豬白細(xì)胞介素10（IL-10）ELISA試劑盒說明書
Porcine Interleukin 10 (IL-10)
ELISA Kit
Catalog Number: PR40137
l For the quantitative in vitro determination of IL-10 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 IL-10 supernates, serum, plasma and tissue.
PRINCIPLEThe kit assay Porcine IL-10 level in the sample，use Purified Porcine IL-10 antibody to coat microtiter plate wells, make solid-phase antibody, then add IL-10 to wells, Combined IL-10 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 IL-10 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×4strips
4. 
Standard 1000pg/ml 0.6ml×1 bottle 5. Standard diluent 2ml×1 bottle
6. Biotinylated anti –IL-10–antibody 0.7ml×1 bottle
7. Chromogen Solution A 3ml×1 bottle
8. Chromogen Solution B 3ml×1 bottle
9. Stop Solution 3ml×1 bottle
10. HRP-Conjugate Reagent 3ml×1 bottle
11. Wash Buffer Concentrate (20ml×20 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. Plasma-Collect plasma using EDTA or heparin 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
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 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 immediately at 405 nm against 620 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 :500 pg/ml,250 pg/m,125 pg/ml, 62.5 pg/ml, 31.25pg/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 –IL-10 -antibody and HRP-Conjugate reagent), testing sample wells. add Sample 40μl to testing sample well, then add Biotinylated anti –IL-10 -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: 20-fold Wash Buffer Concentrate diluted 20-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 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.
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:
豬白細(xì)胞介素10（IL-10）ELISA試劑盒說明書
貨號(hào): PR40137
l 本試劑盒用于體外定量檢測(cè)豬血清、血漿、組織、細(xì)胞上清及相關(guān)液體樣本中白細(xì)胞介素10（IL-10）的含量。
l 有效期：6個(gè)月
l 保存條件：2-8℃
使用目的
本試劑盒用于體外定量檢測(cè)豬血清、血漿、組織、細(xì)胞上清及相關(guān)液體樣本中白細(xì)胞介素10（IL-10）的含量。
實(shí)驗(yàn)原理本試劑盒應(yīng)用雙抗體夾心法測(cè)定標(biāo)本中豬白細(xì)胞介素10（IL-10）水平。用純化的豬白細(xì)胞介素10（IL-10）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白細(xì)胞介素10（IL-10），再與HRP標(biāo)記的白細(xì)胞介素10（IL-10）抗體結(jié)合，形成抗體-抗原-酶標(biāo)抗體復(fù)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(lán)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白細(xì)胞介素10（IL-10）呈正相關(guān)。用酶標(biāo)儀在450nm波長(zhǎng)下測(cè)定吸光度（OD值），通過標(biāo)準(zhǔn)曲線計(jì)算樣品中豬白細(xì)胞介素10（IL-10）濃度。
注意事項(xiàng)
l 本試劑盒僅用于科研，不得用于醫(yī)學(xué)診斷。
l 使用試劑盒前，請(qǐng)仔細(xì)閱讀說明書，以試劑盒內(nèi)的說明書為準(zhǔn)，請(qǐng)務(wù)必理解說明書內(nèi)容。
l 酶標(biāo)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。
l 避免實(shí)驗(yàn)過程中酶標(biāo)板干燥；清洗后盡快加試劑。
l 實(shí)驗(yàn)開始，所有試劑應(yīng)平衡到室溫 (21-26°C) 后方可進(jìn)行。溫度會(huì)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(shí)帶一次性手套，微生物的污染會(huì)影響實(shí)驗(yàn)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(jìn)行。
l 過期的試劑盒切勿再使用。
l TMB 底物對(duì)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(yàn)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(biāo)包被板 12孔×4條
4.標(biāo)準(zhǔn) 1000 pg/ml 0.6ml×1 瓶
5.標(biāo)準(zhǔn)品稀釋液 2ml×1 瓶
6.生物素標(biāo)記的抗IL-10抗體 0.7ml×1瓶
7.顯色劑A液 3ml×1瓶
8.顯色劑B液 3ml×1瓶
9.終止液 3ml×1瓶
10.酶標(biāo)試劑 3ml×1瓶
11. 20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(zhǎng)下檢測(cè)的酶標(biāo)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(biāo)準(zhǔn)和樣品稀釋的試管。
儲(chǔ)存條
u 有效期內(nèi)的試劑如開封后未用完，請(qǐng)2-8°C 保存。
u 過期的試劑請(qǐng)不要用，打開后的試劑2-8°C 保存。
u 酶標(biāo)包被板必須2-8°C 保存，如有未用完的酶標(biāo)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(zhǔn)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(jìn)行實(shí)驗(yàn)，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或者肝素作為抗凝劑，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(fù)凍融。
3. 細(xì)胞上清及相關(guān)液體-收集好的樣本離心后應(yīng)盡快實(shí)驗(yàn)，或者分裝后-20°C 或 -80°C 保存，避免反復(fù)凍融。
操作步驟
u 注意事項(xiàng)
l 所有試劑和樣本使用前必須在室溫下平衡，混勻試劑時(shí)不應(yīng)起泡沫。
l 一旦實(shí)驗(yàn)開始，各操作步驟都應(yīng)盡快完成。
l 避免重復(fù)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(shí)間和溫度成正比。
u 操作步驟
1. 標(biāo)準(zhǔn)品的稀釋：準(zhǔn)備小試管6只，依次編好號(hào)碼，先在各小試管中加入標(biāo)準(zhǔn)品稀釋液100ul，然后取原濃度標(biāo)準(zhǔn)品100ul加入一只已編好號(hào)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(hào)標(biāo)準(zhǔn)品。稀釋后各管濃度分別是：500pg/ml,250 pg/ml,125pg/ml,62.5pg/ml,31.25pg/ml ,0 pg/ml。
在酶標(biāo)包被板上設(shè)標(biāo)準(zhǔn)品孔，依次加入不同濃度的標(biāo)準(zhǔn)品50ul（建議每個(gè)濃度做2個(gè)平行孔）。
2. 加樣：分別設(shè)空白孔（空白對(duì)照孔不加樣品、酶標(biāo)試劑及生物素標(biāo)記的抗IL-10抗體，其余各步操作相同）、待測(cè)樣品孔。在酶標(biāo)包被板上待測(cè)樣品孔中先加樣品40μl，然后再加生物素標(biāo)記的抗IL-10抗體10μl。加樣將樣品加于酶標(biāo)板孔底部，盡量不觸及孔壁，輕輕晃動(dòng)混勻。
3. 溫育：用封板膜封板后置37℃溫育30分鐘。
4. 配液：將20倍濃縮洗滌液用蒸餾水20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(fù)5次，拍干。
6. 加酶：每孔加入酶標(biāo)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(shí)藍(lán)色立轉(zhuǎn)黃色）。
11. 測(cè)定：以空白空調(diào)零，450nm波長(zhǎng)依序測(cè)量各孔的吸光度（OD值）。 測(cè)定應(yīng)在加終止液后15分鐘以內(nèi)進(jìn)行
實(shí)驗(yàn)結(jié)果計(jì)算
以標(biāo)準(zhǔn)物的濃度為橫坐標(biāo)，OD值為縱坐標(biāo)，在坐標(biāo)紙上繪出標(biāo)準(zhǔn)曲線，根據(jù)樣品的OD值由標(biāo)準(zhǔn)曲線查出相應(yīng)的濃度；或用標(biāo)準(zhǔn)物的濃度與OD值計(jì)算出標(biāo)準(zhǔn)曲線的直線回歸方程式，將樣品的OD值代入方程式，計(jì)算出樣品濃度，即為樣品的實(shí)際濃度。
技術(shù)支持: