大鼠（COX-2）酶聯免疫分析試劑盒使用說明書
本試劑僅供研究使用目的：本試劑盒用于測定大鼠血清，細胞上清
及相關液體樣本中環加氧酶-2(COX-2)的含量。
實驗原理：
本試劑盒應用雙抗體夾心法測定標本中大鼠環加氧酶-2(COX-2)水平。用純化的大鼠環加
氧酶-2(COX-2)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環加氧酶
-2(COX-2)，再與HRP 標記的環加氧酶-2(COX-2)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環加氧酶-2(COX-2)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大鼠環加氧酶-2(COX-2)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：180U/L 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
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/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
2
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。
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120 U/L ，80 U/L，40 U/L，20 U/L，10 U/L）。
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. 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，每孔加滿洗滌液，靜置30 秒后棄去，如此
重復5 次，拍干。
6. 加酶：每孔加入酶標試劑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（此時藍色立轉黃色）。
11. 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。
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
3
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。
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。
（此圖僅供參考）
試劑盒性能：
1.樣品線性回歸與預期濃度相關系數R 值為0.990 以上。
2.批內與批見應分別小于9%和11%
檢測范圍：
8 UL– 150 U/L
保存條件及有效期：
1.試劑盒保存：；2-8℃。
2．有效期：6 個月
4
Rat cyclooxygenase-Ⅱ
FOR RESEARCH USE ONLY
Drug Names
Generic Name：Rat cyclooxygenase-Ⅱ (COX-2) ELISA Kit.
Purpose
This kit allows for the determination of COX-2 concentrations in Rat serum,
cell culture supernatant, and other biological fluids.
Principle of the assay
The kit assay Rat COX-2 level in the sample ， use Purified Rat
COX-2antibody to coat microtiter plate wells, make solid-phase antibody, then
add COX-2 to wells, Combined COX-2 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 COX-2 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
Stora
ge
User manual 1 1
Closure plate
membrane
2 2
Sealed bags 1 1
Microelisa stripplate 1 1 2-8℃
Standard：180U/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.
6
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
7
well discard(add Sample 50μl to each well after Diluting ,(density: 120 U/L ，80
U/L，40 U/L，20 U/L，10 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.
8
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.
Calculate
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,
This chartis for reference only
9
Assay range
8 UL– 150 U/L
Storage and validity
1．Storage： 2-8℃.
2．validity： six months.