人17-酮類固醇(17-KS)酶聯免疫分析（ELISA）

試劑盒使用說明書

本(ben)試(shi)劑僅供研究使用(yong)       

目的：本試劑盒用于測定人血清，血漿及相關液體樣本中17-酮類固醇(17-KS)的含量。

實驗原理(li)：

    本(ben)試劑盒應(ying)用雙抗體夾心(xin)法測定標本(ben)中人17-酮(tong)(tong)類固醇(chun)(chun)(17-KS)水(shui)平。用純化的(de)人17-酮(tong)(tong)類固醇(chun)(chun)(17-KS)抗體包被微(wei)孔(kong)板，制成固相抗體，往(wang)包被單(dan)抗的(de)微(wei)孔(kong)中依次加入17-酮類固醇(chun)(17-KS)，再(zai)與HRP標(biao)記的(de)17-酮類(lei)固(gu)醇(17-KS)抗體(ti)結合，形(xing)成抗體(ti)-抗原-酶(mei)(mei)標(biao)抗體(ti)復合物(wu)，經過*洗滌后(hou)加底物(wu)TMB顯色。TMB在(zai)HRP酶(mei)(mei)的(de)催化下轉(zhuan)化成藍(lan)色，并(bing)在(zai)酸的(de)作(zuo)用下轉(zhuan)化成zui終的(de)黃(huang)色。顏色的(de)深淺(qian)和樣品(pin)中的(de)17-酮類(lei)固(gu)醇(17-KS)呈正相關。用酶(mei)(mei)標(biao)儀在(zai)450nm波長下測定吸光(guang)度(du)（OD值），通過標(biao)準(zhun)曲線(xian)計(ji)算(suan)樣品(pin)中人17-酮類(lei)固(gu)醇(17-KS)濃度(du)。

試劑盒組成：

樣(yang)本(ben)處理及要求：

1. 血(xue)清：室(shi)溫血(xue)液(ye)自然凝固(gu)10-20分(fen)(fen)鐘，離心(xin)20分(fen)(fen)鐘左(zuo)右(you)（2000-3000轉/分(fen)(fen)）。仔細收(shou)集上清，保存過(guo)程中如出(chu)現沉淀，應再次離心(xin)。

2. 血漿：應根(gen)據標(biao)本的(de)要求選(xuan)擇EDTA或(huo)檸(ning)檬酸鈉作(zuo)為抗凝劑，混合10-20分鐘后，離心20分鐘左右（2000-3000轉/分）。仔細收集上清，保(bao)存(cun)過(guo)程中(zhong)如有(you)沉淀形成，應該再次離心。

3. 尿液：用無菌(jun)管收集，離(li)(li)心20分鐘左右（2000-3000轉(zhuan)/分）。仔細收集上清，保存(cun)過程(cheng)中(zhong)如有沉淀形成(cheng)，應再次離(li)(li)心。胸腹(fu)水、腦脊液參照實(shi)行。

4. 細(xi)(xi)胞培養上清(qing)：檢測(ce)分(fen)泌性的(de)成份(fen)時(shi)，用無菌管收集(ji)(ji)。離(li)(li)心20分(fen)鐘左右(you)（2000-3000轉/分(fen)）。仔細(xi)(xi)收集(ji)(ji)上清(qing)。檢測(ce)細(xi)(xi)胞內(nei)的(de)成份(fen)時(shi)，用PBS（PH7.2-7.4）稀釋細(xi)(xi)胞懸液(ye)，細(xi)(xi)胞濃度(du)達到100萬/ml左右(you)。通(tong)過反復凍融，以使細(xi)(xi)胞破壞并放出細(xi)(xi)胞內(nei)成份(fen)。離(li)(li)心20分(fen)鐘左右(you)（2000-3000轉/分(fen)）。仔細(xi)(xi)收集(ji)(ji)上清(qing)。保存過程中如有(you)沉(chen)淀形成，應再(zai)次離(li)(li)心。

5. 組織標本(ben)：切割標本(ben)后，稱取重量(liang)。加入一定量(liang)的(de)PBS，PH7.4。用液氮迅(xun)速冷凍保(bao)存備用。標本(ben)融(rong)化后仍然保(bao)持2-8℃的(de)溫度。加入一定量(liang)的(de)PBS（PH7.4），用手工或勻(yun)漿器將標本(ben)勻(yun)漿充分。離(li)心(xin)20分鐘左(zuo)右（2000-3000轉/分）。仔(zi)細收集上清。分裝后一份待檢測(ce)，其余冷凍備用。

6. 標本采集(ji)后盡(jin)早(zao)進(jin)(jin)行(xing)提(ti)(ti)取，提(ti)(ti)取按相關(guan)文(wen)獻進(jin)(jin)行(xing)，提(ti)(ti)取后應盡(jin)快進(jin)(jin)行(xing)實驗(yan)。若不能(neng)馬(ma)上進(jin)(jin)行(xing)試驗(yan)，可將(jiang)標本放于-20℃保存，但應避(bi)免反復凍融(rong).

7. 不能檢測含NaN3的樣(yang)品(pin)，因(yin)NaN3抑制辣根過(guo)氧化物酶的（HRP）活性(xing)。

操作步驟

1. 標準(zhun)品(pin)的稀(xi)(xi)釋(shi)(shi)(shi)與加(jia)樣：在(zai)(zai)酶標包被板(ban)上設標準(zhun)品(pin)孔(kong)(kong)10孔(kong)(kong)，在(zai)(zai)*、第(di)(di)(di)(di)二(er)(er)孔(kong)(kong)中(zhong)(zhong)分(fen)別加(jia)標準(zhun)品(pin)100μl，然(ran)后(hou)(hou)在(zai)(zai)*、第(di)(di)(di)(di)二(er)(er)孔(kong)(kong)中(zhong)(zhong)加(jia)標準(zhun)品(pin)稀(xi)(xi)釋(shi)(shi)(shi)液(ye)50μl，混(hun)(hun)勻(yun)；然(ran)后(hou)(hou)從(cong)*孔(kong)(kong)、第(di)(di)(di)(di)二(er)(er)孔(kong)(kong)中(zhong)(zhong)各(ge)(ge)取(qu)100μl分(fen)別加(jia)到(dao)(dao)第(di)(di)(di)(di)三(san)孔(kong)(kong)和第(di)(di)(di)(di)四孔(kong)(kong)，再在(zai)(zai)第(di)(di)(di)(di)三(san)、第(di)(di)(di)(di)四孔(kong)(kong)分(fen)別加(jia)標準(zhun)品(pin)稀(xi)(xi)釋(shi)(shi)(shi)液(ye)50μl，混(hun)(hun)勻(yun)；然(ran)后(hou)(hou)在(zai)(zai)第(di)(di)(di)(di)三(san)孔(kong)(kong)和第(di)(di)(di)(di)四孔(kong)(kong)中(zhong)(zhong)先各(ge)(ge)取(qu)50μl棄掉(diao)，再各(ge)(ge)取(qu)50μl分(fen)別加(jia)到(dao)(dao)第(di)(di)(di)(di)五、第(di)(di)(di)(di)六(liu)(liu)孔(kong)(kong)中(zhong)(zhong)，再在(zai)(zai)第(di)(di)(di)(di)五、第(di)(di)(di)(di)六(liu)(liu)孔(kong)(kong)中(zhong)(zhong)分(fen)別加(jia)標準(zhun)品(pin)稀(xi)(xi)釋(shi)(shi)(shi)液(ye)50ul，混(hun)(hun)勻(yun)；混(hun)(hun)勻(yun)后(hou)(hou)從(cong)第(di)(di)(di)(di)五、第(di)(di)(di)(di)六(liu)(liu)孔(kong)(kong)中(zhong)(zhong)各(ge)(ge)取(qu)50μl分(fen)別加(jia)到(dao)(dao)第(di)(di)(di)(di)七(qi)、第(di)(di)(di)(di)八(ba)孔(kong)(kong)中(zhong)(zhong)，再在(zai)(zai)第(di)(di)(di)(di)七(qi)、第(di)(di)(di)(di)八(ba)孔(kong)(kong)中(zhong)(zhong)分(fen)別加(jia)標準(zhun)品(pin)稀(xi)(xi)釋(shi)(shi)(shi)液(ye)50μl，混(hun)(hun)勻(yun)后(hou)(hou)從(cong)第(di)(di)(di)(di)七(qi)、第(di)(di)(di)(di)八(ba)孔(kong)(kong)中(zhong)(zhong)分(fen)別取(qu)50μl加(jia)到(dao)(dao)第(di)(di)(di)(di)九、第(di)(di)(di)(di)十孔(kong)(kong)中(zhong)(zhong)，再在(zai)(zai)第(di)(di)(di)(di)九第(di)(di)(di)(di)十孔(kong)(kong)分(fen)別加(jia)標準(zhun)品(pin)稀(xi)(xi)釋(shi)(shi)(shi)液(ye)50μl，混(hun)(hun)勻(yun)后(hou)(hou)從(cong)第(di)(di)(di)(di)九第(di)(di)(di)(di)十孔(kong)(kong)中(zhong)(zhong)各(ge)(ge)取(qu)50μl棄掉(diao)。（稀(xi)(xi)釋(shi)(shi)(shi)后(hou)(hou)各(ge)(ge)孔(kong)(kong)加(jia)樣量都為50μl，濃(nong)度分(fen)別為12μg/L，8μg/L ，4μg/L，2μg/L，1μg/L）。
2. 加樣：分別設空(kong)白(bai)孔(kong)(kong)（空(kong)白(bai)對照孔(kong)(kong)不加樣品(pin)(pin)(pin)及酶標(biao)(biao)試(shi)劑，其余各步操作相(xiang)同(tong)）、待(dai)測(ce)樣品(pin)(pin)(pin)孔(kong)(kong)。在酶標(biao)(biao)包(bao)被板(ban)上待(dai)測(ce)樣品(pin)(pin)(pin)孔(kong)(kong)中先加樣品(pin)(pin)(pin)稀(xi)釋(shi)液40μl，然后再加待(dai)測(ce)樣品(pin)(pin)(pin)10μl（樣品(pin)(pin)(pin)zui終(zhong)稀(xi)釋(shi)度為5倍）。加樣將(jiang)樣品(pin)(pin)(pin)加于(yu)酶標(biao)(biao)板(ban)孔(kong)(kong)底(di)部(bu)，盡量不觸及孔(kong)(kong)壁(bi)，輕輕晃動混(hun)勻(yun)。
3. 溫育：用封板(ban)膜封板(ban)后置37℃溫育30分鐘。
4. 配液：將(jiang)30（48T的(de)20倍）倍濃(nong)縮(suo)洗滌(di)液用蒸(zheng)餾水30（48T的(de)20倍）倍稀釋后備用。
5. 洗滌：小心(xin)揭掉(diao)封板膜(mo)，棄去(qu)(qu)液體，甩干，每孔加滿洗滌液，靜置(zhi)30秒后(hou)棄去(qu)(qu)，如此重(zhong)復5次，拍干。
6. 加(jia)酶(mei)：每孔(kong)加(jia)入(ru)酶(mei)標試劑50μl，空白孔(kong)除外。
7. 溫育：操作(zuo)同3。
8. 洗滌：操作同5。
9. 顯色(se)(se)：每孔(kong)先加(jia)入顯色(se)(se)劑A50μl，再(zai)加(jia)入顯色(se)(se)劑B50μl，輕(qing)輕(qing)震蕩混勻，37℃避光顯色(se)(se)15分鐘.
10. 終止：每孔加終止液50μl，終止反應（此時藍色立(li)轉黃(huang)色）。
11. 測定(ding)：以空(kong)白空(kong)調零，450nm波長依序測量各孔的吸光度（OD值）。 測定(ding)應(ying)在(zai)加(jia)終止液后15分鐘(zhong)以內進行。

注意事項：

1. 試劑盒從冷藏環(huan)境中取出應在室溫平衡15-30分鐘后(hou)方可使用(yong)，酶標包被板開封后(hou)如未用(yong)完，板條應裝入(ru)密(mi)封袋中保存(cun)。
2. 濃洗滌液可(ke)能會(hui)有(you)結晶析(xi)出，稀釋時(shi)可(ke)在水浴中加溫(wen)助溶，洗滌時(shi)不影(ying)響結果。
3. 各(ge)步(bu)加樣均應(ying)使(shi)用加樣器，并經(jing)常校(xiao)對其準(zhun)確性，以(yi)避免試(shi)驗誤差。一次加樣時間控制在5分鐘內(nei)，如標本數(shu)量多，推薦使(shi)用排(pai)槍(qiang)加樣。
4. 請每次測(ce)(ce)定(ding)的(de)同(tong)時(shi)做標(biao)準曲(qu)線，做復孔。如(ru)標(biao)本(ben)(ben)中待測(ce)(ce)物質含量過高（樣本(ben)(ben)OD值(zhi)大(da)于標(biao)準品孔*孔的(de)OD值(zhi)），請先(xian)用樣品稀釋(shi)(shi)液稀釋(shi)(shi)一定(ding)倍數（n倍）后再測(ce)(ce)定(ding)，計(ji)算(suan)時(shi)請zui后乘以(yi)總(zong)稀釋(shi)(shi)倍數（×n×5）。
5. 封(feng)板膜只限一次性使用，以避免交叉污染。
6. 底物(wu)請避(bi)光(guang)保存(cun)。
7. 嚴格按照說明書的操作進行，試驗結果判定必須以酶標儀讀數(shu)為準(zhun).
8. 所有(you)樣品，洗(xi)滌液和各種廢(fei)棄物(wu)都應(ying)按傳染物(wu)處理。
9. 本試(shi)劑不同批號組分不得混用。

10. 如與英文(wen)說明書(shu)有異，以(yi)英文(wen)說明書(shu)為準(zhun)。

計算：

以標(biao)準物的濃(nong)度為橫坐(zuo)標(biao)，OD值為縱坐(zuo)標(biao)，   

在坐標紙(zhi)上繪出(chu)標準曲(qu)線(xian)，根據樣品的OD     

值由標(biao)準(zhun)曲線查出相應的濃(nong)度；再乘(cheng)以稀釋      

倍數；或(huo)用標(biao)準(zhun)物的濃度與OD值計(ji)算出標(biao)      

準曲(qu)線的(de)(de)直線回歸(gui)方(fang)程式，將樣品的(de)(de)OD值      

代入(ru)方程式，計算出樣品(pin)濃(nong)度，再乘以稀釋      

倍數(shu)，即(ji)為樣品的實(shi)際濃度。        ;          ;

                                            

試(shi)劑盒性能：

1.樣品線性回歸與預(yu)期濃(nong)度相關系(xi)數(shu)R值為0.95以上。

2.批(pi)內與批(pi)見應(ying)分別小于9%和11%

檢測范(fan)圍：                                             

0.5μg/L -15μg/L                                       

                           

保存條件及有效期(qi)：

1.試劑盒(he)保存：；2-8℃。

2．有(you)效(xiao)期：6個月

Human 17-ketosteroids

Drug Names

Generic Name：Human 17-ketosteroids (17-KS) ELISA Kit.

Purpose

This kit allows for the determination of 17-KS concentrations in Human serum, blood plasma, and other biological fluids.

Principle of the assay

The kit assay Human17-KS level in the sample，use Purified Human 17-KS antibody to coat microtiter plate wells, make solid-phase antibody, then add 17-KS to wells, Combined 17-KS antibody 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 17-KS 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: 12μg/L,8μg/L ,4μg/L,2μg/L,1μg/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 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

0.5μg/L -15μg/L

Storage and validity

1．Storage：  2-8℃.

2．validity： six months.