大鼠(shu)免疫球蛋白M(IgM)酶聯(lian)免疫分析（ELISA）

試(shi)劑(ji)盒使(shi)用說明書

本試(shi)劑僅供研究使用       

目的：本試劑盒用于測定大鼠血清，組織及相(xiang)關(guan)液體樣本中免(mian)疫球蛋白M(IgM)的含量。

實驗原(yuan)理：

本試劑盒應用雙抗體(ti)夾心法測定標本中大鼠免疫球蛋白M(IgM)水平。用純化的抗-大鼠IgM抗(kang)體(ti)包被微(wei)孔板，制成固相抗(kang)體(ti)，往包被單抗(kang)的微(wei)孔中依(yi)次(ci)加(jia)入大鼠免疫球蛋白(bai)M(IgM)，再與HRP標(biao)記的(de)羊(yang)抗(kang)(kang)鼠抗(kang)(kang)體(ti)結合，形成抗(kang)(kang)體(ti)-抗(kang)(kang)原-酶標(biao)抗(kang)(kang)體(ti)復合物(wu)，經過*洗滌(di)后加底物(wu)TMB顯色。TMB在HRP酶的(de)催(cui)化下轉化成藍(lan)色，并在酸的(de)作(zuo)用下轉化成zui終的(de)黃色。顏色的(de)深(shen)淺和樣品中(zhong)的(de)大鼠免疫球蛋(dan)白M(IgM)呈正(zheng)相(xiang)關。用(yong)酶標儀在450nm波(bo)長下測定吸(xi)光(guang)度（OD值），通過標準曲線計算(suan)樣品中大鼠免疫球蛋(dan)白M(IgM)濃度。

試劑盒(he)組成：

樣(yang)本處理及要(yao)求(qiu)：

1. 血(xue)清(qing)：室溫血(xue)液自然凝固10-20分鐘，離心20分鐘左右（2000-3000轉/分）。仔(zi)細(xi)收集上清(qing)，保存過程中如出(chu)現沉(chen)淀，應再(zai)次離心。

2. 血漿：應根據標本的要求選擇(ze)EDTA或(huo)檸檬酸鈉作(zuo)為抗(kang)凝劑，混(hun)合10-20分(fen)(fen)鐘后，離心20分(fen)(fen)鐘左右（2000-3000轉/分(fen)(fen)）。仔細收(shou)集上清(qing)，保存過(guo)程中(zhong)如有沉淀形成(cheng)，應該再次離心。

3. 尿液：用(yong)無菌管收(shou)集(ji)(ji)，離心20分(fen)鐘左右（2000-3000轉(zhuan)/分(fen)）。仔細收(shou)集(ji)(ji)上清，保存過(guo)程中(zhong)如(ru)有(you)沉(chen)淀形(xing)成，應再(zai)次(ci)離心。胸(xiong)腹水、腦脊(ji)液參照(zhao)實行。

4. 細(xi)胞(bao)培(pei)養上(shang)清：檢測分泌性的成份(fen)時，用無菌管收集。離(li)心20分鐘(zhong)左(zuo)右（2000-3000轉(zhuan)/分）。仔細(xi)收集上(shang)清。檢測細(xi)胞(bao)內(nei)的成份(fen)時，用PBS（PH7.2-7.4）稀(xi)釋細(xi)胞(bao)懸液，細(xi)胞(bao)濃度達(da)到(dao)100萬(wan)/ml左(zuo)右。通過反復凍融，以使(shi)細(xi)胞(bao)破壞并放(fang)出細(xi)胞(bao)內(nei)成份(fen)。離(li)心20分鐘(zhong)左(zuo)右（2000-3000轉(zhuan)/分）。仔細(xi)收集上(shang)清。保存過程(cheng)中(zhong)如有沉淀(dian)形成，應再次離(li)心。

5. 組織(zhi)標本(ben)：切割標本(ben)后(hou)，稱取重(zhong)量。加(jia)入(ru)一定(ding)量的(de)PBS，PH7.4。用液(ye)氮迅速冷凍保存備(bei)用。標本(ben)融化后(hou)仍然保持(chi)2-8℃的(de)溫度。加(jia)入(ru)一定(ding)量的(de)PBS（PH7.4），用手工或勻漿器將標本(ben)勻漿充分(fen)(fen)。離心20分(fen)(fen)鐘(zhong)左右(you)（2000-3000轉/分(fen)(fen)）。仔細收(shou)集(ji)上清。分(fen)(fen)裝后(hou)一份待檢測，其余冷凍備(bei)用。

6. 標本(ben)采集后盡早(zao)進(jin)行(xing)提取(qu)(qu)，提取(qu)(qu)按相關文(wen)獻進(jin)行(xing)，提取(qu)(qu)后應盡快進(jin)行(xing)實(shi)驗。若(ruo)不能馬上(shang)進(jin)行(xing)試驗，可將標本放(fang)于-20℃保存(cun)，但應避免反復凍融.

7. 不(bu)能檢測含NaN3的樣品(pin)，因NaN3抑制辣根過(guo)氧化物酶的（HRP）活(huo)性。

操作(zuo)步驟

1. 標(biao)(biao)準品(pin)(pin)的稀(xi)釋(shi)(shi)與加(jia)(jia)(jia)(jia)(jia)樣：在(zai)(zai)酶標(biao)(biao)包被板上設標(biao)(biao)準品(pin)(pin)孔(kong)10孔(kong)，在(zai)(zai)*、第(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)二孔(kong)中(zhong)(zhong)分(fen)別(bie)加(jia)(jia)(jia)(jia)(jia)標(biao)(biao)準品(pin)(pin)100μl，然(ran)后(hou)在(zai)(zai)*、第(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)二孔(kong)中(zhong)(zhong)加(jia)(jia)(jia)(jia)(jia)標(biao)(biao)準品(pin)(pin)稀(xi)釋(shi)(shi)液50μl，混勻；然(ran)后(hou)從(cong)(cong)*孔(kong)、第(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)二孔(kong)中(zhong)(zhong)各(ge)(ge)(ge)取(qu)100μl分(fen)別(bie)加(jia)(jia)(jia)(jia)(jia)到第(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)三(san)孔(kong)和第(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)四孔(kong)，再在(zai)(zai)第(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)三(san)、第(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)四孔(kong)分(fen)別(bie)加(jia)(jia)(jia)(jia)(jia)標(biao)(biao)準品(pin)(pin)稀(xi)釋(shi)(shi)液50μl，混勻；然(ran)后(hou)在(zai)(zai)第(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)三(san)孔(kong)和第(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)四孔(kong)中(zhong)(zhong)先(xian)各(ge)(ge)(ge)取(qu)50μl棄掉(diao)，再各(ge)(ge)(ge)取(qu)50μl分(fen)別(bie)加(jia)(jia)(jia)(jia)(jia)到第(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)五(wu)(wu)、第(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)六孔(kong)中(zhong)(zhong)，再在(zai)(zai)第(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)五(wu)(wu)、第(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)六孔(kong)中(zhong)(zhong)分(fen)別(bie)加(jia)(jia)(jia)(jia)(jia)標(biao)(biao)準品(pin)(pin)稀(xi)釋(shi)(shi)液50ul，混勻；混勻后(hou)從(cong)(cong)第(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)五(wu)(wu)、第(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)六孔(kong)中(zhong)(zhong)各(ge)(ge)(ge)取(qu)50μl分(fen)別(bie)加(jia)(jia)(jia)(jia)(jia)到第(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)七(qi)、第(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)八孔(kong)中(zhong)(zhong)，再在(zai)(zai)第(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)七(qi)、第(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)八孔(kong)中(zhong)(zhong)分(fen)別(bie)加(jia)(jia)(jia)(jia)(jia)標(biao)(biao)準品(pin)(pin)稀(xi)釋(shi)(shi)液50μl，混勻后(hou)從(cong)(cong)第(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)七(qi)、第(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)(di)八孔(kong)中(zhong)(zhong)分(fen)別(bie)取(qu)50μl加(jia)(jia)(jia)(jia)(jia)到第(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)中(zhong)(zhong)，再在(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)分(fen)別(bie)加(jia)(jia)(jia)(jia)(jia)標(biao)(biao)準品(pin)(pin)稀(xi)釋(shi)(shi)液50μl，混勻后(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)中(zhong)(zhong)各(ge)(ge)(ge)取(qu)50μl棄掉(diao)。（稀(xi)釋(shi)(shi)后(hou)各(ge)(ge)(ge)孔(kong)加(jia)(jia)(jia)(jia)(jia)樣量都為50μl，濃度分(fen)別(bie)為9μg/ ml，6μg/ ml ，3μg/ ml，1.5μg/ ml， 0.75μg/ ml）。
2. 加(jia)樣(yang)(yang)(yang)(yang)：分別設(she)空(kong)白孔(kong)(kong)（空(kong)白對照(zhao)孔(kong)(kong)不(bu)加(jia)樣(yang)(yang)(yang)(yang)品及酶(mei)標(biao)試劑(ji)，其余各步操作(zuo)相同(tong)）、待(dai)測(ce)樣(yang)(yang)(yang)(yang)品孔(kong)(kong)。在酶(mei)標(biao)包被板(ban)上待(dai)測(ce)樣(yang)(yang)(yang)(yang)品孔(kong)(kong)中先加(jia)樣(yang)(yang)(yang)(yang)品稀釋液40μl，然后再加(jia)待(dai)測(ce)樣(yang)(yang)(yang)(yang)品10μl（樣(yang)(yang)(yang)(yang)品zui終稀釋度為5倍）。加(jia)樣(yang)(yang)(yang)(yang)將樣(yang)(yang)(yang)(yang)品加(jia)于酶(mei)標(biao)板(ban)孔(kong)(kong)底(di)部(bu)，盡(jin)量不(bu)觸及孔(kong)(kong)壁(bi)，輕輕晃(huang)動混勻(yun)。
3. 溫育：用封(feng)板膜(mo)封(feng)板后置(zhi)37℃溫育(yu)30分鐘。
4. 配(pei)液：將30（48T的20倍(bei)）倍(bei)濃縮洗(xi)滌液用蒸(zheng)餾水30（48T的20倍(bei)）倍(bei)稀釋后備用。
5. 洗(xi)滌：小心揭掉封板(ban)膜，棄去液(ye)體，甩干，每孔加滿洗(xi)滌液(ye)，靜置30秒后棄去，如此重復5次，拍干。
6. 加(jia)酶：每孔(kong)加(jia)入酶標試劑50μl，空(kong)白孔(kong)除(chu)外。
7. 溫育：操作(zuo)同3。
8. 洗滌：操作同5。
9. 顯色(se)：每孔(kong)先加入(ru)顯色(se)劑A50μl，再加入(ru)顯色(se)劑B50μl，輕輕震蕩混勻，37℃避光顯色15分(fen)鐘.
10. 終止：每孔加終止液50μl，終止反應(ying)（此時藍色(se)立轉(zhuan)黃色(se)）。
11. 測定(ding)：以空白(bai)空調零，450nm波(bo)長依序測量各孔的吸光度（OD值）。 測定(ding)應在加終止液(ye)后15分鐘以內進行。

注意事項(xiang)：

1. 試(shi)劑盒從冷藏(zang)環境中取出應(ying)(ying)在室(shi)溫(wen)平衡15-30分(fen)鐘后(hou)方可使用，酶標包被板開封后(hou)如未用完，板條應(ying)(ying)裝入密封袋(dai)中保存。
2. 濃洗(xi)滌液(ye)可(ke)能會有結晶析出，稀釋時可(ke)在水浴中加溫助(zhu)溶(rong)，洗(xi)滌時不影響結果。
3. 各(ge)步加(jia)樣(yang)均應使用加(jia)樣(yang)器，并經常校對其準確性，以避(bi)免試驗誤差(cha)。一次加(jia)樣(yang)時間控制在(zai)5分(fen)鐘(zhong)內(nei)，如標本數量多，推薦使用排槍(qiang)加(jia)樣(yang)。
4. 請每次測(ce)(ce)定(ding)(ding)的(de)同(tong)時做標準(zhun)曲線，做復孔。如標本(ben)中待測(ce)(ce)物質含量(liang)過高（樣本(ben)OD值(zhi)大于標準(zhun)品孔*孔的(de)OD值(zhi)），請先(xian)用(yong)樣品稀釋液稀釋一定(ding)(ding)倍(bei)數（n倍(bei)）后再測(ce)(ce)定(ding)(ding)，計算(suan)時請zui后乘以(yi)總稀釋倍(bei)數（×n×5）。
5. 封板(ban)膜只限一次(ci)性(xing)使用，以避免交叉污染。
6. 底物請避光保(bao)存。
7. 嚴格按(an)照(zhao)說明(ming)書的(de)操作(zuo)進行，試(shi)驗結(jie)果判定必須以酶(mei)標儀讀數為準.
8. 所有樣(yang)品，洗滌液和(he)各種(zhong)廢棄物都應按傳染物處理。
9. 本試劑(ji)不同批號(hao)組分不得混(hun)用。

10. 如與英(ying)文說明書有(you)異，以(yi)英(ying)文說明書為準。

                                              

試(shi)劑盒性(xing)能：

1.樣(yang)品線性回(hui)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%

檢測范圍：                                             

0.3μg/ ml -11μg/ ml                                      

                           

保(bao)存條件及有效(xiao)期：

1.試劑盒保(bao)存(cun)：；2-8℃。

2．有效期：6個月

FOR RESEARCH USE ONLY

Drug Names

Generic Name：Rat Immunoglobulin M (IgM) ELISA Kit.

Purpose

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

Principle of the assay

The kit assay Rat IgM level in the sample，use Purified Rat IgM antibody to coat microtiter plate wells, make solid-phase antibody, then add IgM 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  IgM 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: 9μg/ ml，6μg/ ml ，3μg/ ml，1.5μg/ ml， 0.75μg/ 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

0.3μg/ ml -11μg/ ml

Storage and validity

1．Storage：  2-8℃.

2．validity： six months.