人結(jié)核桿菌IgGTBIgG酶聯(lián)免疫分析ELISA

1、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(yàn)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，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45

2、0nm波長下測定吸光度（0D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 481X 962-8 C保存陰性對照X 1瓶X 1瓶2-8 C保存陽性對照X 1瓶X 1瓶2-8 C保存酶標(biāo)試劑3 ml X 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

3、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)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

4、，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 （）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,。用液氮迅速冷凍保存?zhèn)溆?。?biāo)本融化后仍然保持 2-8 C的溫度。加入一定量的PBS （）,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

5、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(yàn)。若不能馬上 進(jìn)行試驗(yàn)，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

6、. 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，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。

7、結(jié)果判定：試驗(yàn)有效性：陽性對照孔平均值考陰性對照平均值臨界值（CUT OFF ）計算：臨界值=陰性對照孔平均值+ 陰性判定：樣品 OD值 臨界值（CUT OFF）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(xiàng)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(yàn)結(jié)果判定必須以酶標(biāo)

8、儀讀數(shù)為準(zhǔn)，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：；2-8Co2 有效期：6個月RD /、Human TB-IgGFOR RESEARCH USE ONLYDrug NamesGeneric Nam: Human TB-IgG ELISA Kit.PurposeThis kit allows for the determ in ati on of TB-IgG concen trati ons in Huma n serum, and other biological flu

9、ids.Pr in ciple of the assayThe kit assay TB-IgG level in the sa，pte 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

10、which with HRP labeled becomea ntige n an tibody- en zyme-a ntige n 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 spe

11、ctrophotometrically at a wavele ngth of 450 nm. Compared with the CUTOFF value, according to this to jTdgbgG exist in the sample or not.Materials provided with the kitMaterials provided with the kit48determ in ati ons96 determ in atio nsStorageUser manual1、1Closure plate membra ne2 2Sealed bags11Mic

12、roelisa stripplate112-8 CNegative con trolxi bottle1 bottle2-8 CPositive con trolxi bottle1 bottle2-8 CHRP-Co njugate reagent3ml Xbottle6ml 1 bottle 、2-8 CSample dilue nt3ml 1 bottle6ml 1 bottle2-8 CChromoge n Soluti on A3ml 1 bottle6ml 1 bottle2-8 CChromoge n Soluti on B3ml 1 bottle6ml 1 bottle2-8

13、CStop Soluti on3ml 1 bottle6ml 1 bottle2-8 Cwash solution(20ml x 20 fold)x 1bottle(20ml x 30 fold)x 1 bottle2-8 CSpecime n requireme nts1. serum- coagulation at room temperature 10-20，cieiistrifugation 20-min at the speed of 2000-3000 remove supernatant. If precipitation appeared, Centrifugal again.

14、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 2000-3000 remove supernata nt,I

15、f 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 supernatant,detecthe composition of cells

16、, Dilut cell suspension with )BCell concentration reached 1 million/ ml, repeated freeze-thaw cycles, damage cells and release of in tracellular comp onents, cen trifugatior2 0-min at the speed of 2000-3000 removesuper nata nt,If precipitati on appeared, Cen trifugal aga in.5. Tissue samples After c

17、utting samples, check the weight,add(PB Rapidly frozen with liquid n itroge n, maintain samples at 2-8after melt in g,add PBS) , Homoge ni zed by hand or Grin ders, cen trifugati on 20-min at the speed of 2OOO-e00fiVe super nata nt.6. extract as soon as possibleafter Specime ncollecti on,an daccord

18、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 conaNS, because NaN3 inhibits HRP active.Assay procedure:to samplecorresp on dmicro

19、titrationwell an dNumberSeque nceeach plate shouldbe setfeminine comparison 2 wells, masine comparison 2 wells, blank comparison 1 well(don add sample and HRP-C on jugateeage ntto bla nk comparisorwell, other each step the operati on are same).sample separatelyadd Positive control and Negativecontro

20、l 50 卩 to the Positive andNegative wll . add Sample dilution 40卩 l to testing saadplteweig sample 10 卩 l.add sample to the bottom dELISA plates coated welld on t touch the well wall as far as possible, and Gen tly mix.:After closi ng plate with Closure plate membra ne ,in cubate for 30 nSin at 37liq

21、uid:30-fold(or 20-fold)waslsolutio ndiluted30-fold(or 20-fold)with distilledwater u ntil 600ml,a nd reserve.:Un cover Closure plate membra ne, discard Liquid, dry by swing, add wash ing buffer to every well, still for 30s then drain, repeat 5 times, dry by pat.enzyme Add HRP-Conjugate reagent lto ea

22、ch well, except the blank well.:Operation with 3.:Operati on with 5.: Add Chromogen Solution A 50ul and Chromogen SoldBioneach well, evade the lightpreservati on for 15 min att37the reaction Add Stop Solution) ytbeach well, Stop the reaction(the blue color change to yellow color).11. assay take blan

23、k well as zero , Read absorbanee at 450nm after Adding Stop Solution and within 15mi n.Determi ne the resultTest validity: the average of Positive cowtrtbl sth;average of Negative conwell .Calculate Critical(CUT OFF) : Critical= the average of Negative control well + .Negative con trol: sample OD Calculate Critical(CUT OBFI)gG Positive control.Importa nt no tesaccording to use instruction strictly, Do not mix reagents with those from other lots.kit takes out from the refrigeration environment should be balaneed 15-30 min