本試劑盒只能用于科學(xué)研究奮勇向前，不得用于醫(yī)學(xué)診斷

人（Human）髓鞘相關(guān)生長抑制因子（Nogo-B）

ELISA檢測試劑盒

使用說明書

檢測原理

試劑盒采用雙抗體一步夾心法酶聯(lián)免疫吸附試驗(yàn)（ELISA）。往預(yù)先包被髓鞘相關(guān)生長抑制因子（Nogo-B）抗體的包被微孔中能力建設，依次加入標(biāo)本穩定、標(biāo)準(zhǔn)品效率和安、HRP標(biāo)記的檢測抗體貢獻力量，經(jīng)過溫育并洗滌。用底物TMB顯色規劃，TMB在過氧化物酶的催化下轉(zhuǎn)化成藍(lán)色提高，并在酸的作用下轉(zhuǎn)化成最終的黃色。顏色的深淺和樣品中的髓鞘相關(guān)生長抑制因子（Nogo-B）呈正相關(guān)進入當下。用酶標(biāo)儀在450nm 波長下測定吸光度（OD 值）紮實，計(jì)算樣品濃度。

樣品收集新體系、處理及保存方法

1.  血清：使用不含熱原和內(nèi)毒素的試管投入力度，操作過程中避免任何細(xì)胞刺激，收集血液后尤為突出，3000轉(zhuǎn)離心10分鐘將血清和紅細(xì)胞迅速小心地分離規定。

2.  血漿：EDTA、檸檬酸鹽或肝素抗凝空間載體。3000轉(zhuǎn)離心30分鐘取上清高質量。

3.  細(xì)胞上清液：3000轉(zhuǎn)離心10分鐘去除顆粒和聚合物。

4.  組織勻漿：將組織加入適量生理鹽水搗碎經驗分享。3000轉(zhuǎn)離心10分鐘取上清。

5.  保存：如果樣本收集后不及時(shí)檢測趨勢，請(qǐng)按一次用量分裝有力扭轉，凍存于-20℃，避免反復(fù)凍融一站式服務，在室溫下解凍并確保樣品均勻地充分解凍廣度和深度。

自備物品

1. 酶標(biāo)儀（450nm）

2. 高精度加樣器及槍頭：0.5-10uL、2-20uL引領作用、20-200uL加強宣傳、200-1000uL

3. 37℃恒溫箱

操作注意事項(xiàng)

1.  試劑盒保存在2-8℃，使用前室溫平衡20分鐘用的舒心。從冰箱取出的濃縮洗滌液會(huì)有結(jié)晶技術發展，這屬于正常現(xiàn)象集成，水浴加熱使結(jié)晶溶解后再使用重要手段。

2.  實(shí)驗(yàn)中不用的板條應(yīng)立即放回自封袋中，密封（低溫干燥）保存穩定性。

3.  濃度為0的S0號(hào)標(biāo)準(zhǔn)品即可視為陰性對(duì)照或者空白像一棵樹；按照說明書操作時(shí)樣本已經(jīng)稀釋5倍，最終結(jié)果乘以5才是樣本實(shí)際濃度去突破。

4.  嚴(yán)格按照說明書中標(biāo)明的時(shí)間能運用、加液量及順序進(jìn)行溫育操作達到。

5.  所有液體組分使用前充分搖勻。

試劑盒組成

注：標(biāo)準(zhǔn)品（S0-S5）濃度依次為：可定制

上海軒澤康生物有限公司提供

試劑的準(zhǔn)備

 20×洗滌緩沖液的稀釋：蒸餾水按1：20稀釋不可缺少，即1份的20×洗滌緩沖液加19份的蒸餾水蓬勃發展。

洗板方法

1.  手工洗板：甩盡孔內(nèi)液體，每孔加滿洗滌液重要的角色，靜置1min后甩盡孔內(nèi)液體開放要求，在吸水紙上拍干，如此洗板5次也逐步提升。

2.  自動(dòng)洗板機(jī)：每孔注入洗液350μL記得牢，浸泡1min，洗板5次重要的作用。

操作步驟

1.  從室溫平衡20min后的鋁箔袋中取出所需板條更多可能性，剩余板條用自封袋密封放回4℃。

2.  設(shè)置標(biāo)準(zhǔn)品孔和樣本孔足夠的實力，標(biāo)準(zhǔn)品孔各加不同濃度的標(biāo)準(zhǔn)品50μL緊迫性；

3.  樣本孔先加待測樣本10μL，再加樣本稀釋液40μL更適合；空白孔不加高效。

4.  除空白孔外，標(biāo)準(zhǔn)品孔和樣本孔中每孔加入辣根過氧化物酶（HRP）標(biāo)記的檢測抗體100μL要素配置改革，用封板膜封住反應(yīng)孔體系，37℃水浴鍋或恒溫箱溫育60min。

5.  棄去液體帶動產業發展，吸水紙上拍干責任製，每孔加滿洗滌液，靜置1min倍增效應，甩去洗滌液規則製定，吸水紙上拍干，如此重復(fù)洗板5次（也可用洗板機(jī)洗板）優化服務策略。

6.  每孔加入底物A關規定、B各50μL，37℃避光孵育15min兩個角度入手。

7.  每孔加入終止液50μL安全鏈，15min內(nèi)，在450nm波長處測定各孔的OD值創新為先。

結(jié)果判斷

 繪制標(biāo)準(zhǔn)曲線：在Excel工作表中真正做到，以標(biāo)準(zhǔn)品濃度作橫坐標(biāo)，對(duì)應(yīng)OD值作縱坐標(biāo)創新延展，繪制出標(biāo)準(zhǔn)品線性回歸曲線強化意識，按曲線方程計(jì)算各樣本濃度值長期間。

試劑盒性能

1.  性：標(biāo)準(zhǔn)品線性回歸與預(yù)期濃度相關(guān)系數(shù)R值，大于等于0.9900現場。

2.  靈敏度：檢測濃度小于0.1 ng/mL全過程。

3.  特異性：不與其它可溶性結(jié)構(gòu)類似物交叉反應(yīng)。

4.  重復(fù)性：板內(nèi)探討、板間變異系數(shù)均小于15%不負眾望。

5.  貯藏：2-8℃，避光防潮保存調解製度。

6.  有效期：6個(gè)月

免責(zé)聲明

1.   試劑盒僅供研究使用精準調控，不得用于臨床實(shí)驗(yàn)或人體實(shí)驗(yàn)，否則所產(chǎn)生的一切后果應用的因素之一，由實(shí)驗(yàn)者承擔(dān)解決，本公司概不負(fù)責(zé)。

2.   嚴(yán)格按照說明書操作敢於監督，實(shí)驗(yàn)者違反說明書操作幅度，后果由實(shí)驗(yàn)者承擔(dān)。

FOR RESEARCH USE ONLY. 

NOT FOR USE IN DIAGNOSTIC PROCEDURES.

Human Nogo-B ELISA Kit instruction

Intended use

This Nogo-B 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 Nogo-B in the sample, this Nogo-B 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 Nogo-B concentration. The concentration of Nogo-B in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Sample collection and storages

Serum - Use a serum separator tube and allow samples to clot for 30 minutes before centrifugation for 10 minutes at approximately 3000×g. Remove serum and assay immediately or aliquot and store samples at -20℃ or -80℃.Avoid repeated freeze-thaw cycles

Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 30 minutes at 3000×g at 2-8℃ within 30 minutes of collection. Store samples at -20℃or -80℃. Avoid repeated freeze-thaw cycles.

Cell culture supernates and other biological fluids - Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20℃or -80℃. Avoid repeated freeze-thaw cycles.

Note:  The samples shoule be centrifugated dequately and no hemolysis or granule was allowed.

Materials required but not supplied

1.  Standard microplate reader(450nm)

2.  Precision pipettes and Disposable pipette tips.

3.  37 ℃ incubator

Precautions

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

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

3.  Mix all reagents before using.

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

Materials supplied

Note: Standard (S0 → S5) concentration was followed by:0,0.5,1,2,4,8 ng/ml

Reagent preparation

20×wash solution:Dilute with Distilled or deionized water 1:20.

Assay procedure

1.  Prepare all reagents before starting assay procedure. It is recommended that all Standards and Samples be added in duplicate to the Microelisa Stripplate.

2.  Add standard: Set Standard wells, testing sample wells. Add standard 50μl to standard well.

3.  Add Sample: Add testing sample 10μl then add Sample Diluent 40μl  to testing sample well; Blank well doesn’t add anyting.

4.  Add 100μl of HRP-conjugate reagent to each well, cover with an adhesive strip and incubate for 60 minutes at 37°C. 

5.  Aspirate each well and wash, repeating the process four times for a total of five washes. Wash by filling each well with Wash Solution (400μl) using a squirt bottle, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Solution by aspirating or decanting. Invert the plate and blot it against clean paper towels.

6.  Add chromogen solution A 50μl and chromogen solution B 50μl to each well. Gently mix and incubate for 15 minutes at 37°C. Protect from light.

7.  Add 50μl Stop Solution to each well. The color in the wells should change from blue to yellow. If the color in the wells is green or the color change does not

appear uniform, gently tap the plate to ensure thorough mixing.

8.  Read the Optical Density (O.D.) at 450 nm using a microtiter plate reader within 15 minutes.

Calculation of results

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. (450 nm) 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. To 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 is 0.1 ng/ml

6. Standard curve

Storage：  2-8℃.

validity： six months.

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