上海尊龍凱時生物科技有限公司 www.ruianjituan.com

大鼠中性粒細胞明膠酶相關脂質運載蛋白定量分析酶聯免疫檢測試劑盒

本試劑盒僅供科研使用 。用於體外定量檢測大鼠血清 、血漿或細胞培養上清液中的 NGAL 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 。

NGAL 簡介 ：

中性粒細胞明膠酶相關脂質運載蛋白(NGAL)又名 Lipocalin2 ，屬於 lipocalin 結合蛋白家族 ，也是中性粒細胞的中性粒顆粒的組成部

分 ， NGAL 由 178 個氨基酸殘基組成的單體 ，其分子量大小為 25kD 。NGAL 既能夠自身聚合形成同源二聚體 ，也能夠與 MMP-9 聚合形成異源

二聚體 。

NGAL 在細胞分化 、腫瘤形成以及細胞凋亡等過程中都有一定的作用 。NGAL 是促進多種細胞的生長 、分化的重要因子 ，同時也是一個有

效的抑菌劑 。體內和體外實驗表明 ，NGAL 能夠通過誘導 Ras 的活性控製 Ras 通路 ，從而將上皮細胞改造成間充質 ，影響乳腺腫瘤細胞的侵

襲和轉移行為 。

檢測原理:

本試劑盒采用雙抗體夾心ELISA法檢測樣本中大鼠NGAL的濃度 。大鼠NGAL捕獲抗體已預包被於酶標板上 ，當加入標本或參考品時 ，其

中的大鼠NGAL會與捕獲抗體結合 ，其它遊離的成分通過洗滌的過程被除去 。當加入生物素化的抗大鼠NGAL抗體後 ，抗大鼠NGAL抗體與大鼠

NGAL接合 ，形成夾心的免疫複合物 ，其它遊離的成分通過洗滌的過程被除去 。隨後加入辣根過氧化物酶標記的親合素 。生物素與親合素特

異性結合 ，親合素連接的酶就會與夾心的免疫複合物連接起來 ；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

NGAL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 ，大鼠NGAL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NGAL濃度 。

大鼠NGAL定量分析酶聯免疫檢測試劑盒組成:

組分 規格（96T/48T）

大鼠NGAL預包被板 12條/6條

5×標準品稀釋液 10ml/5ml

大鼠NGAL標準品 2支/1支(凍幹)*

大鼠NGAL生物素化抗體 10ml兩瓶/10ml一瓶

親和素連接的HRP酶 10ml/5ml

濃縮洗滌液 20× 30ml/15ml

TMB底物 10ml/5ml

中止液 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 ；

D.若待測樣本不能及時檢測 ，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 ，剩餘部份請丟棄 。

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5×標準品稀釋液按所需用雙蒸水或去離子水稀釋(1份加4份水) 。

4.標準品:按標簽複溶體積加入1×標準品稀釋液複溶使NGAL終濃度達到5000pg/ml，室溫反應 ，請嚴格控製在25～28℃ ，靜置10~15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5000pg/ml,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 ，取出所需板條置在框架內 ，暫時用不到板條放回鋁箔袋密封 ，保存於4℃ 。

2.建議設置本底較正孔 ，即空白孔,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(100ul/孔)加入相應孔中 ，用封板膠紙封住反應孔 ，室溫(25～28℃)孵育120分鍾 。對於細胞上清標本 ，推

薦稀釋倍數為1-10倍 ，需提前預實驗確定 。對於血清或血漿標本 ，請加入推薦稀釋倍數為100~5000倍 ，根據需要提前預實驗確定。

4.洗板5次 ，且最後一次置厚吸水紙上拍幹 。

5.加入生物素化抗體工作液(100ul/孔) 。用封板膠紙封住反應孔 ，室溫(25～28℃)孵育60分鍾 。

6.洗板5次 ，且最後一次置厚吸水紙上拍幹 。

7.加入親和素連接的HRP酶工作液(100ul/孔) 。用封板膠紙封住反應孔 ，避光室溫(25～28℃)孵育20分鍾。

8.洗板5次 ，且最後一次置厚吸水紙上拍幹 。

9.加入顯色劑TMB100ul/孔 ，避光室溫(25～28℃)孵育20分鍾 。

10.加入中止液50ul/孔,混勻後即刻測量OD450值 。

結果判斷:

上海尊龍凱時生物科技有限公司 www.ruianjituan.com大鼠NGAL參考標準曲線

0

0.5

1

1.5

2

2.5

3

0 156.5 312.5 625 1250 2500 5000

大鼠NGAL濃度(pg/ml)

O

D值

上海尊龍凱時生物科技有限公司 www.ruianjituan.com

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 。

曲線典型數值和參考曲

濃度 pg/ml 平均值 典型數值 1 典型數值 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74pg/ml 。

2.特異性 ：與人Lipocalin-2 、Lipocalin-1及小鼠的Lipocalin-2 、Lipocalin-1等均沒有交叉反應 。與大鼠MMP-9沒有交叉反應 。

3.重複性 ：板內 ，板間變異係數均<10%.

參考文獻:

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