大鼠白細胞介素4（IL-4）酶聯免疫分析(ELISA)

試劑盒使用說(shuo)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，血漿，細胞上清及相關液體樣本中白細胞介素4（IL-4）的含量。

實驗原(yuan)理：

   本試劑盒應用雙抗體夾心法測定標本中大鼠白細胞介素4（IL-4）水平。用純化的大鼠白細胞介素4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白細胞介素4，再與HRP標記的羊抗鼠抗體結合，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IL-4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白細胞介素4（IL-4）濃度。

試劑(ji)盒組成：

樣本處理及要求：

1. 血清：室溫血液自然凝固10-20分(fen)鐘(zhong)，離(li)心(xin)20分(fen)鐘(zhong)左右(you)（2000-3000轉/分(fen)）。仔(zi)細收(shou)集上清，保(bao)存(cun)過(guo)程中(zhong)如出(chu)現沉淀，應再次離(li)心(xin)。

2. 血漿：應(ying)根(gen)據標本的(de)要求選擇EDTA或檸檬酸鈉(na)作為抗凝(ning)劑，混合10-20分(fen)鐘后，離(li)心(xin)20分(fen)鐘左右（2000-3000轉(zhuan)/分(fen)）。仔細收集(ji)上清，保存過程中如有沉淀形成，應(ying)該再次(ci)離(li)心(xin)。

3. 尿液：用無菌(jun)管收集，離(li)心20分鐘左右（2000-3000轉/分）。仔細收集上清，保存(cun)過程中如有(you)沉淀形成，應(ying)再次(ci)離(li)心。胸腹水、腦脊液參照實行。

4. 細(xi)胞(bao)培(pei)養(yang)上(shang)(shang)清：檢(jian)測分(fen)(fen)泌性(xing)的(de)(de)成份(fen)時，用(yong)無菌管收(shou)集(ji)(ji)。離心(xin)20分(fen)(fen)鐘(zhong)左(zuo)右(you)（2000-3000轉/分(fen)(fen)）。仔細(xi)收(shou)集(ji)(ji)上(shang)(shang)清。檢(jian)測細(xi)胞(bao)內(nei)的(de)(de)成份(fen)時，用(yong)PBS（PH7.2-7.4）稀釋細(xi)胞(bao)懸(xuan)液，細(xi)胞(bao)濃度達到100萬(wan)/ml左(zuo)右(you)。通過反(fan)復凍融，以使細(xi)胞(bao)破壞并(bing)放出細(xi)胞(bao)內(nei)成份(fen)。離心(xin)20分(fen)(fen)鐘(zhong)左(zuo)右(you)（2000-3000轉/分(fen)(fen)）。仔細(xi)收(shou)集(ji)(ji)上(shang)(shang)清。保存過程(cheng)中如有沉(chen)淀形成，應再次(ci)離心(xin)。

5. 組織標(biao)本：切割標(biao)本后(hou)，稱取重量。加入一(yi)定量的(de)PBS，PH7.4。用液氮(dan)迅(xun)速冷(leng)凍保(bao)存備用。標(biao)本融化后(hou)仍然保(bao)持2-8℃的(de)溫度。加入一(yi)定量的(de)PBS（PH7.4），用手工或勻漿器將(jiang)標(biao)本勻漿充分。離心20分鐘左右（2000-3000轉/分）。仔細收集(ji)上清。分裝后(hou)一(yi)份待檢測，其余冷(leng)凍備用。

6. 標本(ben)采(cai)集后(hou)盡早進(jin)行(xing)提取，提取按相關(guan)文獻(xian)進(jin)行(xing)，提取后(hou)應盡快進(jin)行(xing)實驗(yan)。若不能馬上進(jin)行(xing)試驗(yan)，可將標本(ben)放于-20℃保存(cun)，但(dan)應避(bi)免(mian)反復凍融.

7. 不能(neng)檢測含NaN3的樣品(pin)，因NaN3抑制辣根過氧化物酶的（HRP）活性。

操作步驟：

1. 標(biao)(biao)準(zhun)(zhun)品(pin)(pin)的稀(xi)釋(shi)與加(jia)(jia)(jia)樣(yang)：在(zai)酶標(biao)(biao)包被板上設標(biao)(biao)準(zhun)(zhun)品(pin)(pin)孔(kong)(kong)(kong)10孔(kong)(kong)(kong)，在(zai)*、第(di)(di)(di)(di)二(er)孔(kong)(kong)(kong)中(zhong)分(fen)(fen)(fen)別(bie)(bie)(bie)(bie)加(jia)(jia)(jia)標(biao)(biao)準(zhun)(zhun)品(pin)(pin)100μl，然后在(zai)*、第(di)(di)(di)(di)二(er)孔(kong)(kong)(kong)中(zhong)加(jia)(jia)(jia)標(biao)(biao)準(zhun)(zhun)品(pin)(pin)稀(xi)釋(shi)液50μl，混(hun)(hun)勻(yun)(yun)；然后從*孔(kong)(kong)(kong)、第(di)(di)(di)(di)二(er)孔(kong)(kong)(kong)中(zhong)各(ge)(ge)取100μl分(fen)(fen)(fen)別(bie)(bie)(bie)(bie)加(jia)(jia)(jia)到(dao)(dao)第(di)(di)(di)(di)三孔(kong)(kong)(kong)和第(di)(di)(di)(di)四孔(kong)(kong)(kong)，再在(zai)第(di)(di)(di)(di)三、第(di)(di)(di)(di)四孔(kong)(kong)(kong)分(fen)(fen)(fen)別(bie)(bie)(bie)(bie)加(jia)(jia)(jia)標(biao)(biao)準(zhun)(zhun)品(pin)(pin)稀(xi)釋(shi)液50μl，混(hun)(hun)勻(yun)(yun)；然后在(zai)第(di)(di)(di)(di)三孔(kong)(kong)(kong)和第(di)(di)(di)(di)四孔(kong)(kong)(kong)中(zhong)先各(ge)(ge)取50μl棄掉，再各(ge)(ge)取50μl分(fen)(fen)(fen)別(bie)(bie)(bie)(bie)加(jia)(jia)(jia)到(dao)(dao)第(di)(di)(di)(di)五、第(di)(di)(di)(di)六(liu)孔(kong)(kong)(kong)中(zhong)，再在(zai)第(di)(di)(di)(di)五、第(di)(di)(di)(di)六(liu)孔(kong)(kong)(kong)中(zhong)分(fen)(fen)(fen)別(bie)(bie)(bie)(bie)加(jia)(jia)(jia)標(biao)(biao)準(zhun)(zhun)品(pin)(pin)稀(xi)釋(shi)液50ul，混(hun)(hun)勻(yun)(yun)；混(hun)(hun)勻(yun)(yun)后從第(di)(di)(di)(di)五、第(di)(di)(di)(di)六(liu)孔(kong)(kong)(kong)中(zhong)各(ge)(ge)取50μl分(fen)(fen)(fen)別(bie)(bie)(bie)(bie)加(jia)(jia)(jia)到(dao)(dao)第(di)(di)(di)(di)七(qi)、第(di)(di)(di)(di)八(ba)(ba)孔(kong)(kong)(kong)中(zhong)，再在(zai)第(di)(di)(di)(di)七(qi)、第(di)(di)(di)(di)八(ba)(ba)孔(kong)(kong)(kong)中(zhong)分(fen)(fen)(fen)別(bie)(bie)(bie)(bie)加(jia)(jia)(jia)標(biao)(biao)準(zhun)(zhun)品(pin)(pin)稀(xi)釋(shi)液50μl，混(hun)(hun)勻(yun)(yun)后從第(di)(di)(di)(di)七(qi)、第(di)(di)(di)(di)八(ba)(ba)孔(kong)(kong)(kong)中(zhong)分(fen)(fen)(fen)別(bie)(bie)(bie)(bie)取50μl加(jia)(jia)(jia)到(dao)(dao)第(di)(di)(di)(di)九、第(di)(di)(di)(di)十孔(kong)(kong)(kong)中(zhong)，再在(zai)第(di)(di)(di)(di)九第(di)(di)(di)(di)十孔(kong)(kong)(kong)分(fen)(fen)(fen)別(bie)(bie)(bie)(bie)加(jia)(jia)(jia)標(biao)(biao)準(zhun)(zhun)品(pin)(pin)稀(xi)釋(shi)液50μl，混(hun)(hun)勻(yun)(yun)后從第(di)(di)(di)(di)九第(di)(di)(di)(di)十孔(kong)(kong)(kong)中(zhong)各(ge)(ge)取50μl棄掉。（稀(xi)釋(shi)后各(ge)(ge)孔(kong)(kong)(kong)加(jia)(jia)(jia)樣(yang)量(liang)都(dou)為(wei)50μl，濃(nong)度分(fen)(fen)(fen)別(bie)(bie)(bie)(bie)為(wei)90 ng/L，60ng/L ，30 ng/L，15ng/L，7.5 ng/L）。
2. 加(jia)樣(yang)(yang)：分別(bie)設空白孔（空白對照孔不(bu)加(jia)樣(yang)(yang)品(pin)及(ji)酶(mei)(mei)標試劑，其(qi)余各步操作相同）、待測樣(yang)(yang)品(pin)孔。在酶(mei)(mei)標包被(bei)板上待測樣(yang)(yang)品(pin)孔中(zhong)先加(jia)樣(yang)(yang)品(pin)稀(xi)釋(shi)液40μl，然后(hou)再加(jia)待測樣(yang)(yang)品(pin)10μl（樣(yang)(yang)品(pin)zui終稀(xi)釋(shi)度(du)為(wei)5倍）。加(jia)樣(yang)(yang)將樣(yang)(yang)品(pin)加(jia)于酶(mei)(mei)標板孔底部，盡量不(bu)觸及(ji)孔壁，輕輕晃動(dong)混(hun)勻。
3. 溫育：用封板膜封板后置37℃溫育30分鐘。
4. 配液：將(jiang)30（48T的(de)20倍）倍濃(nong)縮(suo)洗(xi)滌液用蒸餾水30（48T的(de)20倍）倍稀釋(shi)后備用。
5. 洗滌：小心揭(jie)掉(diao)封板(ban)膜(mo)，棄去(qu)液體，甩干，每孔(kong)加滿(man)洗滌液，靜置30秒(miao)后(hou)棄去(qu)，如此重(zhong)復5次，拍干。
6. 加(jia)酶：每孔加(jia)入(ru)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(se)：每孔先加(jia)(jia)入顯色(se)劑A50μl，再加(jia)(jia)入顯色(se)劑B50μl，輕輕震蕩混勻，37℃避光顯色(se)15分鐘.
10. 終止(zhi)(zhi)：每孔加終止(zhi)(zhi)液50μl，終止(zhi)(zhi)反應（此(ci)時藍色立(li)轉黃色）。
11. 測定：以空白空調零，450nm波長依序測量各(ge)孔的吸光(guang)度（OD值）。 測定應在(zai)加終止(zhi)液后15分鐘以內進行(xing)。

注意(yi)事項：

1. 試劑(ji)盒(he)從冷藏環境中取(qu)出應在室溫平(ping)衡15-30分鐘后(hou)方可使(shi)用，酶標包被板(ban)(ban)開(kai)封后(hou)如未用完，板(ban)(ban)條應裝(zhuang)入密封袋(dai)中保存。
2. 濃洗滌液可能會(hui)有結(jie)晶析(xi)出，稀釋時可在水浴中加(jia)溫(wen)助(zhu)溶，洗滌時不影響結(jie)果。
3. 各步加樣(yang)均應使用加樣(yang)器(qi)，并經(jing)常校對其準確(que)性，以避免試驗誤差。一(yi)次加樣(yang)時間控制(zhi)在5分鐘(zhong)內，如標本數量多(duo)，推薦使用排槍(qiang)加樣(yang)。
4. 請每次測定的同時做(zuo)標準(zhun)曲線，做(zuo)復(fu)孔。如標本(ben)中待測物質含量過高（樣本(ben)OD值大于標準(zhun)品(pin)(pin)孔*孔的OD值），請先用樣品(pin)(pin)稀(xi)釋(shi)液稀(xi)釋(shi)一定倍(bei)數（n倍(bei)）后再測定，計(ji)算時請zui后乘(cheng)以總稀(xi)釋(shi)倍(bei)數（×n×5）。
5. 封板膜只(zhi)限(xian)一次(ci)性使用，以避(bi)免(mian)交叉污染。
6. 底物請避光保存(cun)。
7. 嚴格按照說明(ming)書的操作(zuo)進行(xing)，試(shi)驗(yan)結果判(pan)定必須(xu)以酶標儀讀數為(wei)準.
8. 所(suo)有樣品，洗滌液和各種(zhong)廢棄物(wu)都應按傳染物(wu)處理。
9. 本試劑不同批號組(zu)分不得(de)混用。

10. 如與英文(wen)說明書有異，以英文(wen)說明書為準。

試劑盒性能：

1.樣品線性回歸與預期(qi)濃(nong)度相關(guan)系數(shu)R值(zhi)為(wei)0.92以上。

2.批內與批見應分別小于(yu)9%和15%

檢測范圍：                                             

5ng/L -100ng/L                                      

                           

保(bao)存條件(jian)及(ji)有(you)效期：

1.試劑(ji)盒(he)保存：；2-8℃。

2．有效期：6個月

FOR RESEARCH USE ONLY

Drug Names

Generic Name：Rat interleukin4 (IL-4) ELISA Kit.

Purpose

This kit allows for the determination of IL-4 concentrations in Rat serum, blood plasma, cell culture supernatant and other biological fluids.

Principle of the assay

The kit assay Rat IL-4 level in the sample，use Purified Rat IL-4 to coat microtiter plate wells, make solid-phase antibody, then add IL-4 to wells, Combined antibody which With HRP labeled goat anti-mouse 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 IL-4 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: 90 ng/L，60ng/L ，30 ng/L，15ng/L，7.5 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.
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

5ng/L -100ng/L

Storage and validity

1．Storage：  2-8℃.

2．validity： six months.