人結(jié)核桿菌IgGTB-IgG酶聯(lián)免疫分析ELISA

1、文檔來源為：從網(wǎng)絡(luò)收集整理.word版本可編輯.歡迎下載支持 3文檔收集于互聯(lián)網(wǎng)，如有不妥請聯(lián)系刪除 人結(jié)核桿菌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：本試劑盒用于檢測人血清，血漿及相關(guān) 液體樣本中結(jié)核桿菌IgG（TB-IgG）水平，感染人的血液學(xué)診斷 實(shí)驗原理： 本試劑盒采用雙抗原夾心酶聯(lián)免疫法（ELISA ）測定標(biāo)本中人結(jié)核桿菌IgG（TB-IgG ）。 用純化的抗原包被微孔板，制成固相抗原，可與樣品中結(jié)核桿菌IgG（TB-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)記的抗原結(jié)合，形成抗原-抗體-酶標(biāo)抗原 復(fù)合物

2、，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 的作用下轉(zhuǎn)化成最終的黃色。用酶標(biāo)儀在450nm波長下測定吸光度（OD值），與CUTOFF 值相比較，從而判定標(biāo)本中人結(jié)核桿菌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 圭寸板膜 2 片(48) 2 片(96) 密封袋 1個 1個 酶標(biāo)包被板 1X 48 1X 96 2-8 C保存 陰性對照 0.5ml X 1 瓶 0.5ml X 1 瓶 2-8 C保存 陽性對照 0.5ml X 1 瓶 0.5ml X 1 瓶 2-8 C保存 酶標(biāo)試劑 3 ml X

3、1 瓶 6 ml X 1 瓶 2-8 C保存 樣品稀釋液 3 ml X 1 瓶 6 ml X 1 瓶 2-8 C保存 顯色劑A液 3 ml X 1 瓶 6 ml X 1 瓶 2-8 C保存 顯色劑B液 3 ml X 1 瓶 6 ml X 1 瓶 2-8 C保存 終止液 3ml X 1 瓶 6ml X 1 瓶 2-8 C保存 濃縮洗滌液 (20ml X 20 倍)X 1 瓶 (20ml X 30 倍)X 1 瓶 2-8 C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)份）。仔細(xì)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)

4、根據(jù)標(biāo)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(xì)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)份）。仔細(xì)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(shí)行。 4. 細(xì)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(xì)收集上清。檢測細(xì)胞內(nèi)的成份時，用PBS（ PH7.2-7.4 ）稀釋細(xì)胞懸液，細(xì)胞 濃度達(dá)到100萬/ml左右。通過反復(fù)凍融，以使細(xì)胞破壞并放出細(xì)胞內(nèi)成份。離心20分 鐘左

5、右（2000-3000轉(zhuǎn)/分）。仔細(xì)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(biāo)本：切割標(biāo)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)?用。標(biāo)本融化后仍然保持2-8C的溫度。加入一定量的PBS （ PH7.4）,用手工或勻漿器 將標(biāo)本勻漿充分。離心 20 分鐘左右（ 2000-3000 轉(zhuǎn) /分） 。仔細(xì)收集上清。分裝后一份待 檢測，其余冷凍備用。 6. 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。若不能馬上 進(jìn)行試驗，可將標(biāo)本放于 -20 C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

6、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(biāo)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50卩。然后在待測樣品孔先 加樣品稀釋液 40卩,然后再加待測樣品 10卩。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 C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

7、，拍干。 6. 加酶：每孔加入酶標(biāo)試劑 50卩,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 50 ，再加入顯色劑 B 50卩,輕輕震蕩混勻，37 C避光顯色 15分鐘 10. 終止：每孔加終止液 50卩終止反應(yīng)（此時藍(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。 結(jié)果判定： 試驗有效性：陽性對照孔平均值羽.00；陰性對照平均值 切.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

8、）者為人B 2-GP陰性 陽性判定：樣品 OD值臨界值（CUT OFF ）者為人B 2-GP陽性 注意事項 1操作嚴(yán)格按照說明書進(jìn)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(biāo)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(biāo)儀讀數(shù)為準(zhǔn)，使用雙波長檢測時，參考波長為630nm 7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

9、及有效期 1.試劑盒保存：；2-8Co 2有效期： 6 個月 Human TB-IgG FOR RESEARCH USE ONLY Drug Names Generic Nam： Human TB-IgG ELISA Kit. Purpose This kit allows for the determ in ati on of TB-IgG concen trati ons in Huma n serum, and other biological fluids. Pr in ciple of the assay The kit assay TB-IgG level in the sa，pt

10、e Purified an tige n to coat microtiter plate wells, make solid-phase antibody, then add TB-IgG to wells, Combined With TB-IgG, after washing and rem oving non-comb in ative an tibody and other comp onents ,the n Comb ined an tige n which with HRP labeled becomea ntige n an tibody- en zyme-a ntige n

11、 complex,after washi ng Completely,Add TMB substrate solutio n, TMB substrate becomes blue color At HRP en zyme-catalyzed, reacti on is term in ated by the additi on of a sulphuric acid soluti on and the color cha nge is measured spectrophotometrically at a wavele ngth of 450 nm. Compared with the C

12、UTOFF value, according to this to jTdgbgG exist in the sample or not. Materials provided with the kit Materials provided with the kit 48determ in ati ons 96 determ in atio ns Storage User manual 1 1 Closure plate membra n e2 2 Sealed bags 1 1 Microelisa stripplate 1 1 28C Negative con trol 0.5ml 1Xb

13、ottle 0.5ml Kbottle 28C Positive con trol 0.5ml Xbottle 0.5ml Kbottle 2-8 C HRP-Co njugate reagen t3ml Xbottle 6ml 1 bottle 2-8 C Sample dilue nt 3ml 1 bottle 6ml 1 bottle 2-8 C Chromoge n Soluti on A 3ml 1 bottle 6ml 1 bottle 2-8 C Chromoge n Soluti on B 3ml 1 bottle 6ml 1 bottle 2-8 C Stop Soluti

14、on 3ml 1 bottle 6ml 1 bottle 2-8 C 文檔來源為 :從網(wǎng)絡(luò)收集整理 .word 版本可編輯 .歡迎下載支持 wash solution (20ml x 20 fold x 1bottle (20ml x 30 fold x 1 bottle 28C Specime n requireme nts 1. serum- coagulation at room temperature 10-20，cieiistrifugation 20-min at the speed of 2000-3000 remove supernatant. If precipitation

15、 appeared, Centrifugal again. 2. plasma-use suited EDTA or citrate plasma as an anticoagulant,mix 10-20 mins ,centrifugation20-min at the speed of 2000-3000 remove supernatant,lf precipitation appeared, Centrifugal again. 3. Urine-collect sue a sterile container,centrifugatior20-min at the speed of

16、2000-3000 remove supernata nt,If precipitati on appeared, Cen trifugalaga in. The Operatio n of Hydrothorax and cerebrosp inal fluid Refere nee to it. 4. cell culture supernatan-detect secretorycomponentscollect sue a sterile container, centrifugation20-min at the speed of 2000-3000 remove supernata

17、nt,detecthe compositionof cells, Dilut cell suspensiorwith PBS( PH7.2-7.4 , Cell concentration reached 1 million / ml, repeated freeze-thawcycles, damage cells and release of intracellularcomponents,centrifugation20-min at the speed of 2000-3000 remove supernatant, If precipitation appeared, Centrif

18、ugal again. 5. Tissue samples After cutting samples, check the weight,add PPHE7.2-7.4 , Rapidly froze n with liquid n itroge n, mai ntain samples at2-after melt in g,add PBSPH7.4 , Homoge ni zedby hand or Grin ders,ce ntrifugatior2 0-min at the speed of 2000-3000 remove super nata nt. 6. extract as

19、soon as possibleafter Specime ncollecti on,an daccord in gto the releva nt literature,a nd shouldbe experime ntas soon as possibleafter the extractio n.lf it can specime n can be kept in -20 to preserve, Avoid repeated freeze-thaw cycles. 7. Can t detect the sample which conaNi3, because NaN3 inhibi

20、ts HRP active. Assay procedure 1. Number: to sample correspond microtitration well and Number Sequenee, each plate should be set femi nin ecomparison2 wells, masculi necomparison2 wells, bla nk comparison1 well(d on t add sampHRPC on jugate reage nt to bla nk comparis on well, other each step the op

21、eration are same). 2. add sample separately add Positive control and Negative co5ol l to thPositive and Negativewell . add Sample dilution 40卩 l to testing sampltesteig thample 10 卩 l. add sample to the bottom oEfLISA plates coated welld,ont touch the well wall as far as possible, and Gently mix. 3.

22、ln cubate: After closi ng plate with Closure plate membra ne ,in cubate for 30tmin 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, discard Liquid, dry by swing, add wa

23、shing buffer to every well, still for 30s then drain, repeat 5 times, dry by pat. 6. add enzym: Add HRP-Conjugate reagent 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 Chromogen Solution B to each well, ev

24、ade the light preservati on for 15 min at37 10.Stopthe reaction Add Stop Solutio50 卩1 each well, Stop the react ion (th由lue color change to yellow color). 11. assaytake blank well as zero , Read absorbance at 450nm after Adding Stop Solution and within 15min. Determine the result Test validity: the

25、average of Positive cowtrtbl 1.0l0e average of Negative con trol well 0.10. Calculate Critical(CUT OFF) : Critical= the average of Negative control well + 0.15. Negative control: sample OD Calculate Critical(CUT CTBI-IgG Positive control. Important notes 1. 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 15-3