人(ren)凝血因子IX（FIX)酶聯免(mian)疫分析（ELISA）

試劑(ji)盒使用說明書(shu)

本試劑僅供研(yan)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凝血因子IX（FIX)含量。

實驗原(yuan)理：

  本(ben)試劑盒(he)應(ying)用(yong)雙(shuang)抗(kang)體夾心(xin)法(fa)測定標(biao)本(ben)中人凝(ning)(ning)血(xue)(xue)(xue)因子(zi)(zi)IX（FIX)水平。用(yong)純化(hua)(hua)的(de)(de)人凝(ning)(ning)血(xue)(xue)(xue)因子(zi)(zi)IX（FIX)抗(kang)體包被微孔板，制成(cheng)固相(xiang)抗(kang)體，往(wang)包被單抗(kang)的(de)(de)微孔中依(yi)次加入凝(ning)(ning)血(xue)(xue)(xue)因子(zi)(zi)IX（FIX),再與HRP標(biao)記的(de)(de)凝(ning)(ning)血(xue)(xue)(xue)因子(zi)(zi)IX（FIX)抗(kang)體結合，形成(cheng)抗(kang)體-抗(kang)原(yuan)-酶(mei)標(biao)抗(kang)體復(fu)合物，經過(guo)*洗滌后(hou)加底(di)物TMB顯(xian)色(se)。TMB在(zai)HRP酶(mei)的(de)(de)催化(hua)(hua)下(xia)轉化(hua)(hua)成(cheng)藍(lan)色(se)，并在(zai)酸的(de)(de)作用(yong)下(xia)轉化(hua)(hua)成(cheng)zui終的(de)(de)黃色(se)。顏色(se)的(de)(de)深淺和樣品中的(de)(de)凝(ning)(ning)血(xue)(xue)(xue)因子(zi)(zi)IX（FIX)呈(cheng)正相(xiang)關。用(yong)酶(mei)標(biao)儀在(zai)450nm波長下(xia)測定吸光度(du)（OD值），通過(guo)標(biao)準曲線(xian)計算樣品中人凝(ning)(ning)血(xue)(xue)(xue)因子(zi)(zi)IX（FIX)濃(nong)度(du)。

試(shi)劑盒組成：

樣(yang)本處(chu)理及要求：

1. 血清：室溫血液自然(ran)凝固10-20分(fen)鐘(zhong)(zhong)，離心20分(fen)鐘(zhong)(zhong)左(zuo)右（2000-3000轉/分(fen)）。仔(zi)細收(shou)集上清，保存過程中如(ru)出現沉淀，應再次離心。

2. 血(xue)漿：應根據標本的(de)要求選擇(ze)EDTA或檸檬酸(suan)鈉作為抗凝(ning)劑，混合10-20分鐘(zhong)后，離心(xin)(xin)20分鐘(zhong)左(zuo)右(you)（2000-3000轉/分）。仔(zi)細收集上清(qing)，保存(cun)過程中如有沉淀形成，應該再次離心(xin)(xin)。

3. 尿液：用無菌管收(shou)集，離心20分鐘左右（2000-3000轉(zhuan)/分）。仔細收(shou)集上清，保存過程中如有沉淀形(xing)成，應再次(ci)離心。胸腹水(shui)、腦脊液參照實(shi)行。

4. 細胞(bao)培(pei)養上清(qing)：檢(jian)測分(fen)泌性的(de)成(cheng)(cheng)份時，用無菌管收(shou)集。離心20分(fen)鐘左右(you)（2000-3000轉/分(fen)）。仔細收(shou)集上清(qing)。檢(jian)測細胞(bao)內(nei)的(de)成(cheng)(cheng)份時，用PBS（PH7.2-7.4）稀釋細胞(bao)懸液，細胞(bao)濃度達到100萬/ml左右(you)。通過(guo)反復(fu)凍融，以(yi)使(shi)細胞(bao)破(po)壞并放出細胞(bao)內(nei)成(cheng)(cheng)份。離心20分(fen)鐘左右(you)（2000-3000轉/分(fen)）。仔細收(shou)集上清(qing)。保存過(guo)程中如有沉淀形成(cheng)(cheng)，應再次離心。

5. 組織標(biao)(biao)本：切割標(biao)(biao)本后，稱取重量(liang)。加(jia)(jia)入一(yi)定(ding)量(liang)的PBS，PH7.4。用(yong)(yong)液(ye)氮迅速冷(leng)凍保存備用(yong)(yong)。標(biao)(biao)本融化后仍然(ran)保持2-8℃的溫(wen)度(du)。加(jia)(jia)入一(yi)定(ding)量(liang)的PBS（PH7.4），用(yong)(yong)手工或勻漿器將標(biao)(biao)本勻漿充分(fen)。離心20分(fen)鐘左右（2000-3000轉/分(fen)）。仔細收集上清。分(fen)裝后一(yi)份(fen)待檢(jian)測，其(qi)余(yu)冷(leng)凍備用(yong)(yong)。

6. 標(biao)本采集后(hou)盡(jin)早(zao)進行提取，提取按相關(guan)文獻(xian)進行，提取后(hou)應盡(jin)快進行實驗。若不能馬上進行試驗，可將標本(ben)放于(yu)-20℃保(bao)存，但應避免反復(fu)凍融.

7. 不能(neng)檢測含NaN3的樣品(pin)，因NaN3抑制辣根(gen)過(guo)氧化(hua)物酶的（HRP）活性(xing)。

操作步驟

1. 標(biao)(biao)準(zhun)(zhun)(zhun)品(pin)(pin)的稀(xi)(xi)釋與加(jia)(jia)樣(yang)(yang)：在(zai)酶(mei)標(biao)(biao)包(bao)被板上(shang)設(she)標(biao)(biao)準(zhun)(zhun)(zhun)品(pin)(pin)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)10孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)，在(zai)*、第(di)(di)(di)二(er)(er)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)分(fen)(fen)(fen)別加(jia)(jia)標(biao)(biao)準(zhun)(zhun)(zhun)品(pin)(pin)100μl，然后(hou)(hou)在(zai)*、第(di)(di)(di)二(er)(er)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)加(jia)(jia)標(biao)(biao)準(zhun)(zhun)(zhun)品(pin)(pin)稀(xi)(xi)釋液(ye)50μl，混(hun)勻(yun)；然后(hou)(hou)從*孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)、第(di)(di)(di)二(er)(er)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)各(ge)取(qu)100μl分(fen)(fen)(fen)別加(jia)(jia)到(dao)第(di)(di)(di)三(san)(san)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)和第(di)(di)(di)四(si)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)，再(zai)(zai)在(zai)第(di)(di)(di)三(san)(san)、第(di)(di)(di)四(si)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)分(fen)(fen)(fen)別加(jia)(jia)標(biao)(biao)準(zhun)(zhun)(zhun)品(pin)(pin)稀(xi)(xi)釋液(ye)50μl，混(hun)勻(yun)；然后(hou)(hou)在(zai)第(di)(di)(di)三(san)(san)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)和第(di)(di)(di)四(si)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)先(xian)各(ge)取(qu)50μl棄(qi)掉，再(zai)(zai)各(ge)取(qu)50μl分(fen)(fen)(fen)別加(jia)(jia)到(dao)第(di)(di)(di)五(wu)、第(di)(di)(di)六(liu)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)，再(zai)(zai)在(zai)第(di)(di)(di)五(wu)、第(di)(di)(di)六(liu)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)分(fen)(fen)(fen)別加(jia)(jia)標(biao)(biao)準(zhun)(zhun)(zhun)品(pin)(pin)稀(xi)(xi)釋液(ye)50ul，混(hun)勻(yun)；混(hun)勻(yun)后(hou)(hou)從第(di)(di)(di)五(wu)、第(di)(di)(di)六(liu)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)各(ge)取(qu)50μl分(fen)(fen)(fen)別加(jia)(jia)到(dao)第(di)(di)(di)七、第(di)(di)(di)八(ba)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)，再(zai)(zai)在(zai)第(di)(di)(di)七、第(di)(di)(di)八(ba)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)分(fen)(fen)(fen)別加(jia)(jia)標(biao)(biao)準(zhun)(zhun)(zhun)品(pin)(pin)稀(xi)(xi)釋液(ye)50μl，混(hun)勻(yun)后(hou)(hou)從第(di)(di)(di)七、第(di)(di)(di)八(ba)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)分(fen)(fen)(fen)別取(qu)50μl加(jia)(jia)到(dao)第(di)(di)(di)九(jiu)、第(di)(di)(di)十孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)，再(zai)(zai)在(zai)第(di)(di)(di)九(jiu)第(di)(di)(di)十孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)分(fen)(fen)(fen)別加(jia)(jia)標(biao)(biao)準(zhun)(zhun)(zhun)品(pin)(pin)稀(xi)(xi)釋液(ye)50μl，混(hun)勻(yun)后(hou)(hou)從第(di)(di)(di)九(jiu)第(di)(di)(di)十孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)中(zhong)(zhong)(zhong)各(ge)取(qu)50μl棄(qi)掉。（稀(xi)(xi)釋后(hou)(hou)各(ge)孔(kong)(kong)(kong)(kong)(kong)(kong)(kong)加(jia)(jia)樣(yang)(yang)量都為50μl，濃(nong)度分(fen)(fen)(fen)別為240 U/L，160 U/L ，80 U/L，40 U/L， 20 U/L）。
2. 加樣(yang)：分別設空(kong)白(bai)孔(kong)(kong)(kong)（空(kong)白(bai)對照孔(kong)(kong)(kong)不加樣(yang)品(pin)及(ji)酶(mei)標(biao)試劑，其余各步操作(zuo)相同）、待(dai)測(ce)樣(yang)品(pin)孔(kong)(kong)(kong)。在(zai)酶(mei)標(biao)包被板上待(dai)測(ce)樣(yang)品(pin)孔(kong)(kong)(kong)中先加樣(yang)品(pin)稀(xi)釋液40μl，然后再加待(dai)測(ce)樣(yang)品(pin)10μl（樣(yang)品(pin)zui終(zhong)稀(xi)釋度為(wei)5倍(bei)）。加樣(yang)將樣(yang)品(pin)加于酶(mei)標(biao)板孔(kong)(kong)(kong)底部，盡量(liang)不觸(chu)及(ji)孔(kong)(kong)(kong)壁，輕(qing)輕(qing)晃(huang)動混勻。
3. 溫(wen)育(yu)：用封板膜封板后置37℃溫育30分鐘。
4. 配液：將(jiang)30（48T的(de)20倍(bei)）倍(bei)濃縮洗滌液用蒸餾水(shui)30（48T的(de)20倍(bei)）倍(bei)稀釋后備用。
5. 洗(xi)滌(di)：小心揭掉封板膜，棄(qi)去液(ye)體，甩干，每孔加(jia)滿洗(xi)滌(di)液(ye)，靜置30秒后(hou)棄(qi)去，如此重復5次，拍干。
6. 加酶：每孔加入酶標試劑50μl，空(kong)白(bai)孔除外。
7. 溫育：操(cao)作同3。
8. 洗滌：操作(zuo)同5。
9. 顯(xian)色：每孔(kong)先加入(ru)顯(xian)色劑(ji)A50μl，再加入(ru)顯(xian)色劑(ji)B50μl，輕(qing)輕(qing)震蕩(dang)混勻，37℃避光顯色15分鐘.
10. 終止：每孔加終止液50μl，終止反(fan)應（此時藍色(se)立轉黃色(se)）。
11. 測(ce)定(ding)：以空白孔調零，450nm波(bo)長依序測(ce)量(liang)各孔的吸光(guang)度（OD值）。 測(ce)定(ding)應(ying)在(zai)加終止液后15分鐘以內進行。

注意事項：

1. 試劑盒(he)從冷(leng)藏(zang)環境中(zhong)取出應在室(shi)溫平(ping)衡(heng)15-30分鐘后方可使用，酶標包(bao)被板(ban)開封后如(ru)未用完，板(ban)條應裝入密封袋中(zhong)保存。
2. 濃洗(xi)滌液可(ke)能會有結(jie)晶析出，稀釋時可(ke)在水浴中加溫助(zhu)溶，洗(xi)滌時不影響結(jie)果。
3. 各步加樣(yang)均(jun)應使用加樣(yang)器，并經常校(xiao)對(dui)其準確性，以(yi)避(bi)免試驗誤(wu)差。一(yi)次加樣(yang)時(shi)間控制(zhi)在5分(fen)鐘內(nei)，如標(biao)本數量多，推(tui)薦使用排槍加樣(yang)。
4. 請每次測定(ding)(ding)的(de)同時做標準曲線，做復孔(kong)。如標本(ben)中待測物質(zhi)含量過高（樣本(ben)OD值大于標準品(pin)孔(kong)*孔(kong)的(de)OD值），請先用樣品(pin)稀釋(shi)液稀釋(shi)一定(ding)(ding)倍數（n倍）后再測定(ding)(ding)，計算(suan)時請zui后乘以(yi)總稀釋(shi)倍數（×n×5）。
5. 封(feng)板膜只(zhi)限一次性(xing)使用，以避免交叉污染(ran)。
6. 底(di)物請(qing)避光保存(cun)。
7. 嚴格按照說明書的操作(zuo)進(jin)行，試(shi)驗(yan)結果判定必須以酶標儀讀數(shu)為準.
8. 所有(you)樣(yang)品，洗滌液(ye)和(he)各種廢棄(qi)物都應按傳染物處(chu)理。
9. 本試劑不同批號組分不得混用。

10. 如(ru)與(yu)英(ying)文說明書(shu)有異，以英(ying)文說明書(shu)為準。

計算：

以標準物(wu)的濃度為橫坐標，OD值為縱坐標，   

在坐標(biao)紙上繪出標(biao)準曲線，根(gen)據樣品(pin)的OD     

值由標(biao)準(zhun)曲線(xian)查出相(xiang)應的濃度；再乘以稀釋     

倍(bei)數(shu)；或用標準物的濃(nong)度與OD值計算(suan)出標     

準曲(qu)線的(de)直線回(hui)歸方程(cheng)式，將樣品的(de)OD值     

代入方程式，計算出樣品濃度，再乘以稀釋     

倍(bei)數，即(ji)為樣品(pin)的實際濃(nong)度。                 

                                  ;          

試劑盒性能(neng)：

1.樣品線性回歸(gui)與預(yu)期(qi)濃度相關系數(shu)R值為0.92以上。

2.批內與批間(jian)應分(fen)別小(xiao)于9%和15%

檢測(ce)范圍：                ;                             

10U/L -300U/L                                        

                           

保存條件及有效期：

1.試(shi)劑盒保存：；2-8℃。

2．有效(xiao)期：6個月

FOR RESEARCH USE ONLY

Drug Names

Generic Name：Human coagulation factor IX (FIX) ELISA Kit ELISA Kit.

Purpose

This kit allows for the determination of FIX concentrations in human serum, blood plasma, and other biological fluids.

Principle of the assay

The kit assay human FIX  level in the sample，use Purified human FIX antibody to coat microtiter plate wells, make solid-phase antibody, then add FIX  wells, Combined FIX 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 FIX 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 or 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: 240 U/L，160 U/L ，80 U/L，40 U/L， 20 U/L)

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 min, 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

10U/L -300U/L

Storage and validity

1．Storage：  2-8℃.

2．validity： six months