人P53蛋白酶聯免疫分析（ELISA）試劑盒使用說明書
實驗原理：
本試劑盒用于測定人血清，血漿及相關
液體樣本中P53蛋白的含量。應用雙抗體夾心法測定標本中人 P53蛋白水平。用純化的人 P53蛋白抗體包被微孔板，制成固相抗體，往包被單抗的微孔中依次加入 P53 蛋白再與 HRP 標記的 P53 蛋白抗體結合，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P53 蛋白呈正相關。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 P53 蛋白濃度。
試劑盒組成：
試劑盒組成 48 孔配置 96 孔配置 保存
說明書 1 份 1 份
封板膜 2 片（48） 2 片（96）
密封袋 1 個 1 個
酶標包被板 1×48 1×96 2-8℃保存
標準品：45μg/mL 0.5ml×1 瓶 0.5ml×1 瓶 2-8℃保存
標準品稀釋液 1.5ml×1 瓶 1.5ml×1 瓶 2-8℃保存
酶標試劑 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
人P53蛋白酶聯免疫分析（ELISA）試劑盒使用說明書樣本處理：
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/分）。仔細收集上
清，保存過程中如出現沉淀，應再次離心。
2. 血漿：應根據標本的要求選擇 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/分）。仔細收集上清，保存過程中如有沉淀形成，應該再次
離心。
3. 尿液：用無菌管收集，離心20 分鐘左右（2000-3000 轉/分）。仔細收集上清，保存過程
中如有沉淀形成，應再次離心。胸腹水、腦脊液參照實行。
4. 細胞培養上清：檢測分泌性的成份時，用無菌管收集。離心 20 分鐘左右（2000-3000 轉/
分）。仔細收集上清。檢測細胞內的成份時，用 PBS（PH7.2-7.4）稀釋細胞懸液，細胞
濃度達到 100 萬/ml 左右。通過反復凍融，以使細胞破壞并放出細胞內成份。離心 20 分
鐘左右（2000-3000 轉/分）。仔細收集上清。保存過程中如有沉淀形成，應再次離心。
5. 組織標本：切割標本后，稱取重量。加入一定量的 PBS，PH7.4。用液氮迅速冷凍保存備
用。標本融化后仍然保持 2-8℃的溫度。加入一定量的 PBS（PH7.4），用手工或勻漿器
將標本勻漿充分。離心 20 分鐘左右（2000-3000 轉/分）。仔細收集上清。分裝后一份待
檢測，其余冷凍備用。
6. 標本采集后盡早進行提取，提取按相關文獻進行，提取后應盡快進行實驗。若不能馬上
進行試驗，可將標本放于-20℃保存，但應避免反復凍融.
7. 不能檢測含 NaN3 的樣品，因NaN3 抑制辣根過氧化物酶的（HRP）活性。
實驗操作步驟：
1. 標準品的稀釋與加樣：在酶標包被板上設標準品孔10 孔，在*、第二孔中分別加標
準品 100μl，然后在*、第二孔中加標準品稀釋液 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、第四孔分別加標準品稀釋液 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、第六孔中分別加標準品稀釋液 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、第八孔中分別加標準品稀釋液 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準
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 30μg/mL，20μg/mL ，10μg/mL， 5μg/mL， 2.5μg/mL）。
2. 加樣：分別設空白孔（空白對照孔不加樣品及酶標試劑，其余各步操作相同）、待測樣
品孔。在酶標包被板上待測樣品孔中先加樣品稀釋液 40μl，然后再加待測樣品 10μl（樣
品zui終稀釋度為 5 倍）。加樣將樣品加于酶標板孔底部，盡量不觸及孔壁，輕輕晃動混
勻。
3. 加酶：每孔加入酶標試劑 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 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（此時藍色立轉黃色）。
9. 測定：以空白空調零，450nm 波長依序測量各孔的吸光度（OD 值）。 測定應在加終止
液后 15 分鐘以內進行。
實驗注意事項：
1．試劑盒從冷藏環境中取出應在室溫平衡 15-30 分鐘后方可使用，酶標包被板開封后如未
用完，板條應裝入密封袋中保存。
2．濃洗滌液可能會有結晶析出，稀釋時可在水浴中加溫助溶，洗滌時不影響結果。
3．各步加樣均應使用加樣器，并經常校對其準確性，以避免試驗誤差。一次加樣時間
控制在 5分鐘內，如標本數量多，推薦使用排槍加樣。
4．請每次測定的同時做標準曲線，做復孔。如標本中待測物質含量過高（樣本 OD值
大于標準品孔*孔的 OD 值），請先用樣品稀釋液稀釋一定倍數（n 倍）后再測定，計
算時請zui后乘以總稀釋倍數（×n×5）。
5．封板膜只限一次性使用，以避免交叉污染。
6．底物請避光保存。
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為準.
8．所有樣品，洗滌液和各種廢棄物都應按傳染物處理。
9．本試劑不同批號組分不得混用。
10. 如與英文說明書有異，以英文說明書為準。
人P53蛋白酶聯免疫分析（ELISA）試劑盒使用說明書實驗結果計算 ：
以標準物的濃度為橫坐標，OD 值為縱坐標，在坐標紙上繪出標準曲線，根據樣品的 OD值由標準曲線查出相應的濃度；再乘以稀釋倍數；或用標準物的濃度與 OD 值計算出標準曲線的直線回歸方程式，將樣品的 OD 值代入方程式，計算出樣品濃度，再乘以稀釋倍數，即為樣品的實際濃度。
試劑盒性能：
1.樣品線性回歸與預期濃度相關系數R 值為 0.92 以上。
2.批內與批間應分別小于 9%和 15%
檢測范圍：
1.6μg/mL -40μg/mL
保存條件及有效期：
1.試劑盒保存：2-8℃。
2．有效期：6 個月
Human P53 protein
FOR RESEARCH USE ONLY
Drug Names
Generic Name：Human P53 protein (P53) ELISAKit.
Purpose
This kit allows for the determination of P53 concentrations in Human serum, blood plasma,
and other biological fluids.
Principle of the assay
T he kit assay Human P53 level in the sample，use Purified Human P53 antibody to coat
microtiter plate wells, make solid-phase antibody, then add P53 to wells, Combined P53
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 P53 in thesamples is then determined by comparing the O.D. of the samples
to the standard curve.
Materials provided with the kit
Materials provided with
the kit
48determinations 96 determinations Storage
User manual 1 1
Closure plate
membrane
Sealed bags 1 1
Microelisa stripplate 1 1 2-8℃
Standard ：45μg/mL 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 SolutionA 3ml×1 bottle 6ml×1 bottle 2-8℃
Chromogen SolutionB 3ml×1 bottle 6ml×1 bottle 2-8℃
Stop Solution 3ml×1 bottle 6ml×1 bottle 2-8℃
wash solution
（20ml×20 fold ）
×1 bottle
（20ml×30 fold ）
×1 bottle
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 10-20mins ,centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant, If
precipitation appeared, Centrifugalagain.
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 culturesupernatant-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 atthe speed of 2000-3000 r.p.m. remove
supernatant, If precipitation appeared, Centrifugal again.
5. Tissue samples-After cutting samples, check theweight,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 repeatedfreeze-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 forthwell separay. then add Standard dilution 50μl to the third and the forth
well ,mix ; then take out 50μl from the third and the forth welldiscard, add 50μl tothe fifth and the sixth well ,then add Standarddilution 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 addStandard dilution 50μl to the seventh and the eighth well ,mix ; takeout 50μl from the seventh and the
eighth well and add to the ninthand the tenth well, add Standard dilution 50μl to the ninth and the tenth well, mix , take out50μl from the ninth and the tenth well discard(add Sample 50μl to eachwell after Diluting ,(density: 30μg/mL,20μg/mL,10μg/mL, 5μg/mL, 2.5μg/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
dilution40μl to testing sample well, then add testing sample 10μl (sample final dilution is
5-fold), add sampleto wells , don’t touch the well wall as far as possible, and Gently mix.
3.add enzyme：Add HRP-Conjugate reagent 50μl toeach 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 5times, 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 toeach 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.
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 useup 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 materialcontent is excessively higher (The sample OD is bigger than thefirst
standard well ),please dilute Sample (n-fold), Please diluenteand 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 testresult 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 withthose from other lots.
Calculate
Assay range
1.6μg/mL -40μg/mL
Storage and validity
1．Storage： 2-8℃.
2．validity： six months.
Takethe standard density as the horizontal, the OD
value for the vertical ,draw the standard curve on graph
paper, Find out the correspondingdensity according to the
sample OD value by the Sample curve, multiplied by the
dilution multiple, or calculate the straight line regression
equationof the standard curve with the standard density and
the OD value ,with the sample OD value in the equation,
calculate thesample density, multiplied by the dilution factor,
the result is the sample actual density.
This chart for reference only
