大鼠RAT一氧化氮合成酶NOS酶聯(lián)免疫分析

1、大鼠(RAT) 一氧化氮合成酶 (NOS)酶聯(lián)免疫分析試劑盒使用說(shuō)明書(shū)96T本試劑盒僅供研究使用。檢測(cè)范圍：2.5 mo/lL -80 mo/lL使用目的： 本試劑盒用于測(cè)定大鼠血清、血漿及相關(guān)液體樣本中一氧化氮合成酶 (NOS) 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)本中大鼠一氧化氮合成酶(NOS)水平。 用純化的大鼠一氧化氮合成酶 (NOS) 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一氧 化氮合成酶 (NOS)，再與 HRP 標(biāo)記的一氧化氮合成酶 (NOS)抗體結(jié)合，形成抗體 -抗原 -酶標(biāo) 抗體復(fù)合物，經(jīng)過(guò)徹底洗滌后加底物 TMB 顯色。 TMB 在 HRP 酶的

2、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一氧化氮合成酶 (NOS) 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(guò)標(biāo)準(zhǔn)曲線(xiàn)計(jì)算樣品中大鼠一氧化氮合 成酶 (NOS)濃度。試劑盒組成130 倍濃縮洗滌液20ml× 1瓶7終止液6ml × 1 瓶2酶標(biāo)試劑6ml × 1 瓶8標(biāo)準(zhǔn)品( 160 mo/lL )0.5ml ×1 瓶3酶標(biāo)包被板12孔× 8條9標(biāo)準(zhǔn)品稀釋液1.5ml ×1 瓶4樣品稀釋液6ml × 1 瓶10說(shuō)明書(shū)1份5顯色劑 A 液6ml × 1 瓶

3、11封板膜2張6顯色劑 B 液6ml×1/瓶12密封袋1個(gè)標(biāo)本要求1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，提取后應(yīng)盡快進(jìn)行實(shí)驗(yàn)。若不能馬上進(jìn)行試驗(yàn)，可將標(biāo)本放于 -20保存，但應(yīng)避免反復(fù)凍融2不能檢測(cè)含 NaN3的樣品，因 NaN3 抑制辣根過(guò)氧化物酶的( HRP )活性。操作步驟1. 標(biāo)準(zhǔn)品的稀釋?zhuān)?本試劑盒提供原倍標(biāo)準(zhǔn)品一支， 用戶(hù)可按照下列圖表在小試管中進(jìn)行稀釋。80 mo/lL5 號(hào)標(biāo)準(zhǔn)品150l的原倍標(biāo)準(zhǔn)品加入 150l 標(biāo)準(zhǔn)品稀釋液40 mo/lL4 號(hào)標(biāo)準(zhǔn)品150l的 5 號(hào)標(biāo)準(zhǔn)品加入 150 l 標(biāo)準(zhǔn)品稀釋液20 mo/lL3 號(hào)標(biāo)準(zhǔn)品150l的 4 號(hào)標(biāo)準(zhǔn)品加

4、入 150 l 標(biāo)準(zhǔn)品稀釋液10 mo/lL2 號(hào)標(biāo)準(zhǔn)品150l的 3 號(hào)標(biāo)準(zhǔn)品加入 150 l 標(biāo)準(zhǔn)品稀釋液5 mo/lL1 號(hào)標(biāo)準(zhǔn)品150l的 2 號(hào)標(biāo)準(zhǔn)品加入 150 l 標(biāo)準(zhǔn)品稀釋液2. 加樣： 分別設(shè)空白孔 (空白對(duì)照孔不加樣品及酶標(biāo)試劑， 其余各步操作相同) 、標(biāo)準(zhǔn)孔、 待測(cè)樣品孔。 在酶標(biāo)包被板上標(biāo)準(zhǔn)品準(zhǔn)確加樣 50l，待測(cè)樣品孔中先加樣品稀釋液 40l， 然后再加待測(cè)樣品 10l（樣品最終稀釋度為 5 倍）。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 30 倍濃縮洗滌液用蒸餾水 30 倍稀釋后備

5、用5. 洗滌：小心揭掉封板膜，棄去液體，甩干，每孔加滿(mǎn)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 50l ，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 A50 l，再加入顯色劑 B50l，輕輕震蕩混勻， 37避光顯色15 分鐘 .10. 終止：每孔加終止液 50l，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?xún)?nèi)進(jìn)行。操作程序總結(jié)：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(xiàn)，根

6、據(jù)樣品的OD 值由標(biāo)準(zhǔn)曲線(xiàn)查出相應(yīng)的濃度；再乘以稀釋倍數(shù)；或用標(biāo)準(zhǔn)物的濃度與 OD 值計(jì)算出標(biāo)準(zhǔn)曲線(xiàn)的直線(xiàn)回歸方程式， 將樣品的 OD 值代入方程式， 計(jì)算出樣品濃度， 再乘以稀釋倍數(shù)， 即為樣品的實(shí)際濃度。 注意事項(xiàng) 1試劑盒從冷藏環(huán)境中取出應(yīng)在室溫平衡15-30 分鐘后方可使用，酶標(biāo)包被板開(kāi)封后如未用完，板條應(yīng)裝入密封袋中保存。2濃洗滌液可能會(huì)有結(jié)晶析出，稀釋時(shí)可在水浴中加溫助溶，洗滌時(shí)不影響結(jié)果。 3各步加樣均應(yīng)使用加樣器，并經(jīng)常校對(duì)其準(zhǔn)確性，以避免試驗(yàn)誤差。一次加樣時(shí)間最好控制在 5 分鐘內(nèi)，如標(biāo)本數(shù)量多，推薦使用排槍加樣。4 請(qǐng)每次測(cè)定的同時(shí)做標(biāo)準(zhǔn)曲線(xiàn)，最好做復(fù)孔。如標(biāo)本中待測(cè)物質(zhì)含

7、量過(guò)高（樣本OD 值大于標(biāo)準(zhǔn)品孔第一孔的 OD 值），請(qǐng)先用樣品稀釋液稀釋一定倍數(shù)（ n 倍）后再測(cè)定，計(jì) 算時(shí)請(qǐng)最后乘以總稀釋倍數(shù)（× n× 5）。5 封板膜只限一次性使用，以避免交叉污染。6底物請(qǐng)避光保存。7嚴(yán)格按照說(shuō)明書(shū)的操作進(jìn)行，試驗(yàn)結(jié)果判定必須以酶標(biāo)儀讀數(shù)為準(zhǔn).8所有樣品，洗滌液和各種廢棄物都應(yīng)按傳染物處理。9本試劑不同批號(hào)組分不得混用。10. 如與英文說(shuō)明書(shū)有異，以英文說(shuō)明書(shū)為準(zhǔn)。 保存條件及有效期 1試劑盒保存： ； 2-8。 2有效期： 6 個(gè)月Mouse nitric oxide synthase ( NOS )FOR RESEARCH USE ONLYA

8、ssay range： 2.5 mo/lL -80 mo/lL96 determinationsPurposeThis kit allows for the determinationo f NOS concentrationsin Mouses erum, cell culture supernates and other biological fluidsPrinciple of the assayThe kit assay Mouse NOS level in the sam，pleuse Purified Mouse NOS antibody to coat microtiter pl

9、ate wells, make solid-phasea ntibody,t hen add NOS to wells, Combined antibody which With HRP labeled goat anti- Mouse become antibody - antigen - enzyme-antibodyc omplex, after washing Completely,A dd TMB substrate solution,TMB substrate becomes blue color At HRP enzyme-catalyzed, reaction is termi

10、nated by the addition of a sulphurica cid solution and the color changei s measureds pectrophotometricalalyt a wavelength of 450 nm. The concentration of Mouse NOS in the samples is then determined by comparing the O.D. of the samples to the standard curve.Materials provided with the kit1wash soluti

11、on20ml×1bottle7Stopp Solution6ml×1 bottle2HRP-Conjugate reagent 6ml ×1 bottle8Standard(160mo/lL )0.5ml 1× bottle3Microelisa stripplate12well 8×strips9Standard diluent1.5ml 1×bottle4Sample diluent6ml×1 bottle10Instruction15Chromogen Solution A6ml×1 bottle11Clos

12、ure plate membrane26Chromogen Solution B6ml×1 bottle12Sealed bags1Specimen requirements1. extract as soon as possible after Specimen collection,anda ccordingt o the relevant literature,a nd shouldb e experimenta s soon as possiblea fter the extraction.I f it can't, specimen can be kept in -

13、20 to preserve, Avoid repeated freeze-thaw cycles.2. Can' t detect the sample which coNnataNin3 , because NaN3 inhibits HRP active.Assay procedure1. Dilute and add sample:Dil uOtrieginal density Standard as follow table:80 mo/lL5 Standard150l Original density Standar+d150 l Standard diluent40 mo

14、/lL4 Standard150l 5 Standar+d150 l Standard diluent20 mo/lL3 Standard150l 4 Standar+d150 l Standard diluent10 mo/lL2 Standard150l 3 Standard +150l Standard diluent5 mo/lL1 Standard150l 2 Standard +150l Standard diluent2.add sample：Set blank wells separately( blank comparisonw ells don'atd d samp

15、le and HRP-Conjugate reagent, other each step operation is same). testing sample well. add Sample dilution 40tlo testing sample well, then add testing sample 10(ls amplef inal dilution is 5-fold), add sample to welldso ,n ' t touch the well wall as far as poasnsidb lGe,e ntly mix.3.Incubate: Aft

16、er closing plate with Closure plate membrane ,incubate for 30 m. in at 374. Configurate liquid: 30-fold wash solution diluted 30-fold (or 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

17、 then drain, repeat 5 times, dry by pat.6. add enzym：e Add HRP-Conjugate reage5n0t l to each well, excepbtlank well.7.incubate： Operation with 3.8.washing：Operation with 5.9.color：Add Chromogen Solution A 50ul and Chromogen Solution B to each well, evade the light preservation for 15 min at 3710.Sto

18、pt he reaction： Add Stop Solution50tlo each well, Stop the reaction(theb lue color change to yellow color).11.assay：take blank well as zero , Read absorbance at 450nm after Adding Stop Solution andwithin 15min.Steps descriptionStandard, Sample diluentAdd Standard, Sample diluent, incubate for 30 min

19、 a.t 37Wash 5 time,AdHd RP-Conjugate reagent, incubate for 30 min at. 37Wash 5 times,Add Chromogen Solution A and B, incubate for 30 min. at 37Add Stopp SolutionRead absorbance at 450nm within 15 mincalculateCalculateTaket he standardd ensitya s the horizontal,t he OD value for the vertical, draw th

20、e standard curve on graph paper, Find out the corresponding density according to the sample OD value by the Sample curve, multiplied by the dilution multiple, or calculate the straight line regression equation of the standard curve with the standard density and the OD value ,with the sample OD value

21、 in the equation, calculate the sample density, multiplied by the dilution factor, the result is the sample actual density.Important notes1. The kit takes out from the refrigeration environment should be balanced 15-30 minutes in the room temperature, ELISA plates coated if has not use up after open

22、ed, the plate should be stored in Sealed bag.2. washing buffer will Crystallizations eparation,i t can be heated the water helps dissolve when dilute . Washing does not affect the result.3. add Sample with samplerE ach step, And proofreadi ts accuracyf requently,a voids the experimental error. add sample within 5 min, if the numbe