雞結(jié)核IgGTBIgG酶聯(lián)免疫分析ELISA

1、雞結(jié)核IgG(TB IgG)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：本試劑盒用于檢測雞血清，血漿中結(jié)核IgG(TB IgG)水平。實驗原理： 本試劑盒采用雙抗體夾心酶聯(lián)免疫法（ELISA）測定標本中雞結(jié)核IgG(TB IgG)。用純化的結(jié)核IgG(TB IgG)抗體包被微孔板，制成固相抗體，可與樣品中結(jié)核IgG(TB IgG)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IgG(TB IgG)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450n

2、m波長下測定吸光度（OD值），與CUTOFF值相比較，從而判定標本中雞結(jié)核IgG(TB IgG)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瓶2-8保存顯色劑A液3 ml×1瓶6 ml×1瓶

3、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

4、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)/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（2000-3000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。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?-8

5、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. 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

6、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 50l，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

7、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IgG(TB IgG)陰性 陽性判定：樣品OD值 臨界值（CUT OFF）者為結(jié)核IgG(TB IgG)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

8、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FOR RESEARCH USE ONLYChicken Tuberculosis IgGDrug NamesGeneric Name：Chicken Tuberculosis IgG(TB IgG) ELISA Kit.PurposeThis kit allows fo

9、r the determination of TB IgG concentrations in Chicken serum, and other biological fluids.Principle of the assayThe kit assay TB IgG level in the sample，use Purified TB IgG antibody to coat microtiter plate wells, make solid-phase antigen, then add TB IgG to wells, Combined With TB IgG antibody, af

10、ter washing and removing non-combinative antibody and other components ,then Combined TB IgG antibody which with HRP labeled become antibodyantigen - enzyme- antibody complex, after washing Completely, Add TMB substrate solution, TMB substrate becomes blue color At HRP enzyme-catalyzed, reaction is

11、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 judge TB IgG exist in the sample or not.Materials provided with the kitMaterials provided with the kit48dete

12、rminations96 determinationsStorageUser 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 bottle6ml×1 bottle2-8Sample diluent3ml&

13、#215;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）×1bottle（20ml×30 fold）×1bottle2-8Specimen requirements1.

14、 serum- coagulation at room temperature 10-20 mins，centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant, If precipitation appeared, Centrifugal again.2. plasma-use suited EDTA or citrate plasma as an anticoagulant,mix 10-20 mins ,centrifugation 20-min at the speed of 2000-3000 r

15、.p.m. remove supernatant, If precipitation appeared, Centrifugal again.3. Urine-collect sue a sterile container, centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant, If precipitation appeared, Centrifugal again. The Operation of Hydrothorax and cerebrospinal fluid Reference to

16、it.4. cell culture supernatant-detect secretory components, collect sue a sterile container, centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant,detect the composition of cells, Dilut cell suspension with PBS（PH7.2-7.4）, Cell concentration reached 1 million / ml, repeated freez

17、e-thaw cycles, damage cells and release of intracellular components, centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant, If precipitation appeared, Centrifugal again.5. Tissue samples- After cutting samples, check the weight,add PBS（PH7.2-7.4）, Rapidly frozen with liquid nitro

18、gen, maintain samples at 2-8 after melting,add PBS（PH7.4）, Homogenized by hand or Grinders, centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant.6. extract as soon as possible after Specimen collection,and according to the relevant literature, and should be experiment as soon as

19、 possible after the extraction. If it cant, specimen can be kept in -20 to preserve, Avoid repeated freeze-thaw cycles.7. Cant detect the sample which contain NaN3, because NaN3 inhibits HRP active.Assay procedure1.Number: to sample correspond microtitration well and Number Sequence, each plate shou

20、ld 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, other each step the operation are same).2.add sample：separately add Positive control and Negative control 50l to the Positive and Negative

21、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 as far as possible, and Gently mix.3.Incubate: After closing plate with Closure plate membrane ,incubate for 30 min at 37. 4.Configurate

22、 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, 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-Con

23、jugate 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 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 rea

24、ction(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 resultTest validity: the average of Positive control well1.00; the average of Negative control well 0.10.Calculate Critical(CUT OFF) : C

25、ritical= the average of Negative control well + 0.15.Negative control: sample OD< Calculate Critical(CUT OFF) is TB IgG Negative control.Positive control: ample OD Calculate Critical(CUT OFF) is TB 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