生物素定量測(cè)定試劑盒說(shuō)明書

1、 1st Edition, revised in March, 2015(本試劑盒僅供體外研究使用,不用于臨床診斷!生物素(Biotin定量測(cè)定試劑盒 使用說(shuō)明書Biotin quantitative detection kit 產(chǎn)品貨號(hào):EBD0325 有效期:12個(gè)月使用前請(qǐng)仔細(xì)閱讀說(shuō)明書。如果有任何問(wèn)題,請(qǐng)通過(guò)以下方式聯(lián)系我們:全國(guó)免費(fèi)電話400-660-4808 銷售部電話027-* 技術(shù)部電話027-* 電子郵箱(銷售Perry電子郵箱(技術(shù) techsupportQQ 客服1037150941 網(wǎng)址 聯(lián)系時(shí)請(qǐng)?zhí)峁┊a(chǎn)品貨號(hào)(見(jiàn)試劑盒標(biāo)簽,以便我們更高效地為您服務(wù)。生物素(Biotin

2、定量測(cè)定試劑盒使用說(shuō)明書產(chǎn)品貨號(hào):EBD0325(本試劑盒僅供體外研究使用、不用于臨床診斷!聲明:尊敬的客戶,感謝您選用本公司的產(chǎn)品。本產(chǎn)品適用于體外定量檢測(cè)血清、血漿、乳制品或其它相關(guān)生物液體中生物素濃度。使用前請(qǐng)仔細(xì)閱讀說(shuō)明書并檢查試劑組分!如有疑問(wèn),請(qǐng)及時(shí)聯(lián)系伊萊瑞特生物科技有限公司。試劑盒組成: 特別說(shuō)明:*: 96T/48T(打開包裝后請(qǐng)及時(shí)檢查所有物品是否齊全完整#:一周內(nèi)使用可存于4,需長(zhǎng)時(shí)間存放或多次使用建議存于-20.相關(guān)試劑在分裝時(shí)會(huì)比標(biāo)簽上標(biāo)明的體積稍多一些,請(qǐng)?jiān)谑褂脮r(shí)量取而非直接倒出!檢測(cè)原理:本試劑盒采用競(jìng)爭(zhēng)法。用生物素包被于酶標(biāo)板上,實(shí)驗(yàn)時(shí)樣品或標(biāo)準(zhǔn)品中的生物素與包

3、被的生物素競(jìng)爭(zhēng)辣根過(guò)氧化物酶標(biāo)記的親和素(Avidin-HRP上的結(jié)合位點(diǎn),生物素與親和素特異性結(jié)合而形成復(fù)合物,游離的成分被洗去。加入顯色底物(TMB,TMB在辣根過(guò)氧化物酶的催化下呈現(xiàn)藍(lán)色,加終止液后變成黃色。用酶標(biāo)儀在450nm波長(zhǎng)處測(cè)OD值,生物素濃度與OD450值之間呈反比,通過(guò)繪制標(biāo)準(zhǔn)曲線計(jì)算出樣品中生物素的濃度。試驗(yàn)所需自備物品:1.酶標(biāo)儀(450nm波長(zhǎng)濾光片2.高精度移液器,EP管及一次性吸頭:0.5-10L, 2-20L, 20-200L, 200-1000L3.37恒溫箱,雙蒸水或去離子水4.吸水紙檢測(cè)前準(zhǔn)備工作:1.請(qǐng)?zhí)崆?0分鐘從冰箱中取出試劑盒,平衡至室溫。2.將濃

4、縮洗滌液用雙蒸水稀釋(1:25。未用完的放回4。從冰箱中取出的濃縮洗滌液可能有結(jié)晶,屬于正?，F(xiàn)象,可用40水浴微加熱使結(jié)晶完全溶解后再配制洗滌液(加熱溫度不要超過(guò)50,使用時(shí)洗滌液應(yīng)為室溫。當(dāng)日使用。3.標(biāo)準(zhǔn)品: 于10000×g離心1分鐘,加入標(biāo)準(zhǔn)品&樣品稀釋液1.0mL至凍干標(biāo)準(zhǔn)品中,旋緊管蓋,靜置10分鐘,上下顛倒數(shù)次,待其充分溶解后,輕輕混勻(濃度為20ng/mL。然后根據(jù)需要進(jìn)行倍比稀釋(注:不要直接在反應(yīng)孔中進(jìn)行倍比稀釋。建議配制成以下濃度:20、10、5、2.5、1.25、0.625、0.313、0ng/mL,標(biāo)準(zhǔn)品&樣品稀釋液直接作為空白孔0ng/mL

5、。如配制10ng/mL標(biāo)準(zhǔn)品:取0.5mL 20ng/mL的上述標(biāo)準(zhǔn)品加入含有0.5mL標(biāo)準(zhǔn)品&樣品稀釋液的EP 管中,混勻即可,其余濃度依此類推。4.Avidin-HRP工作液:實(shí)驗(yàn)前計(jì)算當(dāng)次實(shí)驗(yàn)所需用量(以50L/孔計(jì),實(shí)際配制時(shí)應(yīng)多配制100-200L。使用前15分鐘,以酶結(jié)合物稀釋液稀釋濃縮Avidin-HRP (1:100成工作濃度。當(dāng)日使用。標(biāo)準(zhǔn)品稀釋方法圖例:(以500L/管為例,也可根據(jù)實(shí)際用量來(lái)稀釋,如200L/管 1 2 3 4 5 6 7 8 Tube20 10 5 2.5 1.25 0.625 0.313 0 ng/mL洗滌方法:1.自動(dòng)洗板機(jī):每孔加入洗滌液3

6、50L,注入與吸出間隔60秒。2.手工洗板:甩盡孔內(nèi)液體,在潔凈的吸水紙上拍干,每孔加洗滌液350L,浸泡1-2分鐘,吸去(不可觸及板壁或甩掉酶標(biāo)板內(nèi)的液體,在厚的吸水紙上拍干。操作步驟:實(shí)驗(yàn)開始前,各試劑均應(yīng)平衡至室溫;試劑或樣品配制時(shí),均需充分混勻,并盡量避免起泡。1.加樣:分別設(shè)空白孔、標(biāo)準(zhǔn)孔、待測(cè)樣品孔。空白孔加標(biāo)準(zhǔn)品&樣品稀釋液50L,余孔分別加標(biāo)準(zhǔn)品或待測(cè)樣品50L,立即每孔加入配好的Avidin-HRP工作液50L(在使用前15分鐘內(nèi)配制,注意不要有氣泡,加樣時(shí)將樣品加于酶標(biāo)板底部,盡量不觸及孔壁,輕輕晃動(dòng)混勻。給酶標(biāo)板覆膜,37孵育30分鐘。為保證實(shí)驗(yàn)結(jié)果有效性,每次實(shí)

7、驗(yàn)請(qǐng)使用新的標(biāo)準(zhǔn)品溶液。2.棄去孔內(nèi)液體,甩干,洗板5次,每次浸泡1-2分鐘,大約350L/每孔,甩干并在吸水紙上輕拍將孔內(nèi)液體拍干。3.每孔加底物溶液(TMB90L,酶標(biāo)板加上覆膜37避光孵育15分鐘左右(根據(jù)實(shí)際顯色情況酌情縮短或延長(zhǎng),但不可超過(guò)30分鐘。當(dāng)標(biāo)準(zhǔn)孔出現(xiàn)明顯梯度時(shí),即可終止。4.每孔加終止液50L,終止反應(yīng),此時(shí)藍(lán)色立轉(zhuǎn)黃色。終止液的加入順序應(yīng)盡量與底物液的加入順序相同。5.立即用酶標(biāo)儀在450nm波長(zhǎng)測(cè)量各孔的光密度(OD值。應(yīng)提前打開酶標(biāo)儀電源,預(yù)熱儀器,設(shè)置好檢測(cè)程序。6.實(shí)驗(yàn)完畢后將未用完的試劑按規(guī)定的保存溫度放回冰箱保存至有效期結(jié)束。注意事項(xiàng):1.保存:試劑盒中各試

8、劑請(qǐng)按說(shuō)明書提示合理存放。在儲(chǔ)存及溫育過(guò)程中避免將試劑暴露在強(qiáng)光中。所有試劑瓶蓋須旋緊以防止蒸發(fā)和微生物的污染,否則可能會(huì)出現(xiàn)錯(cuò)誤的結(jié)果。2.酶標(biāo)板:剛開啟的酶標(biāo)板孔中可能會(huì)有少許水樣物質(zhì),此為正?，F(xiàn)象,不會(huì)對(duì)實(shí)驗(yàn)結(jié)果造成任何影響。暫時(shí)不用的板條應(yīng)拆卸后放入備用鋁箔袋,按推薦溫度存放!3.加樣:加樣或加試劑時(shí),第一個(gè)孔與最后一個(gè)孔的加樣時(shí)間間隔如果太大,將會(huì)導(dǎo)致不同的“預(yù)溫育”時(shí)間,從而明顯地影響到測(cè)量值的準(zhǔn)確性及重復(fù)性。每次的加樣時(shí)間最好控制在10分鐘內(nèi)。推薦設(shè)置復(fù)孔。4.溫育:為防止樣品蒸發(fā),實(shí)驗(yàn)時(shí)必須給酶標(biāo)板覆膜;洗板后應(yīng)盡快進(jìn)行下步操作,避免酶標(biāo)板處于干燥狀態(tài);嚴(yán)格遵守給定的溫育時(shí)間和

9、溫度。5.洗滌:洗滌過(guò)程中反應(yīng)孔中殘留的洗滌液應(yīng)在吸水紙上拍干,勿將濾紙直接放入反應(yīng)孔中吸水。在讀數(shù)前要注意清除底部殘留的液體和手指印,以免影響酶標(biāo)儀讀數(shù)。6.試劑配制:Concentrated Avidin-HRP體積較小,運(yùn)輸過(guò)程會(huì)使液體沾到管壁或瓶蓋,因此使用前1000轉(zhuǎn)/分離心1min,以使附著管壁或瓶蓋的液體沉積到管底。取用前,請(qǐng)用移液器小心吹打4-5次使溶液混勻。標(biāo)準(zhǔn)品、Avidin-HRP工作液請(qǐng)根據(jù)所需用量配制,并使用相應(yīng)的稀釋液配制,不能混淆。請(qǐng)精確配制標(biāo)準(zhǔn)品及工作液,盡量不要微量配制(如吸取Concentrated Avidin-HRP時(shí),一次不要小于10L,以避免由于不準(zhǔn)

10、確稀釋而造成濃度誤差;請(qǐng)勿重復(fù)使用已稀釋過(guò)的標(biāo)準(zhǔn)品、Avidin-HRP工作液。若需要分次使用標(biāo)準(zhǔn)品應(yīng)按照每一次用量分裝,將其放在-20-80貯存。避免反復(fù)凍融。7.顯色時(shí)間的控制:加入底物后請(qǐng)定時(shí)觀察反應(yīng)孔的顏色變化(比如每隔5分鐘,如梯度已很明顯,請(qǐng)?zhí)崆凹尤虢K止液終止反應(yīng),避免顏色過(guò)深影響酶標(biāo)儀讀數(shù)。8.底物:底物請(qǐng)避光保存,在儲(chǔ)存和溫育時(shí)避免強(qiáng)光直接照射。9.混勻:充分輕微混勻?qū)Ψ磻?yīng)結(jié)果尤為重要,最好使用微量振蕩器(使用最低頻率,如無(wú)微量振蕩器,可在反應(yīng)前手工輕輕敲擊酶標(biāo)板框混勻。10.安全:試驗(yàn)中請(qǐng)穿著實(shí)驗(yàn)服并帶乳膠手套做好防護(hù)工作。特別是檢測(cè)血液或者其他體液樣品時(shí),請(qǐng)按國(guó)家生物試驗(yàn)室

11、安全防護(hù)條例執(zhí)行。11.不同批號(hào)的試劑盒組份不能混用(洗滌液和反應(yīng)終止液除外12.試驗(yàn)中所用的EP管和吸頭均為一次性使用,嚴(yán)禁混用,否則將影響試驗(yàn)結(jié)果!結(jié)果判斷:1.以標(biāo)準(zhǔn)品的濃度為橫坐標(biāo),OD值為縱坐標(biāo),繪制標(biāo)準(zhǔn)曲線。如有設(shè)置復(fù)孔,則應(yīng)取其平均值計(jì)算。以標(biāo)準(zhǔn)品的濃度為橫坐標(biāo),OD值為縱坐標(biāo),繪出標(biāo)準(zhǔn)曲線。亦可以O(shè)D值為橫坐標(biāo),標(biāo)準(zhǔn)品的濃度為縱坐標(biāo),繪出標(biāo)準(zhǔn)曲線。2.推薦使用專業(yè)的曲線制作軟件,如curve expert 1.3或1.4,在軟件界面既可根據(jù)樣品OD值,由標(biāo)準(zhǔn)曲線查出相應(yīng)的濃度,乘以稀釋倍數(shù);亦可將樣品的OD值代入標(biāo)準(zhǔn)曲線的擬合方程式,計(jì)算出樣品濃度,再乘以稀釋倍數(shù),即為樣品的實(shí)

12、際濃度。3.若樣品OD值高于標(biāo)準(zhǔn)曲線上限,應(yīng)適當(dāng)稀釋后重測(cè),計(jì)算濃度時(shí)應(yīng)乘以稀釋倍數(shù)。靈敏度、檢測(cè)范圍、特異性和重復(fù)性:靈敏度:最小可測(cè)0.188ng/mL。檢測(cè)范圍:0.31320ng/mL。 特異性:可檢測(cè)生物素,且與結(jié)構(gòu)類似物無(wú)交叉反應(yīng)。 重復(fù)性:板內(nèi),板間變異系數(shù)均<10%。操作概要 Biotin quantitative determination kitProduct ManualCatalog No: EBD0325(FOR RESEARCH USE ONL Y. DO NOT USE IT IN CLINICAL DIAGONOSIS ! Dear customer, T

13、hank you for choosing our products. Please read the instructions carefully before use and check all the reagent compositions! If in doubt, please contact Elabscience. Valid period: 12 months*: 96T/48T#: Its OK to keep the kit in 4, if the kit is scheduled to be used up in one week. Please keep the r

14、eagent in -20for long-term storage or repeated use.The reagent in each vial is slightly more that its volume written on label, please take out the required volume by certain tools (such as transferpettor, measuring cylinder, rather than pouring directly.Test principleThis kit uses Competitive-method

15、. The microtiter plate provided in this kit has been pre-coated with Biotin. During the reaction, Biotin in the sample or standard competes with a fixed amount of Biotin on the solid phase supporter for sites on the Avidin-HRP specific to Biotin. Excess Avidin-HRP and unbound sample or standard are

16、washed from the plate. Then a TMB substrate solution is added to each well. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of Biotin in the sampl

17、es is then determined by comparing the OD of the samples to the standard curve.Other supplies requiredMicroplate reader with 450nm wavelength filterHigh-precision transferpettor, EP tubes and disposable pipette tips37°C Incubator, Deionized or distilled waterAbsorbent paperReagent preparationBr

18、ing all reagents to room temperature before use.Wash Buffer - Dilute 30mL of Concentrated Wash Buffer into 750 mL of Wash Buffer with deionizedor distilled water. Put unused solution back at 4°C. If crystals have formed in the concentrate, you can warm it with 40°Cwater bath (Heating tempe

19、rature should not exceed 50°C and mix it gently until the crystals have completely dissolved. The solution should be cooled to room temperature before use. Standard -Centrifuge at 10,000×g for 1 minute, and reconstitute the Standard with 1.0mL of Reference Standard &Sample Diluent. Tig

20、hten the lid, let it stand for 10 minutes and turn it upside down for several times. After it dissolves fully, mix it thoroughly with a pipette. This reconstitution produces a stock solution of 20ng/mL. Then make serial dilutions as needed (Making serial dilution inthe wells directly is not permitte

21、d. The recommended concentrations are as follows:20、10、5、2.5、1.25、0.625、0.313、0ng/mL . As if you want to make standard solution at the concentration of 10ng/mL, you can take 0.5mL the standard at 20ng/mL, add it to an EP tube with 0.5mL Reference Standard&Sample Diluent, and mix it. The procedur

22、es of making the remaining concentrations are all the same. The undiluted standard serves as the highest standard (20ng/mL. The Reference Standard &Sample Diluent serves as the zero (0ng/mL.(500L/tube,for example. Can also be diluted according to the actual amount,such as 200L/tube 1 2 3 4 5 6 7

23、 8 Tube20 10 5 2.5 1.25 0.625 0.313 0 ng/mLConcentrated Avidin-HRP Calculate the required amount before experiment (50L/well. In actual preparation you should prepare 100200L more. Dilute the Concentrated Avidin-HRP to the working concentration using HRP Conjugate Diluent (1:100.Washing Procedure:1.

24、 Automated washer: add 350L wash buffer into each well, the interval between injection andsuction should be set about 60s.2.Manual wash: add 350L wash buffer into each well, soak it for 12minutes, suck(no inside walltouching or get rid of liquid within the micro ELISA plate and pat it dry on thick c

25、lean absorbent paper.Assay procedureAllow all reagents to reach room temperature All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming.1.Add Sample and Avidin-HRP : Add 50L of Standard, Blank, or Sample per well. The blank wellis added with Reference Standard

26、 &Sample D iluent. Immediately add 50 L of Avidin-HRP working solution to each well. Cover with the Plate sealer we provided. Gently tap the plate to ensure thorough mixing. Incubate for 30minutes at 37°C. (Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touch

27、ing and foaming to the best of your ability.2.Wash: Aspirate each well and wash, repeating the process five times Wash by filling each wellwith Wash Buffer (approximately 350L using a squirt bottle, multi-channel pipette, manifold dispenser or automated washer. Complete removal of liquid at each ste

28、p is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting.Invert the plate and pat it against thick clean absorbent paper.3.Substrate: Add 90L of Substrate Solution to each well. Cover with a new Plate sealer. Incubatefor about 15 minutes at

29、 37°C. Protect from light. The reaction time can be shortened or extended according to the actual color change, but not more than 30minutes. When apparent gradient appeared in standard wells, you can terminate the reaction.4.Stop: Add 50L of Stop Solution to each well. Color turn to yellow imme

30、diately. The adding orderof stop solution should be as the same as the substrate solution.5.OD Measurement: Determine the optical density (OD value of each well at once, using amicroplate reader set to 450 nm. You should open the microplate reader ahead, preheat the instrument, and set the testing p

31、arameters.6.After experiment, put all the unused reagents back into the refrigerator according to the specifiedstorage temperature respectively until their expiry.Important Note:1.Storage: All the reagents in the kit should be stored following the instructions. Exposure ofreagents to strong light sh

32、ould be avoided in the process of incubation and storage. All the taps of reagents should be tightened to prevent evaporation and microbial contamination, or erroneous results may occur.2.ELISA Plate: Little water-like substance may appear in the ELISA Plate just opened, this isnormal and will not h

33、ave any impact on the experiment results. Keep the remaining plates in spare aluminum foil bag, and keep it in temperature suggested before.3.Add Sample: The interval of sample adding between the first well and the last well should not betoo long, otherwise will cause different pre-incubation time,

34、which will significantly affect the experiments accuracy and repeatability. The interval controlled within 10minutes is good. Parallel measurement is recommended.4.Incubation: To prevent evaporation, proper adhesion of plate sealers during incubation steps isnecessary. Do not allow wells to sit unco

35、vered for extended periods between incubation steps. Do not let the strips dry at any time during the assay. Strict compliance with the given incubation time and temperature.5.Washing: The wash procedure is critical. Insufficient washing will result in poor precision andfalsely elevated absorbance r

36、eadings Residual liquid in the reaction wells should be pat dry against absorbent paper in the washing process. But don't put absorbent paper into reaction wells directly.Note that clear the residual liquid and fingerprint in the bottom before measurement, so as not to affect the microtiter plat

37、e reader.6.Reagent Preparation: As the volume of Concentrated Avidin-HRP is very small, liquid mayadhere to the tube wall or tube cap when being transported. You better hand-throw it or centrifugal it for 1 minute at 1000rpm. Please pipette the solution for 4-5 times before pippeting. Please careful

38、ly reconstitute Standards, working solutions of Avidin-HRP according to the instructions.To minimize imprecision caused by pipetting, ensure that pipettors are calibrated. It is recommended to suck more than 10L for once pipetting. Do not reuse standard solution, working solution of Avidin-HRP, whic

39、h have been diluted. If you need to use standard repeatedly, you can divide the standard into small pack according to the amount of each assay, keep them at -20 -80°C and avoid repeated freezing and thawing7.Reaction Time Control: Please control reaction time strictly following this product des

40、cription!8.Substrate: Substrate Solution is easily contaminated. Please protect it from light.9.Mixing:Youd better use microoscillator at the lowest frequency, as sufficient and gentle mixingis particularly important to reaction result. If there is no microsocillator available, you can knock the ELI

41、SA plate frame gently with your finger before reaction.10.Security: Please wear lab coats and latex gloves for protection. Especially detecting samples ofblood or other body fluid, please perform following the national security columns of biological laboratories.1st Edition, revised in March, 2015 e

42、xception. 12. To avoid cross-contamination, change pipette tips between adding of each standard level, between sample adding, and between reagent adding. Also, use separate reservoirs for each reagent. Otherwise, the results will be inaccurate! Calculation of results Average the duplicate readings for each standard and samples. Create a standard curve by plotting the mean OD value for each standard on the y-axis or x-axis against the concentration on the x-axis or y-axis and draw a best fit curve through the points on t