人幽門螺旋桿菌IgMHP

1、人幽門螺旋桿菌IgM（HP-lgM）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，血漿中幽門螺旋桿菌IgM（HP-IgM）水平。實驗原理：本試劑盒采用雙抗原夾心酶聯(lián)免疫法（ELISA ）測定標本中人幽門螺旋桿菌IgM（HP-IgM）。用純化的抗原包被微孔板，制成固相抗原，可與樣品中幽門螺旋桿菌IgM（HP-lgM）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標記的抗原結(jié)合，形成抗原-抗體-酶標抗原復(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)化成藍色，并在酸的作用下轉(zhuǎn)化成最終的黃色。用酶標儀在450nm波長下測定吸光度（0

2、D值），與CUTOFF值相比較，從而判定標本中人幽門螺旋桿菌IgM（HP-lgM）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個酶標包被板1X 481X 962-8 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

3、 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：用無菌管收集，離心20分鐘左右（2000-3000轉(zhuǎn)份）。仔細收集上清

4、，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?-8C的溫度。加入一定量的PBS （ PH7.4）,用手工或勻漿

5、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 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,

6、然后再加待測樣品10卩。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 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)（此時藍色立轉(zhuǎn)黃色）。11. 測定：以

7、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）者為人幽門螺旋桿菌IgM（HP-lgM）陰性陽性判定：樣品 OD值臨界值（CUT OFF ）者為人幽門螺旋桿菌IgM（HP-lgM）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

8、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Drug NamesGen eric Nam: Human HP-IgM ELISA Kit.PurposeThis kit allows for the determ in ationof HP-IgM concen trati o

9、nSn Huma nserum,a nd other biological fluids.Principle of the assayThe kit assayHP-IgM level in the sample use Purifiechntigen to coat microtiter plate wells, make solid-phasea ntige n,the n add HP-IgM to wells, Comb in edWithHP-IgM , after wash ing and rem ovingnon-comb in ativea ntibody and other

10、comp onen ts,the n Comb ined antigen which with HRP labeled become antigen - antibody - enzyme- antigen complex, after wash ing 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

11、 and the color cha nge is measured spectrophotometrically at a wavele ngth of 450 nm. Compared with the CUTOFF value, according to this to judgeM exist in the sample or not.Materials provided with the kitMaterials provided with the kit48determ in ati ons96 determ in atio nsStorageUser manual11Closur

12、e plate membra ne22Sealed bags11Microelisa stripplate112-8 CNegative con trol0.5ml 1Xbottle0.5ml Kbottle2-8 CPositive con trol0.5ml Xbottle0.5ml Kbottle2-8 CHRP-Co njugate reagent3ml Xbottle6ml 1 bottle2-8 CSample dilue nt3ml Xbottle6ml 1 bottle2-8 CChromoge n Soluti on A3ml Xbottle6ml 1 bottle2-8 C

13、Chromoge n Soluti on B3ml Xbottle6ml 1 bottle2-8 CStop Soluti on3ml 1 bottle6ml 1 bottle2-8 Cwash solution(20ml X 20 fold)x 1bottle(20ml X 30 fold)x 1 bottle2-8 CSpecimen requirements1. serum- coagulation at room temperature 10-20 ,meintrifugation 20-min at the speed of 2000-3000 r.p.m. remove super

14、 nata nt. If precipitati on appeared, Cen trifugal aga in.2. plasma-use suited EDTA or citrate plasma as an anticoagulant,mix 10-20 mins ,cen trifugatior20-min at the speed of 2000-3000r.p.m. remove super nata nt,If precipitation appeared, Centrifugal again.3. Urine collect sue a sterile container,

15、cen trifugati on 20-min at the speed of 2000-3000 r.p.m. 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, cen trifugatior2

16、0-min at the speed of 2000-3000r.p.m. removesuper nata nt,detectie compositionof cells, Dilut cell suspensiorwith PBS (PH7.2-7.4 , Cell concentration reached 1 million / ml, repeated freeze-thawcycles, damage cells and release ofin tracellular comp onen ts, cen trifugati on 20-min at the speed of 20

17、00-3000 r.p.m. remove supernatant, If precipitation appeared, Centrifugal again.5. Tissue samples After cutting samples, check the weight,add (PBS7.2-7.4 , Rapidly froze n with liquid n itroge n, mai ntain samples at2-after melt in g,add PBSPH7.4 ,Homogenized by hand or Grinders, centrifugation 20-m

18、in at the speed of 2000-3000 r.p.m. remove supernatant.6. extract as soon as possibleafter Specimencollection,andaccordingto the relevant literature,and shouldbe experimentas soon as possibleafter the extraction.If it can't, specime n can be kept in -20 to preserve, Avoid repeated freeze-thaw cy

19、cles.7. Can't detect the sample which coNnataNin3, 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 sampHl

20、eRaPn-dConjugate reagent to blank comparison well, other each step the operation are same).2. add sample separately add Positive control and Negative co5®oJ l to thPositive andNegativewell . add Sample dilution 40卩 l to testing saadplteweig sample 10 卩 l.add sample to the bottom oEfLISA plates

21、coated welld,on't touch the well wall as far as possible, and Gently mix.3.ln cubate: After closi ng plate with Closure plate membra ne ,in cubate for 30tmin 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.

22、 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 enzym: Add HRP-Conjugate reage)0tpto each well, except the blank well.7.incubate： Operation with 3.8. washing： Operation with 5.9. colo

23、r：Add Chromogen Solution A 50ul and Chromogen Solution B to each well, evade thelight preservati on for 15 min at37lO.Stopthe reaction Add Stop Solutio50(itb each well, Stop the react ion (th由lue colorchange to yellow color).11. assa：y take blank well as zero , Read absorbance at 450nm after Adding

24、Stop Solution and within 15min.Determine the resultTest validity: the average of Positive con trOHIdOCthe average of Negative con trol well< 0.10.Calculate Critical(CUT OFF) : Critical= the average of Negative control well + C.15.Negative control: sample OD< Calculate Critical(CUT OHFPF-Ig)Mi s Negative control.Positive contr:oalmple OD H CatlceuClaritical(CUT OFF)HisP-IgM 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 balance