大鼠果糖胺（FA）酶聯(lián)免疫分析

1、大鼠果糖胺（大鼠果糖胺（FA）酶聯(lián)免疫分析）酶聯(lián)免疫分析試劑盒使用說明書試劑盒使用說明書本試劑盒僅供研究使用。檢測范圍：檢測范圍： 96T50mol/L -1600mol/L使用目的：使用目的：本試劑盒用于測定大鼠血清、血漿及相關(guān)液體樣本中果糖胺（FA）含量。實(shí)驗(yàn)原理實(shí)驗(yàn)原理本試劑盒應(yīng)用雙抗體夾心法測定標(biāo)本中大鼠果糖胺（FA）水平。用純化的大鼠果糖胺（FA）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果糖胺（FA） ，再與 HRP 標(biāo)記的果糖胺（FA）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

2、下轉(zhuǎn)化成最終的黃色。顏色的深淺和樣品中的果糖胺（FA）呈正相關(guān)。用酶標(biāo)儀在 450nm 波長下測定吸光度（OD 值） ，通過標(biāo)準(zhǔn)曲線計(jì)算樣品中大鼠果糖胺（FA）濃度。 試劑盒組成試劑盒組成 130 倍濃縮洗滌液20ml1 瓶7終止液6ml1 瓶2酶標(biāo)試劑6ml1 瓶8標(biāo)準(zhǔn)品（3200mol/L）0.5ml1 瓶3酶標(biāo)包被板12 孔8 條9標(biāo)準(zhǔn)品稀釋液1.5ml1 瓶4樣品稀釋液6ml1 瓶10說明書1 份5顯色劑 A 液6ml1 瓶11封板膜2 張 6顯色劑 B 液6ml1/瓶12密封袋1 個標(biāo)本要求標(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

3、驗(yàn)，可將標(biāo)本放于-20保存，但應(yīng)避免反復(fù)凍融2不能檢測含 NaN3 的樣品，因 NaN3 抑制辣根過氧化物酶的（HRP）活性。操作步驟操作步驟1.標(biāo)準(zhǔn)品的稀釋：本試劑盒提供原倍標(biāo)準(zhǔn)品一支，用戶可按照下列圖表在小試管中進(jìn)行稀釋。1600mol/L5 號標(biāo)準(zhǔn)品150l 的原倍標(biāo)準(zhǔn)品加入 150l 標(biāo)準(zhǔn)品稀釋液800mol/L4 號標(biāo)準(zhǔn)品150l 的 5 號標(biāo)準(zhǔn)品加入 150l 標(biāo)準(zhǔn)品稀釋液400mol/L3 號標(biāo)準(zhǔn)品150l 的 4 號標(biāo)準(zhǔn)品加入 150l 標(biāo)準(zhǔn)品稀釋液200mol/L2 號標(biāo)準(zhǔn)品150l 的 3 號標(biāo)準(zhǔn)品加入 150l 標(biāo)準(zhǔn)品稀釋液100mol/L1 號標(biāo)準(zhǔn)品150l 的 2 號

4、標(biāo)準(zhǔn)品加入 150l 標(biāo)準(zhǔn)品稀釋液2.加樣：分別設(shè)空白孔（空白對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、待測樣品孔。在酶標(biāo)包被板上標(biāo)準(zhǔn)品準(zhǔn)確加樣 50l，待測樣品孔中先加樣品稀釋液40l，然后再加待測樣品 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(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

5、3。8.洗滌：操作同 5。9.顯色：每孔先加入顯色劑 A50l，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(jìn)行。操作程序總結(jié)：操作程序總結(jié)：計(jì)算計(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；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，再乘以稀釋倍數(shù)

6、，即為樣品的實(shí)際濃度。 注意事項(xiàng)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 請每次測定的同時(shí)做標(biāo)準(zhǔn)曲線，最好做復(fù)孔。如標(biāo)本中待測物質(zhì)含量過高（樣本 OD值大于標(biāo)準(zhǔn)品孔第一孔的 OD 值） ，請先用樣品稀釋液稀釋一定倍數(shù)（n 倍）后再測定，計(jì)算時(shí)請最后乘以總稀釋倍數(shù)（n5） 。5 封板膜只限一次性使用，以避免交叉污

7、染。6底物請避光保存。7嚴(yán)格按照說明書的操作進(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本試劑不同批號組分不得混用。10. 如與英文說明書有異，以英文說明書為準(zhǔn)。保存條件及有效期保存條件及有效期1試劑盒保存：；2-8。2有效期：6 個月Rat froctosamine（FA）FOR RESEARCH USE ONLYAssay range：50mol/L - 1600mol/L 96 determinationsPurposeThis kit allows for the determination of FA concentrations in

8、Rat serum, cell culture supernates and other biological fluidsPrinciple of the assayThe kit assay Rat FA level in the sample，use Purified Rat FA antibody to coat microtiter plate wells, make solid-phase antibody, then add FA to wells, Combined FA antibody which With HRP labeled,become antibody - ant

9、igen - 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 co

10、ncentration of Rat FA in the samples is then determined by comparing the O.D. of the samples to the standard curve.Materials provided with the kit1wash solution20ml1bottle7Stopp Solution6ml1 bottle2HRP-Conjugate reagent6ml1 bottle8Standard（3200mol/L）0.5ml1 bottle3Microelisa stripplate12well8strips9S

11、tandard diluent1.5ml1bottle4Sample diluent6ml1 bottle10Instruction15Chromogen Solution A6ml1 bottle11Closure plate membrane26Chromogen Solution B6ml1 bottle12Sealed bags1Specimen requirementsRD1.extract as soon as possible after Specimen collection,and according to the relevant literature, and shoul

12、d be experiment as soon as possible after the extraction. If it cant, specimen can be kept in -20 to preserve, Avoid repeated freeze-thaw cycles.2.Cant detect the sample which contain NaN3, because NaN3 inhibits HRP active.Assay procedure1.Dilute and add sample:Dilute Original density Standard as fo

13、llow table:1600mol/L5 Standard150l Original density Standard+150l Standard diluent800mol/L4 Standard150l 5 Standard+150l Standard diluent400mol/L3 Standard150l 4 Standard+150l Standard diluent200mol/L2 Standard150l 3 Standard +150l Standard diluent100mol/L1 Standard150l 2 Standard +150l Standard dil

14、uent2.add sample：Set blank wells separately (blank comparison wells dont add sample and HRP-Conjugate reagent, other each step operation is same). testing sample well. add Sample dilution 40l to testing sample well, then add testing sample 10l (sample final dilution is 5-fold), add sample to wells ,

15、 dont 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(or 20-fold) wash solution diluted 30-fold (or 20-fold) with distilled water and reserve.5.washing：Uncover Closure plate me

16、mbrane, 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 50l to each well, except blank well. 7.incubate：Operation with 3.8.washing：Operation with 5.9.color：Add Chromogen Solution A 50ul and Ch

17、romogen Solution B to each well, evade the light preservation for 15 min at 3710.Stop the reaction：Add Stop Solution50l to each well, Stop the reaction(the blue color change to yellow color).11.assay：take blank well as zero , Read absorbance at 450nm after Adding Stop Solution and within 15min.Steps

18、 descriptionStandard, Sample diluentAdd Standard, Sample diluent, incubate for 30 min at 37.Wash 5 time,Add HRP-Conjugate reagent, incubate for 30 min at 37.Wash 5 times,Add Chromogen Solution A and B, incubate for 30 min at 37.Add Stopp SolutionRead absorbance at 450nm within 15 mincalculateCalcula

19、teTake the standard density as the horizontal, the OD value for the vertical ,draw the 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 th

20、e standard curve with the standard density and the OD value ,with the sample OD value 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-

21、30 minutes in the room temperature, ELISA plates coated if has not use up after opened, the plate should be stored in Sealed bag.2.washing buffer will Crystallization separation, it can be heated the water helps dissolve when dilute . Washing does not affect the result.3.add Sample with sampler Each step, And proofread its accuracy frequently, avoids the experimental error. add sample within 5 min, if the number o