獼猴結(jié)核桿菌TB酶聯(lián)免疫分析ELISA

1、獼猴結(jié)核桿菌（ TB ）酶聯(lián)免疫分析（ ELISA）試劑盒使用說明書本試劑僅供研究使用目的：本試劑盒用于檢測獼猴血清，血漿及相關(guān)液體樣本中結(jié)核桿菌（TB）水平。實驗原理：本試劑盒采用雙抗體夾心酶聯(lián)免疫法（ELISA ）測定標本中獼猴結(jié)核桿菌（TB ）。用純化的獼猴結(jié)核桿菌（TB ）抗體包被微孔板，制成固相抗體，可與樣品中結(jié)核桿菌（TB ）相結(jié)合，經(jīng)洗滌除去未結(jié)合的抗體和其他成分后再與HRP 標記的結(jié)核桿菌（ TB ）抗體結(jié)合，形成抗體 -抗原 -酶標抗體復(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)化成藍色，并在酸的作用下轉(zhuǎn)化成最終的黃色。用酶標儀在450nm 波

2、長下測定吸光度（OD 值），與 CUTOFF 值相比較，從而判定標本中獼猴結(jié)核桿菌（TB ）的存在與否。試劑盒組成 ：試劑盒組成48 孔配置96 孔配置保存說明書1 份1 份封板膜2 片（ 48）2 片（ 96）密封袋1 個1 個酶標包被板1× 481× 962-8保存陰性對照0.5ml × 1 瓶0.5ml × 1 瓶2-8保存陽性對照0.5ml × 1 瓶0.5ml × 1 瓶2-8保存酶標試劑3 ml × 1 瓶6 ml × 1 瓶2-8保存樣品稀釋液3 ml × 1 瓶6 ml × 1

3、瓶2-8保存顯色劑 A液3 ml × 1 瓶6 ml × 1 瓶2-8保存顯色劑 B液3 ml × 1 瓶6 ml × 1 瓶2-8保存終止液3ml× 1 瓶6ml × 1 瓶2-8保存濃縮洗滌液（ 20ml× 20 倍）× 1 瓶（20ml ×30 倍）× 1 瓶2-8保存樣本處理及要求：1. 血清：室溫血液自然凝固 10-20 分鐘，離心 20 分鐘左右 （ 2000-3000 轉(zhuǎn) /分）。仔細收集上清，保存過程中如出現(xiàn)沉淀，應(yīng)再次離心。2. 血漿：應(yīng)根據(jù)標本的要求選擇EDTA 或檸檬酸鈉作

4、為抗凝劑，混合10-20 分鐘后，離心20 分鐘左右（ 2000-3000 轉(zhuǎn) /分）。仔細收集上清，保存過程中如有沉淀形成，應(yīng)該再次離心。3. 尿液：用無菌管收集，離心20 分鐘左右（ 2000-3000 轉(zhuǎn) /分）。仔細收集上清，保存過程中如有沉淀形成，應(yīng)再次離心。胸腹水、腦脊液參照實行。4. 細胞培養(yǎng)上清：檢測分泌性的成份時，用無菌管收集。離心20 分鐘左右（ 2000-3000 轉(zhuǎn)/1分）。仔細收集上清。檢測細胞內(nèi)的成份時，用PBS（PH7.2-7.4 ）稀釋細胞懸液，細胞濃度達到100 萬 /ml 左右。通過反復(fù)凍融，以使細胞破壞并放出細胞內(nèi)成份。離心20 分鐘左右（ 2000-30

5、00 轉(zhuǎn)/分）。仔細收集上清。保存過程中如有沉淀形成，應(yīng)再次離心。5. 組織標本：切割標本后，稱取重量。加入一定量的PBS，PH7.4。用液氮迅速冷凍保存?zhèn)溆?。標本融化后仍然保?2-8的溫度。加入一定量的 PBS（ PH7.4），用手工或勻漿器將標本勻漿充分。離心 20 分鐘左右（ 2000-3000 轉(zhuǎn) /分）。仔細收集上清。分裝后一份待檢測，其余冷凍備用。6. 標本采集后盡早進行提取，提取按相關(guān)文獻進行，提取后應(yīng)盡快進行實驗。若不能馬上進行試驗，可將標本放于 -20保存，但應(yīng)避免反復(fù)凍融 .7. 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：1

6、.編號：將樣品對應(yīng)微孔按序編號，每板應(yīng)設(shè)陰性對照2 孔、陽性對照2 孔、空白對照 1孔（空白對照孔不加樣品及酶標試劑，其余各步操作相同）2.加樣：分別在陰、陽性對照孔中加入陰性對照、陽性對照50l。然后在待測樣品孔先加樣品稀釋液 40l，然后再加待測樣品 10l。加樣將樣品加于酶標板孔底部，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（ 48T 的 20 倍）倍濃縮洗滌液加蒸餾水至600ml 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：每孔加入酶標試劑 50l，空白孔

7、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 A 50 l，再加入顯色劑 B 50l，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)（此時藍色立轉(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)進行。結(jié)果判定：試驗有效性：陽性對照孔平均值1.00; 陰性對照平均值0.10臨界值（ CUT OFF ）計算：臨界值=陰性對照孔平均值+0.15陰性判定：樣品OD 值 < 臨界值（ CUT OFF ）者為結(jié)核桿菌（TB ）陰性陽性判定：樣品OD 值

8、臨界值（ CUT OFF ）者為結(jié)核桿菌（TB ）陽性注意事項1操作嚴格按照說明書進行，本試劑不同批號組分不得混用。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，酶標包被板開封后如未用完，板條應(yīng)裝入密封袋中保存。3濃洗滌液可能會有結(jié)晶析出，稀釋時可在水浴中加溫助溶，洗滌時不影響結(jié)果。4 封板膜只限一次性使用，以避免交叉污染。5底物請避光保存。6試驗結(jié)果判定必須以酶標儀讀數(shù)為準，使用雙波長檢測時，參考波長為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，使用時必須2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 個月3Mac

9、aca TBFOR RESEARCH USE ONLYDrug NamesGeneric Name： Macaca TB ELISA Kit.PurposeThis kit allows for thedeterminationof TB concentrationsin Macacaserum, andother biological fluids.Principle of the assayThe kit assayTB level in the sample，use PurifiedTB antibody to coat microtiter plate wells, make soli

10、d-phase antibody, then TBaddto wells, Combined WithTB , after washing and removing non-combinative antibody and other components ,then CombiTBantibodyed which with HRP labeled becomeantibody - antigen - enzyme-antibody complex,after washing Completely,Add TMB substrate solution, TMB substrate become

11、s 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. Compared with the CUTOFF value, according to this to judgeTBexist in the sample or not.4Materials provided with

12、 the kitMaterials provided with48determinations96 determinationsStoragethe kitUser manual11Closure plate membrane22Sealed bags11Microelisa stripplate112-8Negative control0.5ml 1×bottle0.5ml 1×bottle2-8Positive control0.5ml 1×bottle0.5ml 1×bottle2-8HRP-Conjugate reagent3ml×1

13、bottle6ml×1 bottle2-8Sample diluent3ml×1 bottle6ml×1 bottle2-8Chromogen Solution A3ml×1 bottle6ml×1 bottle2-8Chromogen Solution B3ml×1 bottle6ml×1 bottle2-8Stop Solution3ml×1 bottle6ml×1 bottle2-8wash solution（ 20ml × 20 fold）（ 20ml× 30 fold）2-8

14、×1× 1bottlebottleSpecimen requirements1. serum- coagulation at room temperature 10-20，2.3.4. （PH7.2-7.）5supernatant, If precipitation appeared, Centrifugal again.5. Tissue samples- After cutting samples, check the weight,add（PBSPH7.2-7.4） , Rapidly frozen with liquid nitrogen, maintain sam

15、ples at2-8after melting,add PBS（PH7.4）6. extract as soon as possible after Specimen collection,andaccordingto the relevant literature,and shouldbe experimentas soon as possibleafter the extraction.If it cant,specimen can be kept in -20 to preserve, Avoid repeated freeze-thaw cycles.7. Can t detect t

16、he sample which containNaN3, because NaN3 inhibits HRP active.Assay procedure1.Number: to sample correspond microtitration well and Number Sequence, each plate should be set femininecomparison2 wells, masculinecomparison2 wells, blank comparison1 well(don t add sampleHRPand-Conjugate reagent to blan

17、k comparison well, other each step the operation are same).2.add sample：separately add Positive control and Negative control50 l to thePositive andNegative wel. add Sample dilution 40 l to testing sampleaddtestingwell,thensample 10 l.add sample to the bottom ofELISA plates coated well don, t touch t

18、he well wall as far as possible, and Gently mix.3.Incubate: After closing plate with Closure plate membrane ,incubate for 30min. at 374.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 memb

19、rane, 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 reagent50 lto each well, except the blank well.7.incubate： Operation with 3.8.washing：Operation with 5.9.color：Add Chromogen Solution A 50ul and C

20、hromogen Solution B to each well, evade the6light preservation for 15 min at3710.Stopthe reaction： Add Stop Solution50lto each well, Stop the reaction(theblue color change to yellow color).11. assay：take blank well as zero , Read absorbance at 450nm after Adding Stop Solution and within 15min.Determ

21、ine the resultTest validity: the average of Positive controlwell 1.00;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)TBNegativeis control.Positive control: ample

22、OD Calculate Critical(CUT OFF)TBPoisitive 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