大豆凝集素SBA-NEWA

1、本試劑盒只能用于科學(xué)研究，不得用于醫(yī)學(xué)診斷 大豆凝集素（SBA） ELISA檢測(cè)試劑盒 使用說明書檢測(cè)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 ）0往 預(yù)先包被大豆凝集素（SBA）抗體的包被微孔中，依次加入標(biāo)本、標(biāo) 準(zhǔn)品、HRP標(biāo)記的檢測(cè)抗體，經(jīng)過溫育并徹底洗滌。用底物 TMB顯 色，TMB在過氧化物酶的催化下轉(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大豆凝集素（SBA）呈正相關(guān)。 用酶標(biāo)儀在450nm波長(zhǎng)下測(cè)定吸光度（OD值），計(jì)算樣品濃度。 樣品收集、處理及保存方法1 .樣本不能含疊氮鈉（NaN3）,因?yàn)榀B氮鈉（NaN3）是辣根過氧化 物酶（HR

2、P）的抑制劑。2 .標(biāo)本采集后盡早進(jìn)行提取，提取按相關(guān)文獻(xiàn)進(jìn)行。3 .植物萃取液或其它相關(guān)樣本：請(qǐng)1000 x g離心20分鐘，取上清即可檢測(cè)4 .保存：如果樣本收集后不及時(shí)檢測(cè)，請(qǐng)按一次用量分裝，凍存于-20C ,避免反復(fù)凍融，在室溫下解凍并確保樣品均勻地充分解凍。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、20-200uL、200-1000uL3 . 37。叵溫箱操作注意事項(xiàng)1 .試劑盒保存在2-8C,使用前室溫平衡20分鐘。從冰箱取出的濃 縮洗滌液會(huì)有結(jié)晶，這屬于正?，F(xiàn)象，水浴加熱使結(jié)晶完全溶解后 再使用。2 .實(shí)驗(yàn)中不用的板條應(yīng)立即放回自

3、封袋中，密封（低溫干燥）保存。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；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。4 .嚴(yán)格按照說明書中標(biāo)明的時(shí)間、加液量及順序進(jìn)行溫育操作。5 .所有液體組分使用前充分搖勻2.自動(dòng)洗板機(jī)：每孔注入洗液350L,浸泡1min,洗板5次。試劑盒組成名稱96孔配置48孔配置備注微孔酶標(biāo)板12孔X8條12孔X4條無標(biāo)準(zhǔn)品0.3mL*6 管0.3mL*6 管無樣本稀釋液6mL3mL無檢測(cè)抗體-HRP10mL5mL無20 X洗滌緩沖液25mL15mL按說明書進(jìn)行稀釋底物A6mL3mL無底物B6mL3mL無終止液6mL3mL無封板膜2張

4、2張無說明書1份1份無自封袋1個(gè)1個(gè)無注：標(biāo)準(zhǔn)品（S0-S5）濃度依次為：0、50、100、200、400、800 pg/mL試劑的準(zhǔn)備20X洗滌緩沖液的稀釋：蒸儲(chǔ)水按1: 20稀釋，即1份的20X洗滌緩沖液加19份的蒸儲(chǔ)水 洗板方法1.手工洗板：甩盡孔內(nèi)液體，每孔加滿洗滌液，靜置 1min后甩盡操作步驟1 .從室溫平衡20min后的鋁箔袋中取出所需板條，剩余板條用自封 袋密封放回4C。2 .設(shè)置標(biāo)準(zhǔn)品孔和樣本孔，標(biāo)準(zhǔn)品孔各加不同濃度的標(biāo)準(zhǔn)品 50L;3 .樣本孔先加待測(cè)樣本10 pL,再加樣本稀釋液40 pL;空白孔不 加。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）

5、標(biāo)記的檢測(cè)抗體100仙L,用封板膜封住反應(yīng)孔，37c水浴 鍋或恒溫箱溫育60min。5 .棄去液體，吸水紙上拍干，每孔加滿洗滌液，靜置 1min,甩去洗 滌液，吸水紙上拍干，如此重復(fù)洗板 5次（也可用洗板機(jī)洗板）。6 .每孔加入底物A、B各50仙L, 37c避光孵育15min。7 .每孔加入終止液50仙L, 15min內(nèi)，在450nm波長(zhǎng)處測(cè)定各孔的孔內(nèi)液體，在吸水紙上拍干，如此洗板 5次OD值結(jié)果判斷試劑盒性能FOR RESEARCH USE ONLY.繪制標(biāo)準(zhǔn)曲線：在Excel工作表中，以標(biāo)準(zhǔn)品濃度作橫坐標(biāo)，對(duì)應(yīng)OD值作縱坐標(biāo)，繪制出標(biāo)準(zhǔn)品線性回歸曲線，按曲線方程計(jì)算各樣 本濃度值。Y1

6、.準(zhǔn)確性：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0.990002 .靈敏度：最低檢測(cè)濃度小于1.0 pg/mLo3 .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)、板間變異系數(shù)均小于 15%。5 .貯藏：2-8 C,避光防潮保存。6 .有效期：6個(gè)月免責(zé)聲明1 .試劑盒僅供研究使用，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，由實(shí)驗(yàn)者承擔(dān)，本公司概不負(fù)責(zé)。2 .嚴(yán)格按照說明書操作，實(shí)驗(yàn)者違反說明書操作，后果由實(shí)驗(yàn)者承 擔(dān)。NOT FOR USE IN DIAGNOSTIC PROCEDURES.Soybean agglutinin (SBA) ELISA Kit

7、 instructionIntended useThis SBA 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 measu

8、re the concentration of SBA in the sample, this SBA 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 SBA concentration. The concentration of SBA in the

9、 samples is then determined by comparing the O.D. of the samples to the standard curve.Sample collection and storages1. Can;t detect the samples which contain NaN3, because NaN3 inhibits HRP activity of the horseradish peroxidase.2. Extract as soon as possible after Specimen collection, Extracted ac

10、cording to the relevant literature.Cell culture supernates and plant exact fluids - Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20 C or -80 C. Avoid repeated freeze-thaw.Materials required but not supplied1. Standard microplate reader(450nm)2. Precisi

11、on pipettes and Disposable pipette tips.3. 37 incubatorPrecautions1. 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 need

12、ed. 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 suppliedName96 determinations48 determinationsMicroelisa stripplate12*8strips12

13、*4stripsStandard0.3ml*6tubes0.3ml*6tubesSample Diluent6.0ml3.0mlHRP-Conjugate reagent10.0ml5.0ml20X Wash solution25ml15mlChromogen Solution A6.0ml3.0mlChromogen Solution B6.0ml3.0mlStop Solution6.0ml3.0mlClosure plate membrane22User manual11Sealed bags11Note： Standard (S0 - S5) concentration was fol

14、lowed by:0,50,100,200,400,800 pg/mlReagent preparation20 Wash solution:Dilute with Distilled or deionized water 1:20.Assay procedure1. Prepare all reagents before starting assay procedure. It is recommended thatall Standards and Samples be added in duplicate to the Microelisa Stripplate.2. Add stand

15、ard: Set Standard wells, testing sample wells. Add standard 50 standard well.3. Add Sample: Add testing sample 1 0then add Sample Diluent 40 . l to testingsample well; Blank well doesn t add anyting.l to each wel4. Add 100 pl of HRP-conjugate reagent to each well, c over with an adhesive strip and i

16、ncubate 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 (400 (J) using a squirt bottle, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good per

17、formance. 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 50 科 l and chromogen solution B 50科Gently mix and incubate for 15 minutes at 37 C. Protect from light.7. Add 50Stop Soluti

18、on 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.Cal

19、culation of results1to 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 s