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人弗林蛋白酶（FUR）ELISA試劑盒檢測(cè)范圍：
30pg/ml -1800pg/ml
試劑盒性能：
1.樣品線性回歸與預(yù)期濃度相關(guān)系數(shù) R 值為 0.92 以上。
2.批內(nèi)與批間應(yīng)分別小于 9%和 15%
HE041
人弗林蛋白酶（FUR）ELISA試劑盒
Human Furin（FUR)ELISA Kit
實(shí)驗(yàn)原理 ：
本試劑盒用于測(cè)定人血清，血漿及相關(guān)液體樣本中弗林蛋白酶(FUR)含量。應(yīng)用雙抗體夾心法測(cè)定標(biāo)本中人弗林蛋白酶(FUR)水平。用純化的人弗林蛋白酶(FUR)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弗林蛋白酶(FUR)，再與 HRP 標(biāo)記的弗林蛋白酶(FUR)抗體結(jié)合，形成抗體-抗原-酶標(biāo)抗體復(fù)合物，經(jīng)過*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zui終的黃色。顏色的深淺和樣品中的弗林蛋白酶(FUR)呈正相關(guān)。用酶標(biāo)儀在 450nm 波長(zhǎng)下測(cè)定吸光度（OD 值），通過標(biāo)準(zhǔn)曲線計(jì)算樣品中人弗林蛋白酶(FUR)濃度。
人弗林蛋白酶（FUR）ELISA試劑盒樣本處理：
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)/分）。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)/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。胸腹水、腦脊液參照實(shí)行。
4. 細(xì)胞培養(yǎng)上清：檢測(cè)分泌性的成份時(shí)，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(cè)細(xì)胞內(nèi)的成份時(shí)，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。
2
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)?br />用。標(biāo)本融化后仍然保持 2-8℃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(cè)，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℃保存，但應(yīng)避免反復(fù)凍融.
7. 不能檢測(cè)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. 標(biāo)準(zhǔn)品的稀釋與加樣：在酶標(biāo)包被板上設(shè)標(biāo)準(zhǔn)品孔 10 孔，在*、第二孔中分別加標(biāo)
準(zhǔn)品 100μl，然后在*、第二孔中加標(biāo)準(zhǔn)品稀釋液 50μl，混勻；然后從*孔、第二
孔中各取 100μl 分別加到第三孔和第四孔，再在第三、第四孔分別加標(biāo)準(zhǔn)品稀釋液 50μl，
混勻；然后在第三孔和第四孔中先各取 50μl 棄掉，再各取 50μl 分別加到第五、第六孔
中，再在第五、第六孔中分別加標(biāo)準(zhǔn)品稀釋液 50ul，混勻；混勻后從第五、第六孔中各
取 50μl 分別加到第七、第八孔中，再在第七、第八孔中分別加標(biāo)準(zhǔn)品稀釋液 50μl，混
勻后從第七、第八孔中分別取 50μl 加到第九、第十孔中，再在第九第十孔分別加標(biāo)準(zhǔn)
品稀釋液 50μl，混勻后從第九第十孔中各取 50μl 棄掉。（稀釋后各孔加樣量都為 50μl，
濃度分別為 1800pg/ml， 1200pg/ml，600pg/ml，300pg/ml， 150pg/ml）。
2. 加樣：分別設(shè)空白孔（空白對(duì)照孔不加樣品及酶標(biāo)試劑，其余各步操作相同）、待測(cè)樣
品孔。在酶標(biāo)包被板上待測(cè)樣品孔中先加樣品稀釋液 40μl，然后再加待測(cè)樣品 10μl（樣
品zui終稀釋度為 5 倍）。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(dòng)混
勻。
3. 加酶：每孔加入酶標(biāo)試劑 50μl，空白孔除外。
4. 溫育：用封板膜封板后置 37℃溫育 30 分鐘。
5. 配液：將 30（48T 的 20 倍）倍濃縮洗滌液用蒸餾水 30（48T 的 20 倍）倍稀釋后備用。
6. 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。
7. 顯色：每孔先加入顯色劑 A50μl，再加入顯色劑 B50μl，輕輕震蕩混勻，37℃避光顯
色 10 分鐘.
8. 終止：每孔加終止液 50μl，終止反應(yīng)（此時(shí)藍(lán)色立轉(zhuǎn)黃色）。
9. 測(cè)定：以空白孔調(diào)零，450nm 波長(zhǎng)依序測(cè)量各孔的吸光度（OD 值）。 測(cè)定應(yīng)在加終止
液后 15 分鐘以內(nèi)進(jìn)行。
Generic Name：Human Furin(FUR)ELISA Kit.
Purpose
This kit allows for the determination of FUR concentrations in Human serum, blood
plasma, and other biological fluids.
Principle of the assay
T he kit assay Human FUR level in the sample，use Purified Human FUR antibody to coat
microtiter plate wells, make solid-phase antibody, then add FUR to wells, Combined FUR
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 FUR in the samples is then determined by comparing the O.D. of the
samples to the standard curve.
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: 1800pg/ml， 1200pg/ml，600pg/ml，300pg/ml， 150pg/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.add enzyme：Add HRP-Conjugate reagent 50μl to each well, except blank well.
4.Incubate:After closing plate with Closure plate membrane ,incubate for 30 min at 37℃.
5.Configurate liquid: 30-fold (or 20-fold)wash solution diluted 30-fold (or 20-fold) with distilled water and reserve.
6.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.
7.color：Add Chromogen Solution A 50ul and Chromogen Solution B to each well, evade the
light preservation for 10 min at 37℃
8.Stop the reaction：Add Stop Solution50μl to each well, Stop the reaction(the blue color
change to yellow color).
9.assay：take blank well as zero , Read absorbance at 450nm after Adding Stop Solution and
within 15min.
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