人內皮細胞特異性分子-1 (ESM-1)ELISA 試劑盒說明書

本(ben)試劑僅(jin)供研究使用       

目的：本試劑盒用于測定人血清，血漿及相關液體樣本中內皮細胞(bao)特異性分子-1 (ESM-1)的含量。

實(shi)驗原理：

   本試劑盒應用雙抗體夾心法測定標本中人內皮(pi)細胞特異(yi)性分子-1 (ESM-1)水平。用純化的人內皮細(xi)胞特異(yi)性(xing)分(fen)子-1 (ESM-1)抗體包被微孔板，制成固相抗體，往包被單抗的微孔中依次加入內皮細胞特(te)異性分子-1 (ESM-1)，再與HRP標記的內皮細胞特異性分子-1 (ESM-1)抗體結合，形成抗體-抗原-酶標抗體復合物，經過*洗滌后加底物TMB顯色。TMB在HRP酶的催化下轉化成藍色，并在酸的作用下轉化成zui終的黃色。顏色的深淺和樣品中的內皮細(xi)胞(bao)特異性分子-1 (ESM-1)呈正相關。用酶標儀在450nm波長下測定吸光度（OD值），通過標準曲線計算樣品中人內皮細胞(bao)特異(yi)性分(fen)子-1 (ESM-1)濃度。

試劑盒(he)組成：

樣(yang)本處理及要求(qiu)：

1. 血(xue)清：室溫血(xue)液自然(ran)凝固(gu)10-20分(fen)(fen)鐘(zhong)，離(li)心20分(fen)(fen)鐘(zhong)左右（2000-3000轉/分(fen)(fen)）。仔細(xi)收(shou)集(ji)上清，保存過(guo)程中(zhong)如出(chu)現沉淀(dian)，應再次離(li)心。

2. 血漿：應根據(ju)標本的要求選擇EDTA或檸檬酸鈉作為(wei)抗凝劑，混合10-20分(fen)鐘后，離心20分(fen)鐘左右（2000-3000轉/分(fen)）。仔細(xi)收(shou)集上清(qing)，保存過(guo)程中如有沉淀形成，應該再次離心。

3. 尿液：用無(wu)菌管收(shou)集(ji)，離心(xin)20分鐘左右（2000-3000轉/分）。仔細(xi)收(shou)集(ji)上清，保存過程(cheng)中如(ru)有沉淀(dian)形成，應再次(ci)離心(xin)。胸腹(fu)水、腦脊液參照(zhao)實行。

4. 細胞(bao)培(pei)養上(shang)清：檢測分(fen)(fen)泌(mi)性的成份時(shi)，用無(wu)菌管收集。離心20分(fen)(fen)鐘左(zuo)右（2000-3000轉/分(fen)(fen)）。仔(zi)細收集上(shang)清。檢測細胞(bao)內的成份時(shi)，用PBS（PH7.2-7.4）稀釋細胞(bao)懸液，細胞(bao)濃度達到100萬/ml左(zuo)右。通過反復凍融，以使細胞(bao)破壞并放出細胞(bao)內成份。離心20分(fen)(fen)鐘左(zuo)右（2000-3000轉/分(fen)(fen)）。仔(zi)細收集上(shang)清。保存過程中(zhong)如有沉淀形成，應(ying)再次(ci)離心。

5. 組織(zhi)標本：切割標本后，稱取(qu)重量。加入(ru)(ru)一定量的PBS，PH7.4。用(yong)液氮迅速冷(leng)凍(dong)(dong)保存(cun)備用(yong)。標本融化(hua)后仍然保持2-8℃的溫度。加入(ru)(ru)一定量的PBS（PH7.4），用(yong)手工或(huo)勻漿(jiang)器將(jiang)標本勻漿(jiang)充(chong)分。離心20分鐘(zhong)左右(you)（2000-3000轉/分）。仔細收(shou)集上清。分裝后一份待(dai)檢測，其余(yu)冷(leng)凍(dong)(dong)備用(yong)。

6. 標本采(cai)集后盡早進(jin)行(xing)(xing)提取(qu)(qu)，提取(qu)(qu)按相關文獻進(jin)行(xing)(xing)，提取(qu)(qu)后應盡快(kuai)進(jin)行(xing)(xing)實驗。若不能(neng)馬上進(jin)行(xing)(xing)試驗，可將(jiang)標本放于-20℃保存(cun)，但應避免反(fan)復凍融.

7. 不能(neng)檢測含(han)NaN3的樣品，因NaN3抑(yi)制辣根過(guo)氧化物(wu)酶(mei)的(de)（HRP）活性。

操作步(bu)驟：

1. 標(biao)(biao)準(zhun)品(pin)(pin)(pin)的(de)稀釋(shi)(shi)(shi)與(yu)加(jia)(jia)(jia)(jia)樣(yang)：在酶標(biao)(biao)包被板上設(she)標(biao)(biao)準(zhun)品(pin)(pin)(pin)孔(kong)(kong)(kong)(kong)(kong)10孔(kong)(kong)(kong)(kong)(kong)，在*、第(di)(di)(di)(di)(di)(di)(di)(di)(di)二孔(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)分(fen)(fen)(fen)(fen)別(bie)加(jia)(jia)(jia)(jia)標(biao)(biao)準(zhun)品(pin)(pin)(pin)100μl，然后(hou)(hou)(hou)在*、第(di)(di)(di)(di)(di)(di)(di)(di)(di)二孔(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)加(jia)(jia)(jia)(jia)標(biao)(biao)準(zhun)品(pin)(pin)(pin)稀釋(shi)(shi)(shi)液(ye)50μl，混勻(yun)；然后(hou)(hou)(hou)從(cong)*孔(kong)(kong)(kong)(kong)(kong)、第(di)(di)(di)(di)(di)(di)(di)(di)(di)二孔(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)各(ge)取100μl分(fen)(fen)(fen)(fen)別(bie)加(jia)(jia)(jia)(jia)到(dao)第(di)(di)(di)(di)(di)(di)(di)(di)(di)三孔(kong)(kong)(kong)(kong)(kong)和第(di)(di)(di)(di)(di)(di)(di)(di)(di)四孔(kong)(kong)(kong)(kong)(kong)，再(zai)(zai)在第(di)(di)(di)(di)(di)(di)(di)(di)(di)三、第(di)(di)(di)(di)(di)(di)(di)(di)(di)四孔(kong)(kong)(kong)(kong)(kong)分(fen)(fen)(fen)(fen)別(bie)加(jia)(jia)(jia)(jia)標(biao)(biao)準(zhun)品(pin)(pin)(pin)稀釋(shi)(shi)(shi)液(ye)50μl，混勻(yun)；然后(hou)(hou)(hou)在第(di)(di)(di)(di)(di)(di)(di)(di)(di)三孔(kong)(kong)(kong)(kong)(kong)和第(di)(di)(di)(di)(di)(di)(di)(di)(di)四孔(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)先各(ge)取50μl棄掉(diao)，再(zai)(zai)各(ge)取50μl分(fen)(fen)(fen)(fen)別(bie)加(jia)(jia)(jia)(jia)到(dao)第(di)(di)(di)(di)(di)(di)(di)(di)(di)五(wu)、第(di)(di)(di)(di)(di)(di)(di)(di)(di)六(liu)孔(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)，再(zai)(zai)在第(di)(di)(di)(di)(di)(di)(di)(di)(di)五(wu)、第(di)(di)(di)(di)(di)(di)(di)(di)(di)六(liu)孔(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)分(fen)(fen)(fen)(fen)別(bie)加(jia)(jia)(jia)(jia)標(biao)(biao)準(zhun)品(pin)(pin)(pin)稀釋(shi)(shi)(shi)液(ye)50ul，混勻(yun)；混勻(yun)后(hou)(hou)(hou)從(cong)第(di)(di)(di)(di)(di)(di)(di)(di)(di)五(wu)、第(di)(di)(di)(di)(di)(di)(di)(di)(di)六(liu)孔(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)各(ge)取50μl分(fen)(fen)(fen)(fen)別(bie)加(jia)(jia)(jia)(jia)到(dao)第(di)(di)(di)(di)(di)(di)(di)(di)(di)七、第(di)(di)(di)(di)(di)(di)(di)(di)(di)八(ba)孔(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)，再(zai)(zai)在第(di)(di)(di)(di)(di)(di)(di)(di)(di)七、第(di)(di)(di)(di)(di)(di)(di)(di)(di)八(ba)孔(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)分(fen)(fen)(fen)(fen)別(bie)加(jia)(jia)(jia)(jia)標(biao)(biao)準(zhun)品(pin)(pin)(pin)稀釋(shi)(shi)(shi)液(ye)50μl，混勻(yun)后(hou)(hou)(hou)從(cong)第(di)(di)(di)(di)(di)(di)(di)(di)(di)七、第(di)(di)(di)(di)(di)(di)(di)(di)(di)八(ba)孔(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)分(fen)(fen)(fen)(fen)別(bie)取50μl加(jia)(jia)(jia)(jia)到(dao)第(di)(di)(di)(di)(di)(di)(di)(di)(di)九、第(di)(di)(di)(di)(di)(di)(di)(di)(di)十孔(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)，再(zai)(zai)在第(di)(di)(di)(di)(di)(di)(di)(di)(di)九第(di)(di)(di)(di)(di)(di)(di)(di)(di)十孔(kong)(kong)(kong)(kong)(kong)分(fen)(fen)(fen)(fen)別(bie)加(jia)(jia)(jia)(jia)標(biao)(biao)準(zhun)品(pin)(pin)(pin)稀釋(shi)(shi)(shi)液(ye)50μl，混勻(yun)后(hou)(hou)(hou)從(cong)第(di)(di)(di)(di)(di)(di)(di)(di)(di)九第(di)(di)(di)(di)(di)(di)(di)(di)(di)十孔(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)各(ge)取50μl棄掉(diao)。（稀釋(shi)(shi)(shi)后(hou)(hou)(hou)各(ge)孔(kong)(kong)(kong)(kong)(kong)加(jia)(jia)(jia)(jia)樣(yang)量都為50μl，濃度分(fen)(fen)(fen)(fen)別(bie)為48ng/mL，32 ng/mL ，16 ng/mL，8 ng/mL，4 ng/mL）。
2. 加(jia)樣(yang)：分(fen)別設空白孔(kong)(kong)（空白對照孔(kong)(kong)不(bu)加(jia)樣(yang)品(pin)(pin)(pin)及酶標試劑，其余各步操作相同）、待測(ce)樣(yang)品(pin)(pin)(pin)孔(kong)(kong)。在酶標包被板上待測(ce)樣(yang)品(pin)(pin)(pin)孔(kong)(kong)中(zhong)先加(jia)樣(yang)品(pin)(pin)(pin)稀釋液(ye)40μl，然后再加(jia)待測(ce)樣(yang)品(pin)(pin)(pin)10μl（樣(yang)品(pin)(pin)(pin)zui終稀釋度為5倍）。加(jia)樣(yang)將樣(yang)品(pin)(pin)(pin)加(jia)于酶標板孔(kong)(kong)底(di)部，盡(jin)量不(bu)觸及孔(kong)(kong)壁，輕(qing)輕(qing)晃動(dong)混勻。
3. 溫育：用封(feng)板膜封(feng)板后(hou)置37℃溫育30分鐘。
4. 配液：將30（48T的20倍）倍濃(nong)縮(suo)洗滌液用(yong)蒸餾水30（48T的20倍）倍稀釋后(hou)備用(yong)。
5. 洗滌：小(xiao)心揭掉封板膜，棄去液(ye)體(ti)，甩干，每孔加滿洗滌液(ye)，靜(jing)置(zhi)30秒(miao)后棄去，如此重復(fu)5次，拍干。
6. 加酶：每(mei)孔加入酶標(biao)試劑50μl，空白孔除外。
7. 溫育：操(cao)作同3。
8. 洗滌：操作同5。
9. 顯色：每孔先加入(ru)顯色劑(ji)A50μl，再加入(ru)顯色劑(ji)B50μl，輕(qing)輕(qing)震蕩混(hun)勻，37℃避(bi)光顯色15分鐘.
10. 終止(zhi)：每孔加終止(zhi)液50μl，終止(zhi)反應(ying)（此時藍色立(li)轉黃色）。
11. 測定(ding)：以空白空調零(ling)，450nm波長(chang)依序測量各孔的(de)吸光度（OD值）。 測定(ding)應(ying)在加終止液(ye)后15分鐘(zhong)以內(nei)進行。

注意事項：

1. 試(shi)劑盒從冷藏環(huan)境中(zhong)取出應在室(shi)溫平衡15-30分鐘后方可(ke)使用，酶標包被(bei)板(ban)開(kai)封(feng)后如未用完，板(ban)條應裝入密封(feng)袋(dai)中(zhong)保存。
2. 濃(nong)洗滌(di)液可(ke)能會有結(jie)(jie)晶析出，稀(xi)釋時可(ke)在水浴中(zhong)加溫助溶，洗滌(di)時不影響(xiang)結(jie)(jie)果。
3. 各步(bu)加樣均應使用(yong)加樣器(qi)，并(bing)經常校對其準確性，以避(bi)免試驗(yan)誤差。一次加樣時(shi)間(jian)控制在5分鐘內(nei)，如標本數(shu)量多，推薦使用(yong)排槍加樣。
4. 請(qing)(qing)每次測定(ding)的同時做標(biao)準曲線，做復孔。如標(biao)本中待測物質含量過(guo)高（樣本OD值(zhi)大于標(biao)準品孔*孔的OD值(zhi)），請(qing)(qing)先用樣品稀(xi)釋液稀(xi)釋一定(ding)倍(bei)數（n倍(bei)）后再測定(ding)，計算時請(qing)(qing)zui后乘以總稀(xi)釋倍(bei)數（×n×5）。
5. 封板(ban)膜只限一次性使用，以避(bi)免交(jiao)叉污染。
6. 底物(wu)請避(bi)光保存。
7. 嚴格(ge)按(an)照說明(ming)書(shu)的操作(zuo)進行，試(shi)驗結果判定必須以酶標(biao)儀讀數為準(zhun).
8. 所有(you)樣品，洗滌液和各種廢棄物(wu)都應按傳(chuan)染物(wu)處理。
9. 本試(shi)劑(ji)不同批號組分不得混用(yong)。

10. 如與英(ying)文(wen)說明(ming)書有異，以英(ying)文(wen)說明(ming)書為準。

計算：

以(yi)標(biao)準物的濃度為橫坐標(biao)，OD值(zhi)為縱坐標(biao)，   

在坐(zuo)標紙上繪(hui)出標準曲線，根據(ju)樣品的OD     

值由標(biao)準曲(qu)線查出相應的濃(nong)度；再乘以稀釋(shi)      

倍數(shu)；或用標準物的濃度與OD值計算出標      

準曲線(xian)的直線(xian)回歸(gui)方程(cheng)式，將樣品的OD值      

代入方程式，計算出樣品濃度，再乘以稀釋(shi)      

倍數(shu)，即為樣品的實際濃度。                  

                                            

試劑盒(he)性能：

1.樣品線性回歸與預期濃度相關(guan)系數R值為(wei)0.92以上。

2.批內與批見應分別小于9%和(he)15%

檢測范(fan)圍：                                             

0 ng/mL -60 ng/mL                                      

                            ;

保存條(tiao)件(jian)及有效期：

1.試劑盒保存：；2-8℃。

2．有效期：6個月(yue)

Human ESM-1

Drug Names

Generic Name：Human ESM-1 ELISA Kit.

Purpose

This kit allows for the determination of ESM-1 concentrations in Human serum, blood plasma, and other biological fluids.

Principle of the assay

The kit assay Human ESM-1 level in the sample，use Purified Human ESM-1 to coat microtiter plate wells, make solid-phase antibody, then add ESM-1 to wells, Combined ESM-1antibody which With HRP labeled , become antibody - antigen - enzyme-antibody complex, after washing Compley, Add TMB substrate solution,TMB substrate becomes 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. The concentration of ESM-1 in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Materials provided with the kit

Specimen requirements

1. 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.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 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 freeze-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 nitrogen, 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 possible after the extraction. If it can’t, specimen can be kept in -20 ℃ to preserve, Avoid repeated freeze-thaw cycles.
7. Can’t detect the sample which contain NaN3, because NaN3 inhibits HRP active.

Assay procedure

1.Dilute and add sample to Standard: set 10 Standard wells on the ELISA plates coated, add Standard 100μl to the first and the second well, then add Standard dilution 50μl to the first and the second well, mix; take out 100μl form the first and the second well then add it to the third and the forth well separay. then add Standard dilution 50μl to the third and the forth well ,mix ; then take out 50μl from the third and the forth well discard, add 50μl to the fifth and the sixth well ,then add Standard dilution 50μl to the fifth and the sixth well, mix ; take out 50μl from the fifth and the sixth well and add to the seventh and the eighth well, then add Standard dilution 50μl to the seventh and the eighth well ,mix ; take out 50μl from the seventh and the eighth well and add to the ninth and the tenth well, add Standard dilution 50μl to the ninth and the tenth well, mix , take out 50μl from the ninth and the tenth well discard(add Sample 50μl to each well after Diluting ,(density: 48ng/mL，32 ng/mL ，16ng/mL，8ng/mL，4 ng/mL)

2.add sample：Set blank wells separay (blank comparison wells don’t add sample and HRP-Conjugate reagent, other each step operation is same). testing sample well. add Sample dilution 40μl to testing sample well, then add testing sample 10μl (sample final dilution is 5-fold), add sample to wells , don’t 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 liquid: 30-fold (or 20-fold)wash solution diluted 30-fold (or 20-fold) with distilled water 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-Conjugate reagent 50μl to each well, except  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 37℃

10.Stop the reaction：Add Stop Solution50μl to each well, Stop the reaction(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.

Important notes

1. The kit takes out from the refrigeration environment should be balanced 15-30 minutes in the room temperature, ELISA plates coated if has not use up after opened, the plate should be stored in Sealed bag.
2. washing buffer will Crystallization separation, it can be heated the water helps dissolve when dilute . Washing does not affect the result.
3. add Sample with sampler Each step, And proofread its accuracy frequently, avoids the experimental error. add sample within 5 mins, if the number of sample is much , recommend to use Volley .
4. if the testing material content is excessively higher (The sample OD is bigger than the first standard well ),please dilute Sample (n-fold), Please diluente and multiplied by the dilution factor.（×n×5）.
5. Closure plate membrane only limits the disposable use, to avoid cross-contamination.
6. The substrate evade the light preservation.
7. Please according to use instruction strictly, The test result determination must take the microtiter plate reader as a standard.
8. All samples, washing buffer and each kind of reject should according to infective material process.
9. Do not mix reagents with those from other lots.
   

Assay range

0 ng/mL -60 ng/mL

Storage and validity

1．Storage：  2-8℃.

2．validity： six months.