单体，其分子量大小为 25kD。NGAL 既能够自身聚合形成同源二聚体，也能够与 MMP-9 聚合形成异源

二聚体。

NGAL 在细胞分化、肿瘤形成以及细胞凋亡等过程中都有一定的作用。NGAL 是促进多种细胞的生长、分化的重要因子，同时也是一个有

效的抑菌剂。体内和体外实验表明，NGAL 能够通过诱导 Ras 的活性控制 Ras 通路，从而将上皮细胞改造成间充质，影响乳腺肿瘤细胞的侵

袭和转移行为。

检测原理:

本试剂盒采用双抗体夹心ELISA法检测样本中大鼠NGAL的浓度。大鼠NGAL捕获抗体已预包被于酶标板上，当加入标本或参考品时，其

中的大鼠NGAL会与捕获抗体结合，其它游离的成分通过洗涤的过程被除去。当加入生物素化的抗大鼠NGAL抗体后，抗大鼠NGAL抗体与大鼠

NGAL接合，形成夹心的免疫复合物，其它游离的成分通过洗涤的过程被除去。随后加入辣根过氧化物酶标记的亲合素。生物素与亲合素特

异性结合，亲合素连接的酶就会与夹心的免疫复合物连接起来；其它游离的成分通过洗涤的过程被除去。最后加入显色剂，若样本中存在

NGAL将会形成免疫复合物，辣根过氧化物酶会催化无色的显色剂氧化成蓝色物质，在加入终止液后呈黄色。通过酶标仪检测，读其450nm

处的OD值，大鼠NGAL浓度与OD450值之间呈正比，通过参考品绘制标准曲线，对照未知样本中OD值，即可算出标本中NGAL浓度。

大鼠NGAL定量分析酶联免疫检测试剂盒组成:

组分 规格（96T/48T）

大鼠NGAL预包被板 12条/6条

5×标准品稀释液 10ml/5ml

大鼠NGAL标准品 2支/1支(冻干)*

大鼠NGAL生物素化抗体 10ml两瓶/10ml一瓶

亲和素连接的HRP酶 10ml/5ml

浓缩洗涤液 20× 30ml/15ml

TMB底物 10ml/5ml

中止液 5ml/3ml

封板胶纸 3/2张

说明书 1份

标本收集:

1.标本的收集请按下列流程进行操作；

A.细胞上清标本离心去除悬浮物后即可；

B.血清标本应是自然凝固后，取上清，避免在冰箱中凝固血液；

C.血浆标本，推荐用EDTA的方法收集；

D.若待测样本不能及时检测，标本收集后请分装，冻存于－20℃，避免反复冻融。

2.血清标本不应添加任何防腐剂或抗凝剂；

3.标本应清澈透明，检测前样本中如有悬浮物应通过离心去除。

4.请勿使用溶血，高血脂或污染的标本检测，否则结果将不准确。

注意事项:1.试剂盒请保存在2～8℃。

2.浓缩洗涤液因在低温下可能有结晶，请水浴加热使结晶完全溶解后再配制工作液。

3.标准品复溶加样后，剩余部份请丢弃。

4.底物请勿接触氧化剂和金属。

5.加样时，请及时更换枪头，避免交叉污染。

6.严禁混用不同批号的试剂盒组份。

7.充分混匀对保证反应结果的准性很重要，在加液后请轻轻叩击边缘以保证混匀。

8.室温反应，请严格控制在25～28℃。

9.洗涤过程是至关重要的，洗涤不充分会使精确度下降并导致结果误差较大。

10.试验中标准品和样本检测时建议作双复孔。

11.加样过程中避免气泡的产生。

12.血清和血浆标本的检测时，检测抗体的孵育时间应适当延长。

检测前准备工作:

1.试剂盒自冰箱中取出后应置室温(25～28℃)平衡20分钟；每次检测后剩余试剂请及时于2～8℃保存。

2.将浓缩洗涤液用双蒸水或去离子水稀释(1份加19份水)。

3. 如有5×标准品稀释液按所需用双蒸水或去离子水稀释(1份加4份水)。

4.标准品:按标签复溶体积加入1×标准品稀释液复溶使NGAL终浓度达到5000pg/ml，室温反应，请严格控制在25～28℃，静置10~15分钟后轻

轻混悬（建议抽吸几次）待彻底溶解，用标准品稀释液倍比梯度稀释后依次加入检测孔中。（标准

曲线取七个点，最高浓度为5000pg/ml,标准品稀释液直接加入作为0浓度.）

洗涤方法:

自动洗板机或人工洗板：每孔洗涤液为300ul，注入与吸出间隔15-30秒。洗板5次。最后一次洗板完成后将板倒扣着在厚吸水纸上用力拍干。

实验过程需自备的材料：

1.不同规格的加样枪及相应的枪头；

2.酶标仪；

3.自动洗板机；

4.去离子水或双蒸水；

操作步骤:

1.通过计算并确定一次性实验所需的板条数，取出所需板条置在框架内，暂时用不到板条放回铝箔袋密封，保存于4℃。

2.建议设置本底较正孔，即空白孔,方法为该孔只加TMB显色液和中止液。每次实验均需做标准品对照并画出标准曲线。

3.分别将标本或不同浓度标准品(100ul/孔)加入相应孔中，用封板胶纸封住反应孔，室温(25～28℃)孵育120分钟。对于细胞上清标本，推

荐稀释倍数为1-10倍，需提前预实验确定。对于血清或血浆标本，请加入推荐稀释倍数为100~5000倍，根据需要提前预实验确定。

4.洗板5次，且最后一次置厚吸水纸上拍干。

5.加入生物素化抗体工作液(100ul/孔)。用封板胶纸封住反应孔，室温(25～28℃)孵育60分钟。

6.洗板5次，且最后一次置厚吸水纸上拍干。

7.加入亲和素连接的HRP酶工作液(100ul/孔)。用封板胶纸封住反应孔，避光室温(25～28℃)孵育20分钟。

8.洗板5次，且最后一次置厚吸水纸上拍干。

9.加入显色剂TMB100ul/孔，避光室温(25～28℃)孵育20分钟。

10.加入中止液50ul/孔,混匀后即刻测量OD450值。

结果判断:

上海传秋生物科技有限公司 www.chuanqiubio.com大鼠NGAL参考标准曲线

0

0.5

1

1.5

2

2.5

3

0 156.5 312.5 625 1250 2500 5000

大鼠NGAL浓度(pg/ml)

O

D值

上海传秋生物科技有限公司 www.chuanqiubio.com

1.复孔的值在20%的差异范围内结果才有效，复孔的值平均后可作为测量值。

2.每个标准品或标本的OD值应减去本底校正孔的OD值。

3.手工绘制标准曲线。以标准品浓度作横坐标，OD值作纵坐标，以平滑线连接各标准品的坐标点。通过标本的OD值可在标准曲线上查出其

浓度。

4.若标本OD值高于标准曲线上限，应适当稀释后重测，计算浓度时应乘以稀释倍数。

曲线典型数值和参考曲

浓度 pg/ml 平均值 典型数值 1 典型数值 2

0 0.09 0.074 0.106

156.5 0.274 0.252 0.296

312.5 0.466 0.452 0.48

625 0.718 0.701 0.735

1250 1.124 1.085 1.163

2500 1.75 1.697 1.803

5000 2.564 2.368 2.76

大鼠NGAL参考标准曲线

注意：本图仅供参考，应以同次试验标准品所绘标准曲线计算标本含量。

灵敏度，特异性和重复性:

1.灵敏度：多次重复结果表明，最小检出量为74pg/ml。

2.特异性：与人Lipocalin-2、Lipocalin-1及小鼠的Lipocalin-2、Lipocalin-1等均没有交叉反应。与大鼠MMP-9没有交叉反应。

3.重复性：板内，板间变异系数均<10%.

参考文献:

1. Kjeldsen, L. et al. (2000) Biochim. Biophys. Acta 1482:272.

2. Mishra, J. et al. (2005) Lancet 365:1231.

3. Berger, T. et al. (2006) Proc. Natl. Acad. Sci. USA 103:1834.

4. Reghunathan, R. et al. (2005) BMC Immunol. 6:2.

5. Friedl, A. et al. (1999) Histochem. J. 31:433.

6. Flo, T.H. et al. (2004) Nature 432:917.

7. Tong, Z. et al. (2005) Biochem. J. 391:441.

ELISA Kit for the Quantitative Analysis of Rat NGAL 上海传秋生物科技有限公司 www.chuanqiubio.com

The rat NGAL ELISA (enzyme-linked immunosorbent assay) kit is used for detection of rat NGAL in cell culture supernatants, rat

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

Neutrophil gelatinase-associated lipocalin (NGAL) also known as Lipocalin2(LCN2). Which is a constituent of neutrophil granules and be

a member of the lipocalin family of binding proteins.

NGAL has been encoded with 178 amino acid(aa) and the molecular weight is 25kD. It can be formation the homologous dimmers by

itself and become the heterologous dimmers with MMP–9.

NGAL plays a role in a variety of processes including cell differentiation, tumorigenesis, and apoptosis. Acting as a growth and

differentiation factor in multiple cell types, NGAL also acts as a potent bacteriostatic reagents. NGAL can alter the invasive and

metastatic behavior of Ras transformed breast cancer cells in vitro and in vivo by reversing epithelial to mesenchymal transition inducing

activity of Ras

Principles of the Test

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

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

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

rat NGAL biotin-conjugated antibody were added and binds to rat NGAL captured by the first antibody, which formed a sandwich.

Streptavidin-HRP would be added and binds to the biotin conjugated antibody, then free Streptavidin-HRP would be removed during a

wash step. 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 mouse NGAL in the original specimen

Materials provided with the kits:

Reagent 96/48Test Kit

Rat NGAL Antibody-Coated Wells 12 strips/6 strips

5×Standard Diluent 20ml/10ml

Rat NGAL Standard 2/1vial(s)

Rat NGAL Detetion Antibody 10ml/5ml

Streptavidin-HRP 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 remains after use of the dissolved standard.

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. Dilute 1ml of 5×sample diluent into 4ml deionized or distilled water to 1×sample diluent.

4. Add standard diluent to the bottle according to the volume of the label and wait 15 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 by aspirating. 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 100ul of standard or sample.Cover with the Plate Covers provided.Incubate for 2 hours at room temperature. If assay the serum

sample,you should diluent the sample to 100~5000 times by preliminary experiment before add into the wells while 1~10 times if you

assay the Cell supernatant sample.

4.Five times wash process were repeated.

5.Add 100ul of detetion antibody. Cover with the Plate Covers provided.Incubate for 1 hour at room temperature.

6.Five times wash process were repeated.

7.Add 100ul of Streptavidin-HRP. Cover with the Plate Covers provided. Lucifugal incubation for 20 minutes at room temperature.

8.Five times wash process were repeated.

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

10.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 percent 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

(pg/ml)

Typical data

1

Typical data 2 Average

0 0.09 0.074 0.106

156.5 0.274 0.252 0.296

312.5 0.466 0.452 0.48

625 0.718 0.701 0.735

1250 1.124 1.085 1.163

2500 1.75 1.697 1.803

5000 2.564 2.368 2.76

Rat NGAL Standard Curve

Sensitivity, Specificity, Repeatability

Sensitivity: repeated assays were evaluated and the minimum detectable dose was74pg/ml.

Specificity : No significant cross-reactivity or interference with human or mouse Lipocalin-2, Lipocalin-1. and has no significant

cross-reactivity with rat MMP-9.

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

REFERENCES:

1. Clauss, M. (2000) Semin. Thromb. Hemost. 26:561.

2. Shibuya, M. (2001) Cell Struct. Funct. 26:25.

3. Woolard, J. et al. (2004) Cancer Res. 64:7822.

4. Yamane, A. et al. (1994) Oncogene 9:2683.

5. Namiki, A. et al. (1995) J. Biol. Chem. 270:31189.

6. Fuh, G. et al. (2000) J. Biol. Chem. 275:26690