1
魚(Cortisol)酶聯(lián)免疫分析（ELISA）
試劑盒使用說明書
本試劑僅供研究使用 目的：本試劑盒用于測定魚血清，血漿及相關(guān)
液體樣本中(Cortisol)的含量。
實驗原理：
本試劑盒應(yīng)用雙抗體夾心法測定標(biāo)本中魚(Cortisol)水平。用純化的魚
(Cortisol)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(Cortisol)，
再與 HRP 標(biāo)記的抗體結(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(Cortisol)呈正相關(guān)。 用酶標(biāo)儀在450nm波長下測定吸光度 （OD
值） ，通過標(biāo)準(zhǔn)曲線計算樣品中魚(Cortisol)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(biāo)包被板 1×48 1×96 2-8℃保存
標(biāo)準(zhǔn)品：2250ng/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、腦脊液參照實行。
4. 細(xì)胞培養(yǎng)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(zhuǎn)/
分） 。仔細(xì)收集上清。檢測細(xì)胞內(nèi)的成份時，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，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)行實驗。若不能馬上
進(jìn)行試驗，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 1500 ng/L，1000 ng/L ，500 ng/L，250 ng/L，125 ng/L） 。
2. 加樣：分別設(shè)空白孔（空白對照孔不加樣品及酶標(biāo)試劑，其余各步操作相同） 、待測樣
品孔。在酶標(biāo)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(biāo)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(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)（此時藍(lán)色立轉(zhuǎn)黃色） 。
11. 測定：以空白空調(diào)零，450nm波長依序測量各孔的吸光度（OD 值） 。 測定應(yīng)在加終止
液后 15分鐘以內(nèi)進(jìn)行。
注意事項：
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．濃洗滌液可能會有結(jié)晶析出，稀釋時可在水浴中加溫助溶，洗滌時不影響結(jié)果。
3．各步加樣均應(yīng)使用加樣器，并經(jīng)常校對其準(zhǔn)確性，以避免試驗誤差。一次加樣時間
控制在 5 分鐘內(nèi)，如標(biāo)本數(shù)量多，使用排槍加樣。
4．請每次測定的同時做標(biāo)準(zhǔn)曲線，做復(fù)孔。如標(biāo)本中待測物質(zhì)含量過高（樣本 OD 值
大于標(biāo)準(zhǔn)品孔*孔的 OD 值） ，請先用樣品稀釋液稀釋一定倍數(shù)（n倍）后再測定，計
算時請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．底物請避光保存。3
7．嚴(yán)格按照說明書的操作進(jìn)行，試驗結(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．本試劑不同批號組分不得混用。
10. 如與英文說明書有異，以英文說明書為準(zhǔn)。
計算：
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，根據(jù)樣品的 OD
值由標(biāo)準(zhǔn)曲線查出相應(yīng)的濃度；再乘以稀釋
倍數(shù)；或用標(biāo)準(zhǔn)物的濃度與 OD 值計算出標(biāo)
準(zhǔn)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(shù)，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(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%
檢測范圍：
100 ng/L -1800 ng/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
FOR RESEARCH USE ONLY
Fish Cortisol
Drug Names
Generic Name：Fish Cortisol ELISA Kit.
Purpose
This kit allows for the determination of Cortisol concentrations in Fish
serum, blood plasma, and other biological fluids.
Principle of the assay
The kit assay Fish Cortisol level in the sample，use Purified Fish Cortisol
antibody to coat microtiter plate wells, make solid-phase antibody, then add
Cortisol to wells, Combined Cortisol 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 Cortisol 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：2250ng/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 suspension6
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: 15007
ng/L,1000 ng/L ,500 ng/L,250 ng/L,125 ng/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.8
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
Assay range
100 ng/L -1800 ng/L
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
This chart for reference only9
Storage and validity
1．Storage： 2-8℃.
2．validity： six months.