大鼠白細胞介素17（IL-17）酶聯免疫分析(ELISA)

試劑盒使用說明書

本試劑僅(jin)供研(yan)究使(shi)用       

目的：本試劑盒用于測定大鼠血清，組織及相關液體樣本中白細胞介素17（IL-17）的含量。

實(shi)驗原理(li)：

  本試劑盒應用雙抗體夾心法測定標本中大鼠白細胞介素17（IL-17）水平。用純化的大鼠白細胞介素17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17，再與HRP標記的白細胞介素17（IL-17）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-17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17（IL-17）濃度。

試劑(ji)盒(he)組成(cheng)：

樣本處理(li)及要求：

1. 血清：室(shi)溫(wen)血液自然(ran)凝固10-20分鐘(zhong)，離心20分鐘(zhong)左右（2000-3000轉(zhuan)/分）。仔細收集上(shang)清，保(bao)存過程(cheng)中(zhong)如出現沉淀，應再次(ci)離心。

2. 血漿：應根據標本的(de)要求選擇EDTA或檸檬酸鈉作為抗凝劑，混合10-20分(fen)鐘后，離心20分(fen)鐘左(zuo)右(you)（2000-3000轉/分(fen)）。仔細收集上清，保(bao)存過程中如有沉淀形成(cheng)，應該再次離心。

3. 尿液(ye)：用無菌管收集，離(li)心20分(fen)鐘左右（2000-3000轉/分(fen)）。仔細收集上清(qing)，保存(cun)過程(cheng)中如有沉淀(dian)形(xing)成，應再次離(li)心。胸腹水(shui)、腦脊液(ye)參(can)照實行。

4. 細胞培養上清：檢(jian)測分(fen)(fen)(fen)泌性(xing)的成份(fen)(fen)時，用無(wu)菌管收集(ji)。離(li)心(xin)20分(fen)(fen)(fen)鐘左右（2000-3000轉/分(fen)(fen)(fen)）。仔細收集(ji)上清。檢(jian)測細胞內(nei)(nei)的成份(fen)(fen)時，用PBS（PH7.2-7.4）稀(xi)釋細胞懸(xuan)液，細胞濃度達到100萬/ml左右。通過(guo)反(fan)復凍融，以使細胞破壞并放出細胞內(nei)(nei)成份(fen)(fen)。離(li)心(xin)20分(fen)(fen)(fen)鐘左右（2000-3000轉/分(fen)(fen)(fen)）。仔細收集(ji)上清。保存過(guo)程(cheng)中如有沉淀(dian)形成，應再次離(li)心(xin)。

5. 組織標(biao)本(ben)：切割標(biao)本(ben)后，稱取重(zhong)量(liang)(liang)。加入一定(ding)量(liang)(liang)的(de)PBS，PH7.4。用液(ye)氮(dan)迅速(su)冷(leng)凍保(bao)存備用。標(biao)本(ben)融化(hua)后仍然保(bao)持(chi)2-8℃的(de)溫度。加入一定(ding)量(liang)(liang)的(de)PBS（PH7.4），用手工或(huo)勻(yun)漿(jiang)器將標(biao)本(ben)勻(yun)漿(jiang)充分。離心20分鐘左右（2000-3000轉/分）。仔(zi)細收集上清。分裝后一份待檢測，其(qi)余冷(leng)凍備用。

6. 標本采(cai)集后盡(jin)早(zao)進(jin)行(xing)提(ti)取，提(ti)取按相關文獻進(jin)行(xing)，提(ti)取后應盡(jin)快進(jin)行(xing)實驗(yan)。若不能馬上進(jin)行(xing)試(shi)驗(yan)，可將標本放于-20℃保存，但應避免反(fan)復凍融.

7. 不能檢測含NaN3的樣(yang)品，因NaN3抑制辣根(gen)過氧化物酶(mei)的（HRP）活性。

操作步驟：

1. 標(biao)準(zhun)(zhun)品(pin)的稀(xi)釋(shi)與加(jia)(jia)樣(yang)：在(zai)(zai)(zai)酶(mei)標(biao)包被板上設(she)標(biao)準(zhun)(zhun)品(pin)孔(kong)(kong)10孔(kong)(kong)，在(zai)(zai)(zai)*、第(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)二孔(kong)(kong)中(zhong)(zhong)(zhong)(zhong)分(fen)(fen)別(bie)(bie)加(jia)(jia)標(biao)準(zhun)(zhun)品(pin)100μl，然后(hou)(hou)(hou)(hou)在(zai)(zai)(zai)*、第(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)二孔(kong)(kong)中(zhong)(zhong)(zhong)(zhong)加(jia)(jia)標(biao)準(zhun)(zhun)品(pin)稀(xi)釋(shi)液50μl，混(hun)(hun)(hun)(hun)勻(yun)(yun)(yun)；然后(hou)(hou)(hou)(hou)從(cong)(cong)*孔(kong)(kong)、第(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)二孔(kong)(kong)中(zhong)(zhong)(zhong)(zhong)各取100μl分(fen)(fen)別(bie)(bie)加(jia)(jia)到(dao)第(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)三孔(kong)(kong)和(he)第(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)四孔(kong)(kong)，再(zai)在(zai)(zai)(zai)第(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)三、第(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)四孔(kong)(kong)分(fen)(fen)別(bie)(bie)加(jia)(jia)標(biao)準(zhun)(zhun)品(pin)稀(xi)釋(shi)液50μl，混(hun)(hun)(hun)(hun)勻(yun)(yun)(yun)；然后(hou)(hou)(hou)(hou)在(zai)(zai)(zai)第(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)三孔(kong)(kong)和(he)第(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)四孔(kong)(kong)中(zhong)(zhong)(zhong)(zhong)先各取50μl棄掉(diao)，再(zai)各取50μl分(fen)(fen)別(bie)(bie)加(jia)(jia)到(dao)第(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)五、第(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)六(liu)(liu)孔(kong)(kong)中(zhong)(zhong)(zhong)(zhong)，再(zai)在(zai)(zai)(zai)第(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)五、第(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)六(liu)(liu)孔(kong)(kong)中(zhong)(zhong)(zhong)(zhong)分(fen)(fen)別(bie)(bie)加(jia)(jia)標(biao)準(zhun)(zhun)品(pin)稀(xi)釋(shi)液50ul，混(hun)(hun)(hun)(hun)勻(yun)(yun)(yun)；混(hun)(hun)(hun)(hun)勻(yun)(yun)(yun)后(hou)(hou)(hou)(hou)從(cong)(cong)第(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)五、第(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)六(liu)(liu)孔(kong)(kong)中(zhong)(zhong)(zhong)(zhong)各取50μl分(fen)(fen)別(bie)(bie)加(jia)(jia)到(dao)第(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)七、第(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)八孔(kong)(kong)中(zhong)(zhong)(zhong)(zhong)，再(zai)在(zai)(zai)(zai)第(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)七、第(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)八孔(kong)(kong)中(zhong)(zhong)(zhong)(zhong)分(fen)(fen)別(bie)(bie)加(jia)(jia)標(biao)準(zhun)(zhun)品(pin)稀(xi)釋(shi)液50μl，混(hun)(hun)(hun)(hun)勻(yun)(yun)(yun)后(hou)(hou)(hou)(hou)從(cong)(cong)第(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)七、第(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)八孔(kong)(kong)中(zhong)(zhong)(zhong)(zhong)分(fen)(fen)別(bie)(bie)取50μl加(jia)(jia)到(dao)第(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)九(jiu)、第(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)十孔(kong)(kong)中(zhong)(zhong)(zhong)(zhong)，再(zai)在(zai)(zai)(zai)第(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)九(jiu)第(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)十孔(kong)(kong)分(fen)(fen)別(bie)(bie)加(jia)(jia)標(biao)準(zhun)(zhun)品(pin)稀(xi)釋(shi)液50μl，混(hun)(hun)(hun)(hun)勻(yun)(yun)(yun)后(hou)(hou)(hou)(hou)從(cong)(cong)第(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)九(jiu)第(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)十孔(kong)(kong)中(zhong)(zhong)(zhong)(zhong)各取50μl棄掉(diao)。（稀(xi)釋(shi)后(hou)(hou)(hou)(hou)各孔(kong)(kong)加(jia)(jia)樣(yang)量(liang)都為(wei)50μl，濃度分(fen)(fen)別(bie)(bie)為(wei)90 pg/mL，60 pg/mL，30 pg/mL，15 pg/mL，7.5 pg/mL）。
2. 加樣(yang)(yang)：分(fen)別設空(kong)白(bai)孔(kong)（空(kong)白(bai)對照孔(kong)不(bu)加樣(yang)(yang)品(pin)及酶(mei)標(biao)(biao)試劑，其(qi)余各步操作(zuo)相同）、待(dai)(dai)測樣(yang)(yang)品(pin)孔(kong)。在(zai)酶(mei)標(biao)(biao)包被板(ban)上待(dai)(dai)測樣(yang)(yang)品(pin)孔(kong)中先加樣(yang)(yang)品(pin)稀釋(shi)液(ye)40μl，然后再加待(dai)(dai)測樣(yang)(yang)品(pin)10μl（樣(yang)(yang)品(pin)zui終稀釋(shi)度(du)為5倍）。加樣(yang)(yang)將樣(yang)(yang)品(pin)加于(yu)酶(mei)標(biao)(biao)板(ban)孔(kong)底(di)部，盡量(liang)不(bu)觸及孔(kong)壁，輕輕晃動(dong)混勻(yun)。
3. 溫育：用封板膜封板后置37℃溫育(yu)30分鐘。
4. 配液：將30（48T的(de)(de)20倍(bei)）倍(bei)濃縮洗滌液用(yong)蒸餾水30（48T的(de)(de)20倍(bei)）倍(bei)稀釋(shi)后備用(yong)。
5. 洗滌：小心揭掉(diao)封板(ban)膜，棄(qi)去(qu)液體，甩干(gan)，每孔加滿洗滌液，靜置30秒后(hou)棄(qi)去(qu)，如此重復5次(ci)，拍干(gan)。
6. 加酶：每孔加入(ru)酶標試劑50μl，空白孔除(chu)外。
7. 溫育：操作同3。
8. 洗滌(di)：操作同5。
9. 顯色：每孔先加(jia)入顯色劑A50μl，再加(jia)入顯色劑B50μl，輕(qing)輕(qing)震蕩混勻，37℃避光顯色15分鐘.
10. 終(zhong)止(zhi)：每孔加終(zhong)止(zhi)液50μl，終(zhong)止(zhi)反應（此時藍色(se)(se)立轉黃色(se)(se)）。
11. 測定：以空(kong)白空(kong)調零(ling)，450nm波長依序測量各孔的(de)吸光度(du)（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(zang)環(huan)境中取出應在(zai)室溫平(ping)衡15-30分鐘(zhong)后方可使(shi)用(yong)(yong)，酶標包被板(ban)開封(feng)后如未用(yong)(yong)完，板(ban)條應裝入密封(feng)袋中保存(cun)。
2. 濃洗(xi)滌液可能(neng)會有結晶析出，稀釋(shi)時可在水(shui)浴(yu)中(zhong)加(jia)溫助溶，洗(xi)滌時不影(ying)響(xiang)結果(guo)。
3. 各步加樣均應(ying)使用加樣器，并(bing)經(jing)常校對(dui)其準確性，以(yi)避免試驗(yan)誤差。一次(ci)加樣時間控制在5分鐘內(nei)，如標(biao)本數量(liang)多，推薦使用排槍加樣。
4. 請(qing)每次測(ce)(ce)定的(de)(de)同時(shi)做標(biao)(biao)準(zhun)曲線(xian)，做復孔(kong)。如標(biao)(biao)本中(zhong)待測(ce)(ce)物(wu)質(zhi)含(han)量過高（樣本OD值(zhi)大于標(biao)(biao)準(zhun)品孔(kong)*孔(kong)的(de)(de)OD值(zhi)），請(qing)先用樣品稀釋(shi)液(ye)稀釋(shi)一(yi)定倍數(shu)（n倍）后(hou)再測(ce)(ce)定，計算時(shi)請(qing)zui后(hou)乘以總稀釋(shi)倍數(shu)（×n×5）。
5. 封(feng)板膜只限(xian)一(yi)次性(xing)使用(yong)，以避免交(jiao)叉污染。
6. 底物請避光(guang)保存。
7. 嚴格按照說明書的(de)操作進(jin)行，試驗(yan)結果判定必(bi)須以(yi)酶標儀讀數為準(zhun).
8. 所有樣品，洗滌液和(he)各種廢棄物都應按傳(chuan)染物處理。
9. 本試劑不(bu)同批號組分(fen)不(bu)得混(hun)用。

10. 如與英文說明(ming)書有異，以英文說明(ming)書為(wei)準。

試劑盒性能：

1.樣(yang)品(pin)線性回歸與預期濃(nong)度相關系(xi)數R值為(wei)0.92以上(shang)。

2.批內與批見應(ying)分別小于(yu)9%和15%

檢(jian)測范圍：        ;                                     

5pg/mL -100 pg/mL                                      

                       ;    

保存條(tiao)件及有效期：

1.試劑盒保(bao)存：；2-8℃。

2．有效(xiao)期(qi)：6個(ge)月

 Rat interleukin 17

Drug Names

Generic Name：Rat interleukin17(IL-17) ELISA Kit.

Purpose

This kit allows for the determination of IL-17 concentrations in Rat serum, tissue, and other biological fluids.

Principle of the assay

The kit assay Rat IL-17 level in the sample，use Purified Rat IL-17 to coat microtiter plate wells, make solid-phase antibody, then add IL-17 to wells, Combined IL-17antibody 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 IL-17 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 pg/mL,60 pg/mL ,30 pg/mL,15 pg/mL,7.5 pg/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 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

5 pg/mL -100 pg/mL

Storage and validity

1．Storage：  2-8℃.

2．validity： six months.