大鼠補體(ti)4(C4)酶聯免疫分(fen)析(ELISA)

試劑盒(he)使用說明書

本試劑(ji)僅供(gong)研究使用       

目的：本試劑盒用于測定大鼠血清，血漿,組織及相關液體樣本中補體4(C4)的含量。

實驗原理：

    本(ben)試劑盒(he)應用雙抗體(ti)夾(jia)心(xin)法(fa)測定標本(ben)中(zhong)大鼠補(bu)體(ti)4(C4)水平。用純化的大鼠補體(ti)4(C4)抗體包(bao)被微孔板(ban)，制成固相抗體(ti)，往包(bao)被單抗的微孔中依次加(jia)入(ru)補(bu)體4(C4)，再(zai)與HRP標記的補體4(C4)抗(kang)體(ti)結合，形成抗(kang)體(ti)-抗原-酶(mei)標抗體(ti)復(fu)合(he)物(wu)，經過*洗滌后加底物(wu)TMB顯色。TMB在HRP酶(mei)的催化下轉(zhuan)化成藍色(se)(se)，并在酸的作用(yong)下轉(zhuan)化成zui終(zhong)的黃色(se)(se)。顏色(se)(se)的深淺和樣品中的補(bu)體(ti)4(C4)呈正相關(guan)。用(yong)酶標儀在450nm波長下(xia)測定(ding)吸光度(du)（OD值），通過標準曲線計算樣品(pin)中大(da)鼠補體(ti)(ti)4(C4)含(han)量。

試劑盒組成：

樣本(ben)處理及要求：

1. 血清：室(shi)溫血液自然凝固10-20分鐘(zhong)，離心20分鐘左右(you)（2000-3000轉(zhuan)/分(fen)）。仔(zi)細收集(ji)上清，保存過程中如出(chu)現沉(chen)淀，應再(zai)次離心。

2. 血漿：應根據標本的要求(qiu)選擇(ze)EDTA或(huo)檸檬酸鈉作為抗凝劑，混合10-20分鐘(zhong)后，離心20分(fen)鐘(zhong)左右（2000-3000轉/分）。仔細(xi)收集上清(qing)，保存過程中如(ru)有沉(chen)淀形成，應(ying)該再次離心(xin)。

3. 尿(niao)液：用無(wu)菌管收(shou)集，離心20分鐘左右（2000-3000轉/分）。仔(zi)細收(shou)集上清，保存過程中如(ru)有沉(chen)淀形(xing)成，應再次離心。胸腹(fu)水、腦脊液參(can)照(zhao)實(shi)行。

4. 細胞培養上清：檢測分泌(mi)性的成份時，用無菌管收集(ji)。離(li)心20分鐘左右（2000-3000轉/分）。仔細(xi)收集上清。檢測細(xi)胞(bao)內(nei)的(de)成份時，用PBS（PH7.2-7.4）稀釋細胞(bao)懸液，細胞(bao)濃(nong)度達到100萬/ml左右。通過反復凍融，以使細(xi)胞破壞并放出細(xi)胞內成份。離心20分鐘左右（2000-3000轉(zhuan)/分）。仔(zi)細收集上清。保存(cun)過程(cheng)中如有沉淀形成，應(ying)再次離(li)心。

5. 組織標本(ben)：切割標本(ben)后，稱取(qu)重量。加(jia)入一定量的PBS，PH7.4。用(yong)(yong)液(ye)氮(dan)迅(xun)速冷(leng)凍保存(cun)備用(yong)(yong)。標(biao)本融化后仍然保持2-8℃的溫度。加入一定量(liang)的PBS（PH7.4），用手(shou)工或(huo)勻(yun)漿器(qi)將標本勻(yun)漿充(chong)分。離(li)心20分鐘左右（2000-3000轉/分）。仔細收集上清。分裝后一(yi)份待檢(jian)測，其余冷(leng)凍備用(yong)。

6. 標本采集后盡(jin)早進(jin)(jin)行(xing)提取(qu)，提取(qu)按相關文(wen)獻進(jin)(jin)行(xing)，提取(qu)后應盡(jin)快進(jin)(jin)行(xing)實驗。若不能馬上(shang)進(jin)(jin)行(xing)試驗，可(ke)將標本放于-20℃保存，但應避(bi)免反復(fu)凍融.

7. 不能檢測含(han)NaN3的(de)樣品，因NaN3抑制(zhi)辣根(gen)過氧化物酶的（HRP）活性。

操(cao)作步驟：

1. 標(biao)(biao)準(zhun)品的稀釋與加樣：在(zai)酶標(biao)(biao)包被板上設標(biao)(biao)準(zhun)品孔10孔(kong)，在*、第二孔(kong)中分別加標(biao)準品100μl，然后在*、第二孔中(zhong)加(jia)標準品稀釋液50μl，混勻；然(ran)后從*孔、第(di)二孔中各取100μl分別(bie)加(jia)到(dao)第(di)三孔(kong)和第(di)四孔(kong)，再在第(di)三、第(di)四孔(kong)分別(bie)加(jia)標準品稀(xi)釋液50μl，混勻；然后在第(di)三孔和(he)第(di)四孔中(zhong)先各取50μl棄(qi)掉，再各取50μl分別(bie)加到第五、第六孔中(zhong)(zhong)，再在第五、第六孔中(zhong)(zhong)分別(bie)加標準品稀釋(shi)液(ye)50ul，混(hun)勻；混(hun)勻后從第五、第六孔中各取50μl分別(bie)加(jia)到(dao)第七(qi)(qi)、第八(ba)孔中，再在第七(qi)(qi)、第八(ba)孔中分別(bie)加(jia)標準品(pin)稀釋液50μl，混勻后(hou)從第七、第八孔(kong)中分別取50μl加到第(di)九(jiu)、第(di)十孔中，再在第(di)九(jiu)第(di)十孔分別加標(biao)準品稀釋液50μl，混勻后(hou)從第(di)(di)九第(di)(di)十孔中各取50μl棄(qi)掉。（稀(xi)釋后各孔加樣量都為(wei)50μl，濃(nong)度分別為480μg/ml，320μg/ml ，160μg/ml，80μg/ml， 40μg/ml）。
2. 加樣：分別(bie)設空(kong)白孔（空(kong)白對照孔不加樣品及酶標試劑(ji)，其(qi)余各步操作相同(tong)）、待測樣品(pin)孔。在酶標包被板(ban)上(shang)待測樣品(pin)孔中(zhong)先加樣品(pin)稀釋液40μl，然(ran)后再加待測樣品10μl（樣品zui終(zhong)稀釋度為5倍）。加(jia)樣將樣品加(jia)于酶標板孔底部，盡量不觸及(ji)孔壁(bi)，輕輕晃動混勻。
3. 溫育：用封板膜封板后置37℃溫育30分鐘(zhong)。
4. 配液：將30（48T的(de)20倍）倍濃縮洗(xi)滌(di)液用蒸(zheng)餾水30（48T的20倍(bei)）倍(bei)稀釋(shi)后備用。
5. 洗滌：小心揭掉(diao)封板膜，棄去液體(ti)，甩干，每孔加滿洗滌液，靜置30秒后棄去(qu)，如(ru)此重復(fu)5次，拍干。
6. 加酶：每(mei)孔加入酶標試劑50μl，空白孔(kong)除(chu)外。
7. 溫育：操(cao)作同3。
8. 洗滌：操(cao)作同5。
9. 顯(xian)色(se)：每孔先(xian)加入顯(xian)色(se)劑A50μl，再(zai)加入顯色劑B50μl，輕輕震蕩(dang)混(hun)勻，37℃避光(guang)顯色15分(fen)鐘.
10. 終止(zhi)：每孔加終止(zhi)液(ye)50μl，終止反應(ying)（此(ci)時藍色(se)立轉(zhuan)黃色(se)）。
11. 測定：以空(kong)白空(kong)調零，450nm波(bo)長依(yi)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(jin)行。

注意(yi)事項：

1. 試(shi)劑(ji)盒從冷(leng)藏環境中取出應在室溫平(ping)衡15-30分鐘后方可(ke)使用，酶標包被(bei)板(ban)開封后如(ru)未用完，板(ban)條(tiao)應裝入密封袋中保存。
2. 濃(nong)洗(xi)(xi)滌(di)液(ye)可(ke)能會有結晶析出(chu)，稀釋時(shi)可(ke)在水浴中(zhong)加溫助溶，洗(xi)(xi)滌(di)時(shi)不影響結果(guo)。
3. 各步加樣(yang)(yang)均應使用加樣(yang)(yang)器(qi)，并(bing)經常校對其(qi)準確性，以避免(mian)試(shi)驗(yan)誤差。一(yi)次加樣(yang)(yang)時間控制在5分鐘內，如(ru)標本數(shu)量多，推薦使用排槍加樣。
4. 請(qing)每次(ci)測定的同時做標(biao)準(zhun)曲(qu)線(xian)，做復孔。如標(biao)本中待測物質含量過(guo)高（樣本OD值(zhi)大于標準品孔*孔的OD值(zhi)），請先用樣品稀(xi)釋液稀(xi)釋一定倍數（n倍）后(hou)再測(ce)定，計算時請zui后(hou)乘以總稀(xi)釋倍數(shu)（×n×5）。
5. 封(feng)板膜(mo)只(zhi)限一次性使用(yong)，以避免交叉污染。
6. 底物請(qing)避光保存。
7. 嚴格按照說(shuo)明書的(de)操作進行，試驗結果判定必須(xu)以(yi)酶標儀讀數為準.
8. 所有樣品，洗滌(di)液(ye)和各種廢棄物都應按傳染物處理。
9. 本試劑不同批號組分(fen)不得混用。

10. 如與英文(wen)說明(ming)書有異，以(yi)英文(wen)說明(ming)書為準。

                                              

試劑(ji)盒(he)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.批內與批見應分別小于9%和(he)15%

檢測(ce)范圍：                                             

20μg/ml -500μg/ml                                      

                     ;      ;

保存條件及有效期：

1.試劑盒(he)保存(cun)：；2-8℃。

2．有效期：6個月

 Rat Complement 4

Drug Names

Generic Name：Rat Complement 4(C4) ELISA Kit.

Purpose

This kit allows for the determination of C4 concentrations in Rat serum, blood plasma, tissue and other biological fluids.

Principle of the assay

The kit assay Rat C4 level in the sample，use Purified Rat C4 to coat microtiter plate wells, make solid-phase antibody, then add C4 to wells, Combined C4 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 C4 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: 480μg/ml，320μg/ml ，160μg/ml，80μg/ml， 40μg/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

20μg/ml -500μg/ml

Storage and validity

1．Storage：  2-8℃.

2．validity： six months.