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Human NSE 酶聯(lián)免疫分析（ELISA）
試劑盒使用說明書
貨號: HY10786
本試劑盒用于體外定量檢測人血清、血漿、組織、細胞上清及相關(guān)液體樣本中NSE的含量。
本試劑盒用于體外定量檢測人血清、血漿、組織、細胞上清及相關(guān)液體樣本中NSE的含量。
本試劑盒應用雙抗體夾心法測定標本中人NES水平。用純化的人NES抗體包被微孔板，制成固相抗體，往包被單抗的微孔中依次加入NES，再與HRP標記的NSE抗體結(jié)合，形成抗體-抗原-酶標抗體復合物，經(jīng)過*洗滌后加底物TMB顯色。TMB在HRP酶的催化下轉(zhuǎn)化成藍色，并在酸的作用下轉(zhuǎn)化成zui終的黃色。顏色的深淺和樣品中的NSE呈正相關(guān)。用酶標儀在450nm波長下測定吸光度（OD值），通過標準曲線計算樣品中人NSE濃度。
l 本試劑盒僅用于科研，不得用于醫(yī)學診斷。
l 使用試劑盒前，請仔細閱讀說明書，以試劑盒內(nèi)的說明書為準，請務必理解說明書內(nèi)容。
l 酶標包被板屬于可拆型，開封后如未用完，板條應裝入鋁箔袋密封并2°C-8°C保存。
l 加樣樣本和試劑應盡快，每一步加樣都根據(jù)相同的序列。Pipetting of samples and reagents must be done as quickly as possible and in the same sequence for each step.
l Use reservoirs only for single reagents. This especially applies to the substrate reservoirs. Using a reservoir for dispensing a substrate solution that had previously been used for the conjugate solution may turn solution colored. Do not pour reagents back into vials as reagent contamination may occur.
l Mix the contents of the microplate wells thoroughly to ensure good test results. Do not reuse microwells.
l 避免實驗過程中酶標板干燥；清洗后盡快加試劑。Do not let wells dry during assay; add reagents immediay after completing the rinsing steps.
l 實驗開始，所有試劑應平衡到室溫 （21-26°C） 后方可進行。溫度會影響吸光度值，但不影響樣本值。Allow the reagents to reach room temperature （21-26°C） before starting the test. Temperature will affect the absorbance readings of the assay. However, values for the patient samples will not be affected.
l 切忌用口加樣，以免試劑和樣本粘到皮膚和粘膜上。Never pipet by mouth and avoid contact of reagents and specimens with skin and mucous membranes.
l 不要在處理樣本或試劑的區(qū)域抽煙、吃東西、喝酒或化妝。Do not smoke, eat, drink or apply cosmetics in areas where specimens or kit reagents are handled.
l 操作時帶一次性手套，微生物的污染會影響實驗結(jié)果的正確性。Wear disposable latex gloves when handling specimens and reagents. Microbial contamination of reagents or specimens may give false results.
l 操作應按照國家規(guī)定的微生物安全條列進行。Handling should be done in accordance with the procedures defined by an appropriate national biohazard safety guideline or regulation.
l 過期的試劑盒切勿再使用。Do not use reagents beyond expiry date as shown on the kit labels.
l All indicated volumes have to be performed according to the protocol. Optimal test results are only obtained when using calibrated pipettes and microtiterplate readers.
l Do not mix or use components from kits with different lot numbers. It is advised not to exchange wells of different plates even of the same lot. The kits may have been shipped or stored under different conditions and the binding characteristics of the plates may result slightly different.
l Avoid contact with Stop Solution containing 0.5 M H2SO4. It may cause skin irritation and burns.
l 含有防腐劑、BND或者類似防腐劑MIT的試劑Some reagents contain Proclin, BND and/or MIT as preservatives. In case of contact with eyes or skin, flush immediay with water.
l TMB 底物對皮膚有刺激性，不慎入眼，立即用大量水沖洗，皮膚上不慎接觸到也應立即用肥皂，并用大量的水沖洗。試驗中被污染的物品在下次使用前應盡快清洗。TMB substrate has an irritant effect on skin and mucosa. In case of possible contact, wash eyes with an abundant volume of water and skin with soap and abundant water. Wash contaminated objects before reusing them. If inhaled, take the person to open air.
l 配置的化學品或使用后的試劑，根據(jù)國家微生物安全條列都視為危險廢物。Chemicals and prepared or used reagents have to be treated as hazardous waste according to the national biohazard safety guideline or regulation.
l 參照材料安全系數(shù)表知道試劑盒里的有害物質(zhì)。For information on hazardous substances included in the kit please refer to Material Safety Data Sheets
| 說明書 | 1 | |
| | 2 | |
| 酶標包被板 | 12孔×8條 | 2-8℃ |
| 標準 | 0.5ml×1 瓶 0 ng/ml 0.5ml×1瓶 6.0 ng/ml 0.5ml×1瓶 12 ng/ml 0.5ml×1瓶 24 ng/ml 0.5ml×1瓶 48 ng/ml 0.5ml×1瓶 96 ng/m | 2-8℃ |
| 樣本親和素 | 1.5ml×1瓶 | 2-8℃ |
| 顯色劑A液 | 6ml×1瓶 | 2-8℃ |
| 顯色劑B液 | 6ml×1瓶 | 2-8℃ |
| 終止液 | 6ml×1瓶 | 2-8℃ |
| 酶標試劑 | 6ml×1瓶 | 2-8℃ |
| 30倍濃縮洗滌液 | 20ml×1瓶 | 2-8℃ |
l 吸光度值在450nm波長下檢測的酶標儀。
l 1-2ml的加樣器。
l 100 ml和1L的量筒。
l Calibrated adjustable precision pipettes, preferably with disposable plastic tips. （A manifold multi-channel pipette is desirable for large assays.）
l 吸水紙。
l 37°C 恒溫箱。
l 蒸餾水或去離子水。
l 數(shù)據(jù)分析和繪圖軟件，圖紙：線性（笛卡爾），Data analysis and graphing software. Graph paper: linear （Cartesian）,log-log or semi-log, or log-logit as desired.
l 標準和樣品稀釋的試管。Tubes to prepare standard or sample dilutions.
u 有效期內(nèi)的試劑如開封后未用完，請2-8°C 保存。
u 過期的試劑請不要用，打開后的試劑2-8°C 保存。
u 酶標包被板必須2-8°C 保存，如有未用完的酶標包被板，打開的鋁箔袋須密封并2-8°C 保存。
u 開封后的試劑盒2-8°C只能保存8周。
試劑盒從冷藏環(huán)境中取出應在室溫平衡后方可使用。
血清-用采血管收集血液，室溫血液自然凝固30分鐘，離心15分鐘左右（1000轉(zhuǎn)/分），收集上清，盡早進行實驗，或者分裝后-20°C 或 -80°C 保存。Use a serum separator tube（SST） and allow samples to clot for 30minutes before centrifugation for 15minutes at approximay 1000 xg.Remove serum and assay immediay or aliquot and store samples at -20°C or -80°C.
血漿-收集好的血漿根據(jù)要求選擇EDTA或者肝素作為抗凝劑，2-8°C離心15分鐘左右（1000轉(zhuǎn)/分），30分鐘內(nèi)收集好，-20°C 或 -80°C 保存，避免反復凍融。
細胞上清及相關(guān)液體-收集好的樣本離心后應盡快實驗，或者分裝后-20°C 或 -80°C 保存，避免反復凍融。
u 注意事項
l 所有試劑和樣本使用前必須在室溫下平衡，混勻試劑時不應起泡沫。All reagents and specimens must be allowed to come to room temperature before use. All reagents must be mixed without foaming.
l 一旦實驗開始，各操作步驟都應盡快完成。Once the test has been started, all steps should be completed without interruption.
l 避免重復使用手中的吸頭和試管，以免交叉污染。Use new disposal plastic pipette tips for each standard, control or sample in order to avoid cross contamination.
l 吸光度值是溫育時間和溫度的函數(shù)，實驗開始前，準備好所有的試劑，Absorbance is a function of the incubation time and temperature. Before starting the assay, it is recommended that all reagents are ready, caps removed, all needed wells secured in holder, etc. This will ensure equal elapsed time for each pipetting step without interruption.
l 一般情況，酶的反應與時間和溫度成正比。As a general rule the enzymatic reaction is linearly proportional to time and temperature.
l Determine absorption with an ELISA reader at 450 nm against 620 nm as reference. If no reference wavelength is available, read only at 450nm. If the extinction of the highest standard exceeds the measurement range of the photometer, absorption must be measured immediay at 405 nm against 620 nm as reference.
u 操作步驟
1. 加標準品：在酶標包被板上設標準品孔，依次加入不同濃度的標準品50ul（建議每個濃度做2個平行孔）。
2. 加樣：分別設空白孔（空白對照孔不加樣品及酶標試劑，其余各步操作相同）、待測樣品孔。在酶標包被板上待測樣品孔中先加樣品40μl，然后再加樣品親和素10μl。加樣將樣品加于酶標板孔底部，盡量不觸及孔壁，輕輕晃動混勻。
3. 溫育：用封板膜封板后置37℃溫育30分鐘。
4. 配液：將30倍濃縮洗滌液用蒸餾水30倍稀釋后備用。
5. 洗滌：小心揭掉封板膜，棄去液體，甩干，每孔加滿洗滌液，靜置30秒后棄去，如此重復5次，拍干。
6. 加酶：每孔加入酶標試劑50μl，空白孔除外。
7. 溫育：操作同3。
8. 洗滌：操作同5。
9. 顯色：每孔先加入顯色劑A50μl，再加入顯色劑B50μl，輕輕震蕩混勻，37℃避光顯色15分鐘.
10. 終止：每孔加終止液50μl，終止反應（此時藍色立轉(zhuǎn)黃色）。
11. 測定：以空白空調(diào)零，450nm波長依序測量各孔的吸光度（OD值）。 測定應在加終止液后15分鐘以內(nèi)進行
l Calculate the average absorbance values for each set of standards, controls and patient samples.
l Construct a standard curve by plotting the mean absorbance obtained from each standard against its.
l Concentration with absorbance value on the vertical（Y） axis and concentration on the horizontal （X） axis.
l Using the mean absorbance value for each sample determine the corresponding concentration from the standard curve.
l Automated method: The results in the IFU have been calculated automatically using a 4 PL.
l （4 Parameter Logistics） curve fit. 4 Parameter Logistics is the preferred calculation method. Other data.
l Reduction functions may give slightly different results.
l The concentration of the samples can be read directly from this standard curve. Samples with.
l Concentrations higher than that of the highest standard have to be further diluted. For the calculation of.
l The concentrations this dilution factor has to be taken into account.
REF : Cat.-No.: / Kat.-Nr.: / No.- Cat.: / Cat.-No.: / N.º Cat.: / N.–Cat
LOT : Lot-No.: / Chargen-Bez.: / No. Lot: / Lot-No.: / Lote N.º: / Lotto n.:
:No. of Tests: / Kitgröße: / Nb. de Tests: / No. de Determ.: / N.º de Testes: / Quantità dei tests:
: Keep away from heat or direct sun light. / Vor Hitze und direkter Sonneneinstrahlung schützen. /
Garder à l’abri de la chaleur et de toute exposition lumineuse. / Manténgase alejado del calor o la
luz solar directa. / Manter longe do calor ou luz solar directa. / Non esporre ai raggi solari.
: Read instructions before use. / Arbeitsanleitung lesen. / Lire la fiche technique avant emploi. /Lea las instrucciones antes de usar. / Ler as instruções antes de usar. / Leggere le istruzioni prima dell’uso.
: Store at: / Lagern bei: / Stocker à: / Almacene a :/ Armazenar a :/ Conservare a:
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