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人外周蛋白抗體(PRPH Ab)酶聯(lián)免疫分析（ELISA）
試劑盒使用說(shuō)明書(shū)
本試劑僅供研究使用 目的：本試劑盒用于測(cè)定人血清，血漿及相關(guān)
液體樣本中外周蛋白抗體(PRPH Ab)的含量。
實(shí)驗(yàn)原理：
本試劑盒應(yīng)用雙抗原夾心法測(cè)定標(biāo)本中人外周蛋白抗體(PRPH Ab)水平。用純化的抗原
包被微孔板，制成固相抗原，往包被的微孔中依次加入外周蛋白抗體(PRPH Ab)，再與 HRP
標(biāo)記的黑色素細(xì)胞抗原結(jié)合，形成抗原-抗體-抗原復(fù)合物，經(jīng)過(guò)*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
和樣品中的外周蛋白抗體(PRPH Ab)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(guò)標(biāo)準(zhǔn)曲線(xiàn)計(jì)算樣品中人外周蛋白抗體(PRPH Ab)濃度。
試劑盒組成：
試劑盒組成 48孔配置 96孔配置 保存
說(shuō)明書(shū) 1份 1 份
封板膜 2片（48） 2 片（96）
密封袋 1個(gè) 1 個(gè)
酶標(biāo)包被板 1×48 1×96 2-8℃保存
標(biāo)準(zhǔn)品：1800ng/L 0.5ml×1瓶 0.5ml×1 瓶 2-8℃保存
標(biāo)準(zhǔn)品稀釋液 1.5ml×1瓶 1.5ml×1 瓶 2-8℃保存
酶標(biāo)試劑 3 ml×1瓶 6 ml×1 瓶 2-8℃保存
樣品稀釋液 3 ml×1瓶 6 ml×1 瓶 2-8℃保存
顯色劑 A 液 3 ml×1瓶 6 ml×1 瓶 2-8℃保存
顯色劑 B 液 3 ml×1瓶 6 ml×1 瓶 2-8℃保存
終止液 3ml×1瓶 6ml×1 瓶 2-8℃保存
濃縮洗滌液 （20ml×20 倍）×1 瓶 （20ml×30 倍）×1 瓶 2-8℃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)/分）。仔細(xì)收集上
清，保存過(guò)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(guò)程中如有沉淀形成，應(yīng)該再次
離心。
3. 尿液：用無(wú)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(guò)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(wú)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(wàn)/ml 左右。通過(guò)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(guò)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(chēng)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(guò)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 1200ng/L，800ng/L ，400ng/L，200ng/L， 100ng/L）。
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. 溫育：用封板膜封板后置 37℃溫育 30分鐘。
4. 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。
5. 洗滌：小心揭掉封板膜，棄去液體，甩干，每孔加滿(mǎn)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(biāo)試劑 50μl，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 A50μl，再加入顯色劑 B50μl，輕輕震蕩混勻，37℃避光顯色
15分鐘.
10. 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）。
11. 測(cè)定：以空白空調(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?xún)?nèi)進(jìn)行。
注意事項(xiàng)：
1．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(biāo)包被板開(kāi)封后如未
用完，板條應(yīng)裝入密封袋中保存。
2．濃洗滌液可能會(huì)有結(jié)晶析出，稀釋時(shí)可在水浴中加溫助溶，洗滌時(shí)不影響結(jié)果。
3．各步加樣均應(yīng)使用加樣器，并經(jīng)常校對(duì)其準(zhǔn)確性，以避免試驗(yàn)誤差。一次加樣時(shí)間
控制在 5 分鐘內(nèi)，如標(biāo)本數(shù)量多，使用排槍加樣。
4．請(qǐng)每次測(cè)定的同時(shí)做標(biāo)準(zhǔn)曲線(xiàn)，做復(fù)孔。如標(biāo)本中待測(cè)物質(zhì)含量過(guò)高（樣本 OD 值
大于標(biāo)準(zhǔn)品孔*孔的 OD 值），請(qǐng)先用樣品稀釋液稀釋一定倍數(shù)（n 倍）后再測(cè)定，計(jì)
算時(shí)請(qǐng)zui后乘以總稀釋倍數(shù)（×n×5）。
5．封板膜只限一次性使用，以避免交叉污染。
6．底物請(qǐng)避光保存。3
7．嚴(yán)格按照說(shuō)明書(shū)的操作進(jìn)行，試驗(yàn)結(jié)果判定必須以酶標(biāo)儀讀數(shù)為準(zhǔn).
8．所有樣品，洗滌液和各種廢棄物都應(yīng)按傳染物處理。
9．本試劑不同批號(hào)組分不得混用。
10. 如與英文說(shuō)明書(shū)有異，以英文說(shuō)明書(shū)為準(zhǔn)。
計(jì)算：
以標(biāo)準(zhǔn)物的濃度為橫坐標(biāo)，OD值為縱坐標(biāo)，
在坐標(biāo)紙上繪出標(biāo)準(zhǔn)曲線(xiàn)，根據(jù)樣品的 OD
值由標(biāo)準(zhǔn)曲線(xiàn)查出相應(yīng)的濃度；再乘以稀釋
倍數(shù)；或用標(biāo)準(zhǔn)物的濃度與 OD 值計(jì)算出標(biāo)
準(zhǔn)曲線(xiàn)的直線(xiàn)回歸方程式，將樣品的 OD 值
代入方程式，計(jì)算出樣品濃度，再乘以稀釋
倍數(shù)，即為樣品的實(shí)際濃度。
（此圖僅供參考）
試劑盒性能：
1.樣品線(xiàn)性回歸與預(yù)期濃度相關(guān)系數(shù) R 值為 0.95 以上。
2.批內(nèi)與批見(jiàn)應(yīng)分別小于 9%和 11%
檢測(cè)范圍：
50ng/L -1500ng/L
保存條件及有效期：
1.試劑盒保存：；2-8℃。
2．有效期：6個(gè)月4
Human PRPH Ab
FOR RESEARCH USE ONLY
Drug Names
Generic Name：Human PRPH Ab ELISA Kit.
Purpose
This kit allows for the determination of PRPH Ab concentrations in Human serum, blood
plasma, and other biological fluids.
Principle of the assay
The kit assay Human PRPH Ab level in the sample，use Purified Human MC antigen to
coat microtiter plate wells, make solid-phase antibgen, then add PT Ab to wells, Combined MC
antigen which With HRP labeled ， become antigen - antibody - enzyme-antigen 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 PRPH Ab in the samples is then determined by comparing the O.D. of the
samples to the standard curve.5
Materials provided with the kit
Materials provided with
the kit
48determinations 96 determinations Storage
User manual 1 1
Closure plate membrane 2 2
Sealed bags 1 1
Microelisa stripplate 1 1 2-8℃
Standard：1800ng/L 0.5ml×1 bottle 0.5ml×1 bottle 2-8℃
Standard diluent 1.5ml×1 bottle 1.5ml×1 bottle 2-8℃
HRP-Conjugate reagent 3ml×1 bottle 6ml×1 bottle 2-8℃
Sample diluent 3ml×1 bottle 6ml×1 bottle 2-8℃
Chromogen Solution A 3ml×1 bottle 6ml×1 bottle 2-8℃
Chromogen Solution B 3ml×1 bottle 6ml×1 bottle 2-8℃
Stop Solution 3ml×1 bottle 6ml×1 bottle 2-8℃
wash solution
（20ml×20 fold）
×1bottle
（20ml×30 fold）
×1bottle
2-8℃
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 of6
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: 1200ng/L，800ng/L ，400ng/L，200ng/L， 100ng/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.7
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 material8
process.
9. Do not mix reagents with those from other lots.
Calculate
Assay range
50ng/L -1500ng/L
Storage and validity
1．Storage： 2-8℃.
2．validity： six months.
Take the standard density as the horizontal, the OD
value for the vertical ,draw the standard curve on graph
paper, Find out the corresponding density according to the
sample OD value by the Sample curve, multiplied by the
dilution multiple, or calculate the straight line regression
equation of the standard curve with the standard density and
the OD value ,with the sample OD value in the equation,
calculate the sample density, multiplied by the dilution factor,
the result is the sample actual density.
This chartis for reference only