豬腫瘤壞死因子α（TNF-α）ELISA試劑盒PorcineTumor necrosis factor α (TNF-α)ELISA Kit
Catalog Number: PR40133
l For the quantitative in vitro determination of TNF-α concentrations in Porcine culture supernates, serum, plasma and tissue.
l Expiration date：six months .
l Storage： 2-8℃.
INTENDED USE
An enzyme immunoassay quantitative measurement in cell culture in vitro Porcine TNF-α supernates, serum, plasma and tissue.
PRINCIPLE
The kit assay Porcine TNF-α level in the sample，use Purified Porcine TNF-α antibody to coat microtiter plate wells, make solid-phase antibody, then add TNF-α to wells, Combined TNF-α antibody which With HRP labeled , become antibody - antigen - enzyme- antibody complex, after washing Completely, Add TMB substrate solution,TMB substrate becomes blue color At HRP enzyme-catalyzed, reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm. The concentration of TNF-α in the samples is then determined by comparing the O.D. of the samples to the standard curve.
WARNINGS AND PRECAUTIONS
l This kit is only for scientific research, and shall not be used as a clinical diagnosis of use.
l Before starting the assay, read the instructions completely and carefully. Use the valid version of the package insert provided with the kit. Be sure that everything is understood.
l The microplate contains snap-off strips. Unused wells must be stored at 2 °C to 8 °C in the sealed foil pouch and used in the frame provided.
l 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 Do not let wells dry during assay; add reagents immediately after completing the rinsing steps.
l 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 Never pipet by mouth and avoid contact of reagents and specimens with skin and mucous membranes.
l Do not smoke, eat, drink or apply cosmetics in areas where specimens or kit reagents are handled.
l Wear disposable latex gloves when handling specimens and reagents. Microbial contamination of reagents or specimens may give false results.
l Handling should be done in accordance with the procedures defined by an appropriate national biohazard safety guideline or regulation.
l 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 Some reagents contain Proclin, BND and/or MIT as preservatives. In case of contact with eyes or skin, flush immediately with water.
l 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 Chemicals and prepared or used reagents have to be treated as hazardous waste according to the national biohazard safety guideline or regulation.
l For information on hazardous substances included in the kit please refer to Material Safety Data Sheets
MATERIALS PROVIDED WITH THE KIT
1. Instruction 1
2. Closure Plate Membrane 2
3. Microelisa Stripplate 12well×8strips
4. Standard 1600pg/ml 0.6ml×1 bottle
5. Standard diluent 2ml×1 bottle
6. Biotinylated anti –TNF-α–antibody 1.5ml×1 bottle
7. Chromogen Solution A 6ml×1 bottle
8. Chromogen Solution B 6ml×1 bottle
9. Stop Solution 6ml×1 bottle
10. HRP-Conjugate Reagent 6ml×1 bottle
11. Wash Buffer Concentrate (20ml×30 fold)×1bottle
MATERIALS REQUIRED BUT NOT PROVIDED
l Microplate reader capable of measuring absorbance at 450 nm.
l Precision pipettes to deliver 2 ml to 1 ml volumes.
l 100 ml and 1 liter graduated cylinders.
l Calibrated adjustable precision pipettes, preferably with disposable plastic tips. (A manifold multi-channel pipette is desirable for large assays.)
l Absorbent paper.
l 37°C incubator.
l Distilled or deionized water.
l Data analysis and graphing software. Graph paper: linear (Cartesian),log-log or semi-log, or log-logit as desired.
l Tubes to prepare standard or sample dilutions.
STORAGE CONDITIONS
u When stored at 2 °C to 8 °C unopened reagents will retain reactivity until expiration date.
u Do not use reagents beyond this date. Opened reagents must be stored at 2 °C to 8 °C.
u Microtiter wells must be stored at 2 °C to 8 °C. Once the foil bag has been opened, care should be taken to close it tightly again.
u Opened kits retain activity for 8 weeks if stored as described above.
REAGENT PREPARATION
Bring all reagents to room temperature before use
SPECIMEN COLLECTION AND PREPARATION
1. Serum-Use a serum separator tube(SST) and allow samples to clot for 30minutes before centrifugation for 15minutes at approximately 1000 xg.Remove serum and assay immediately or aliquot and store samples at -20°C or -80°C.
2. PlasmaCollect plasma using EDTA or Citric acid sodium as an anticoagulant.Centrifuge samples for 15 minutes at 1000 x g at 2-8°C within 30minutes of collection. Store samples at -20°C or -80°C.Avoid repeated freeze-thaw cycles.
3. Cell culture fluid and other biological fluids-Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20°C or -80°C.Avoid repeated freeze-thaw cycles.
ASSAY PROCEDURE
u General Remarks豬腫瘤壞死因子α（TNF-α）ELISA試劑盒
l All reagents and specimens must be allowed to come to room temperature before use. All reagents must be mixed without foaming.
l Once the test has been started, all steps should be completed without interruption.
l Use new disposal plastic pipette tips for each standard, control or sample in order to avoid cross contamination.
l 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 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 630 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 immediately at 450 nm against 630 nm as reference.
u Assay Procedure
1. Dilute standard: Prepare sixs test tube ,make number successively,add Standard diluent 100ul to ervery test tube,add Original density Standard 100ul to the first test tube, Gently mix;then take out 100ul from the first test tube and add to the second test tube, Gently mix; then take out 100ul from the second test tube and add to the third test tube, Gently mix; then take out 100ul from the third test tube and add to the forth test tube, Gently mix; then take out 100ul from the forth test tube and add to the fifth test tube, Gently mix; take out 100ul from the fifth test tube and Discard,make the sixth test tube as standard 0. the density Standard of every test tube is :800 pg/ml,400 pg/m,200 pg/ml,100 pg/ml,50pg/ml,0 pg/ml.
set Standard wells on the Microelisa Stripplate , add different concentrations of standard 50 ul successively .
2. Add samples: Set blank wells separately (blank comparison wells don’t add samples ,Biotinylated anti –TNF-α -antibody and HRP-Conjugate reagent), testing sample wells. add Sample 40μl to testing sample well, then add Biotinylated anti –TNF-α -antibody 10μl , don’t touch the well wall as far as possible, and Gently mix.
3. Incubate: After closing plate with Closure plate membrane ,incubate for 30 min at 37℃.
4. Configurate liquid: 30-fold Wash Buffer Concentrate diluted 30-fold with distilled water and reserve.
5. washing：Uncover Closure plate membrane, discard Liquid, dry by swing, add washing buffer to every well, still for 30s then drain, repeat 5 times, dry by pat.
6. add enzyme：Add HRP-Conjugate Reagent 50μl to each well, except the blank well.
7. incubate：Operation with 3.
8. washing：Operation with 5.
9. color：add Chromogen Solution A 50μl and Chromogen Solution B 50μl to each well. Gently mix, incubate for 15 min at 37℃.
10. Stop the reaction：Add Stop Solution50μl to each well, Stop the reaction(the blue color change to yellow color Immediately).
11. assay：take blank well as zero , measure the optical densit (OD) at 450 nm after Adding Stop Solution and within 15min.
CALCULATION OF RESULTS
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 verticalY) 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.
REFERENCES
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:
豬腫瘤壞死因子α（TNF-α）酶聯(lián)免疫分析(ELIA)
試劑盒使用說明書
貨號: PR40133
l 本試劑盒用于體外定量檢測豬血清、血漿、組織、細胞上清及相關(guān)液體樣本中腫瘤壞死因子α（TNF-α）的含量。
l 有效期：6個月
l 保存條件：2-8℃
使用目的
本試劑盒用于體外定量檢測豬血清、血漿、組織、細胞上清及相關(guān)液體樣本中腫瘤壞死因子α（TNF-α）的含量。
實驗原理
本試劑盒應(yīng)用雙抗體夾心法測定標本中豬腫瘤壞死因子α（TNF-α）水平。用純化的豬腫瘤壞死因子α（TNF-α）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腫瘤壞死因子α（TNF-α），再與HRP標記的腫瘤壞死因子α（TNF-α）抗體結(jié)合，形成抗體-抗原-酶標抗體復合物，經(jīng)過徹底洗滌后加底物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)化成最終的黃色。顏色的深淺和樣品中的腫瘤壞死因子α（TNF-α）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豬腫瘤壞死因子α（TNF-α）濃度。
注意事項
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(wù)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(yīng)裝入鋁箔袋密封并2-8°C保存。
l 加樣樣本和試劑應(yīng)盡快完成。豬腫瘤壞死因子α（TNF-α）ELISA試劑盒
l 避免實驗過程中酶標板干燥；清洗后盡快加試劑。
l 實驗開始，所有試劑應(yīng)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。
l 切忌用口加樣，以免試劑和樣本粘到皮膚和粘膜上。
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。
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。
l 操作應(yīng)按照國家規(guī)定的安全條列進行。
l 過期的試劑盒切勿再使用。
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(yīng)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(yīng)盡快清洗。
試劑盒組成
1.說明書 1份
2.封板膜 2張
3.酶標包被板 12孔×8條
4.標準 1600 pg/ml 0.6ml×1 瓶
5.標準品稀釋液 2ml×1 瓶
6.生物素標記的抗TNF-α抗體 1.5ml×1瓶
7.顯色劑A液 6ml×1瓶
8.顯色劑B液 6ml×1瓶
9.終止液 6ml×1瓶
10.酶標試劑 6ml×1瓶
11. 30倍濃縮洗滌液 20ml×1瓶
需要而未提供的試劑和器材
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 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。
l 標準和樣品稀釋的試管。
儲存條件
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。
試劑準備
試劑盒從冷藏環(huán)境中取出應(yīng)在室溫平衡后方可使用。
樣本處理及要求
1. 血清-用采血管收集血液，室溫血液自然凝固30分鐘，1000 xg離心15分鐘左右，收集上清，盡早進行實驗，或者分裝后-20°C 或 -80°C 保存。
2. 血漿-收集好的血漿根據(jù)要求選擇EDTA或者檸檬酸鈉作為抗凝劑，2-8°C 1000 xg離心15分鐘左右，30分鐘內(nèi)收集好，-20°C 或 -80°C 保存，避免反復凍融。
3. 細胞上清及相關(guān)液體-收集好的樣本離心后應(yīng)盡快實驗，或者分裝后-20°C 或 -80°C 保存，避免反復凍融。
操作步驟
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(yīng)起泡沫。
l 一旦實驗開始，各操作步驟都應(yīng)盡快完成。
l 避免重復使用手中的吸頭和試管，以免交叉污染。
l 一般情況，酶的反應(yīng)與時間和溫度成正比。
u 操作步驟
1. 標準品的稀釋：準備小試管6只，依次編好號碼，先在各小試管中加入標準品稀釋液100ul，然后取原濃度標準品100ul加入一只已編好號的試管中，充分混勻;再在該試管中取100ul加入第二支試管中，充分混勻；再在該試管中取100ul加入第三只試管中，充分混勻；再在該試管中取100ul加入第四只試管中，充分混勻；再在該試管中取100ul加入第五只試管中，充分混勻；然后在該試管中取100ul，棄掉。第六只試管作為0號標準品。稀釋后各管濃度分別是：800pg/ml,400 pg/ml,200 pg/ml,100pg/ml,50pg/ml ,0 pg/ml。
在酶標包被板上設(shè)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(shè)空白孔（空白對照孔不加樣品、酶標試劑及生物素標記的抗TNF-α抗體，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，然后再加生物素標記的抗TNF-α抗體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(yīng)（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(yīng)在加終止液后15分鐘以內(nèi)進行
實驗結(jié)果計算 豬腫瘤壞死因子α（TNF-α）ELISA試劑盒
以標準物的濃度為橫坐標，OD值為縱坐標，在坐標紙上繪出標準曲線，根據(jù)樣品的OD值由標準曲線查出相應(yīng)的濃度；或用標準物的濃度與OD值計算出標準曲線的直線回歸方程式，將樣品的OD值代入方程式，計算出樣品濃度，即為樣品的實際濃度。
技術(shù)支持:阿儀網(wǎng)
