人髓過(guò)氧化物酶特異性抗中性粒細(xì)胞胞質(zhì)抗體IgG

1、人髓過(guò)氧化物酶特異性抗中性粒細(xì)胞胞質(zhì)抗體IgG（MPO-ANCA IgG）酶聯(lián)免疫分析試劑盒使用說(shuō)明書本試劑盒僅供研究使用。 96T使用目的：本試劑盒用于測(cè)定人血清、血漿及相關(guān)液體樣本中髓過(guò)氧化物酶特異性抗中性粒細(xì)胞胞質(zhì)抗體IgG（MPO-ANCA IgG）表達(dá)。實(shí)驗(yàn)原理本試劑盒應(yīng)用雙抗體夾心法測(cè)定標(biāo)本中人髓過(guò)氧化物酶特異性抗中性粒細(xì)胞胞質(zhì)抗體IgG（MPO-ANCA IgG）表達(dá)。用純化的抗體包被微孔板，制成固相抗體，可與樣品中髓過(guò)氧化物酶特異性抗中性粒細(xì)胞胞質(zhì)抗體IgG（MPO-ANCA IgG）相結(jié)合，經(jīng)洗滌除去未結(jié)合的抗原和其他成分后再與HRP標(biāo)記的抗體結(jié)合，形成抗體-抗原-酶標(biāo)抗體復(fù)

2、合物，經(jīng)過(guò)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。用酶標(biāo)儀在450nm波長(zhǎng)下測(cè)定吸光度（OD值），與CUTOFF值相比較，從而判定標(biāo)本中人髓過(guò)氧化物酶特異性抗中性粒細(xì)胞胞質(zhì)抗體IgG（MPO-ANCA IgG）的存在與否。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陽(yáng)性對(duì)照0.5ml×1瓶3酶標(biāo)包被板12孔×8條9陰性對(duì)照0.5ml×1瓶4樣品稀釋液6ml×1瓶10說(shuō)明書1份5顯色劑A液6ml×1瓶11封板膜2張 6

3、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. 編號(hào)：將樣品對(duì)應(yīng)微孔按序編號(hào)，每板應(yīng)設(shè)陰性對(duì)照2孔、陽(yáng)性對(duì)照2孔、空白對(duì)照1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）2. 加樣：分別在陰、陽(yáng)性對(duì)照孔中加入陰性對(duì)照、陽(yáng)性對(duì)照50l。然后在待測(cè)樣品孔先加樣品稀釋液40l，然后再加待測(cè)樣品10l。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

4、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(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。操作程序總結(jié)：計(jì)算和結(jié)果判定： 試驗(yàn)有效性：陽(yáng)性

5、對(duì)照孔平均值1.00; 陰性對(duì)照平均值0.10 臨界值（CUT OFF）計(jì)算：臨界值=陰性對(duì)照孔平均值+0.15 陰性判定：樣品OD值< 臨界值（CUT OFF）者為髓過(guò)氧化物酶特異性抗中性粒細(xì)胞胞質(zhì)抗體IgG（MPO-ANCA IgG）陰性 陽(yáng)性判定：樣品OD值 臨界值（CUT OFF）者為髓過(guò)氧化物酶特異性抗中性粒細(xì)胞胞質(zhì)抗體IgG（MPO-ANCA IgG）陽(yáng)性。 注意事項(xiàng)1操作嚴(yán)格按照說(shuō)明書進(jìn)行，本試劑不同批號(hào)組分不得混用。2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。3濃洗滌液可能會(huì)有結(jié)晶析出，稀釋時(shí)可在水浴中加

6、溫助溶，洗滌時(shí)不影響結(jié)果。4 封板膜只限一次性使用，以避免交叉污染。5底物請(qǐng)避光保存。6試驗(yàn)結(jié)果判定必須以酶標(biāo)儀讀數(shù)為準(zhǔn)，使用雙波長(zhǎng)檢測(cè)時(shí)，參考波長(zhǎng)為630nm7所有樣品，洗滌液和各種廢棄物都應(yīng)按傳染物處理。終止液為2M的硫酸，使用時(shí)必須注意安全。保存條件及有效期1試劑盒保存：；2-8。2有效期：6個(gè)月RDHuman MPO-ANCA IgGFOR RESEARCH USE ONLY 96 determinationsPurposeThis kit allows for the determination of MPO-ANCA IgG concentrations in Human seru

7、m, and other biological fluids.Principle of the assayThe kit assay MPO-ANCA IgG level in the sample，use Purified antibody to coat microtiter plate wells, make solid-phase antibody, then add MPO-ANCA IgG to wells, Combined With MPO-ANCA IgG, after washing and removing non-combinative antibody and oth

8、er components ,then Combined 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

9、 color change is measured spectrophotometrically at a wavelength of 450 nm. Compared with the CUTOFF value, according to this to judge MPO-ANCA IgG exist in the sample or not.Materials provided with the kit1wash solution20ml×1bottle7Stopp Solution6ml×1 bottle2HRP-Conjugate reagent6ml×

10、1 bottle8Positive control0.5ml×1 bottle3Microelisa stripplate12well×8strips9Negative control0.5ml×1bottle4Sample diluent6ml×1 bottle10Instruction15Chromogen Solution A6ml×1 bottle11Closure plate membrane26Chromogen Solution B6ml×1 bottle12Sealed bags1Specimen requiremen

11、ts1. extract as soon as possible after Specimen collection,and according to the relevant literature, and should 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,

12、 because NaN3 inhibits HRP active.Assay procedure1.Number: to sample correspond microtitration well and Number Sequence, each plate should be set feminine comparison 2 wells, masculine comparison 2 wells, blank comparison 1 well(dont add sample and HRP-Conjugate reagent to blank comparison well, oth

13、er each step the operation are same).2.add sample：separately add Positive control and Negative control 50l to the Positive and Negative well . add Sample dilution 40l to testing sample well, then add testing sample 10l. add sample to the bottom of ELISA plates coated well , dont touch the well wall

14、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 until 600ml,and reserve.5.washing：Uncover Closure plate membrane, disca

15、rd 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 50lto each well, except the blank well. 7.incubate：Operation with 3.8.washing：Operation with 5.9.color：Add Chromogen Solution A 50ul and Chromogen So

16、lution 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.Determine the

17、resultTest validity: the average of Positive control well1.00; the average of Negative control well 0.10.Calculate Critical(CUT OFF) : Critical= the average of Negative control well + 0.15.Negative control: sample OD< Calculate Critical(CUT OFF) is MPO-ANCA IgG Negative control.Positive control:

18、ample OD Calculate Critical(CUT OFF) is MPO-ANCA IgG Positive control.Important notes1.Please according to use instruction strictly, Do not mix reagents with those from other lots.2.The kit takes out from the refrigeration environment should be balanced 15-30 minutes in the room temperature then use, ELISA plates coated if has not use up after opened, the plate should be stored in Sealed bag.3.washing buffer will Crystallization separation, it can be he