實驗原理:
本(ben)試(shi)劑盒應用雙抗體(ti)夾心(xin)法(fa)測(ce)定(ding)標(biao)本(ben)中(zhong)大(da)鼠CD31分子(zi)(CD31)水平。用純(chun)化(hua)的(de)(de)大(da)鼠CD31分子(zi)(CD31)捕(bu)獲抗體(ti)包(bao)被微孔板(ban),制(zhi)成(cheng)固相抗體(ti),往包(bao)被的(de)(de)微孔中(zhong)依次加(jia)入大(da)鼠CD31分子(zi)(CD31),再(zai)與HRP標(biao)記的(de)(de)檢(jian)測(ce)抗體(ti)結(jie)合(he),形成(cheng)抗體(ti)-抗原-酶標(biao)抗體(ti)復合(he)物(wu),經過洗滌后(hou)加(jia)底物(wu)TMB顯色。TMB在HRP酶的(de)(de)催化(hua)下轉化(hua)成(cheng)藍色,并在酸的(de)(de)作用下轉化(hua)成(cheng)最(zui)終的(de)(de)黃(huang)色。顏色的(de)(de)深淺和(he)樣(yang)品中(zhong)的(de)(de)大(da)鼠CD31分子(zi)(CD31)呈正(zheng)相關。用酶標(biao)儀在450nm波長(chang)下測(ce)定(ding)吸光度(OD值),通過標(biao)準曲線計算樣(yang)品中(zhong)大(da)鼠CD31分子(zi)(CD31)含量。
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 | 2片 | |
密封袋 | 1個 | 1個 | |
酶標包被板 | 1×48 | 1×96 | 2-8℃保存 |
標準品 | 0.3ml×6管 | 0.3ml×6管 | 2-8℃保存 |
酶標試劑 | 5 ml×1瓶 | 10 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 | 3 ml×1瓶 | 6 ml×1瓶 | 2-8℃保存 |
20×濃縮洗滌液 | 15ml×1瓶 | 25ml×1瓶 | 2-8℃保存 |
注:標準(zhun)品濃度依(yi)次為:200、100、50、25、12.5、0 ng/mL.
樣本處理及要求:
1. 血清:室溫血液自(zi)然凝(ning)固(gu)10-20分鐘,離心(xin)20分鐘左右(2000-3000轉/分)。仔細收集(ji)上清,保存(cun)過程中如出(chu)現沉淀,應再次離心(xin)。
2. 血漿:應根據標本的要求選擇EDTA或檸檬酸(suan)鈉作為抗凝劑,混合10-20分鐘(zhong)后,離心20分鐘(zhong)左右(you)(2000-3000轉(zhuan)/分)。仔細收集上(shang)清,保存(cun)過(guo)程中如有沉(chen)淀(dian)形成,應該再次離心。
3. 尿(niao)液:用無菌管收集,離(li)心20分鐘左(zuo)右(2000-3000轉/分)。仔細收集上清,保存過程中(zhong)如有沉淀形成,應再(zai)次離(li)心。胸腹水(shui)、腦脊液參照實行。
4. 細胞(bao)(bao)(bao)(bao)(bao)培養上(shang)清:檢測(ce)分(fen)(fen)泌性的(de)成(cheng)份時,用無(wu)菌管收(shou)集。離(li)心(xin)20分(fen)(fen)鐘(zhong)左右(2000-3000轉/分(fen)(fen))。仔細收(shou)集上(shang)清。檢測(ce)細胞(bao)(bao)(bao)(bao)(bao)內(nei)的(de)成(cheng)份時,用PBS(PH7.2-7.4)稀釋細胞(bao)(bao)(bao)(bao)(bao)懸液,細胞(bao)(bao)(bao)(bao)(bao)濃度(du)達(da)到100萬/ml左右。通過反復凍融,以使細胞(bao)(bao)(bao)(bao)(bao)破壞并(bing)放(fang)出細胞(bao)(bao)(bao)(bao)(bao)內(nei)成(cheng)份。離(li)心(xin)20分(fen)(fen)鐘(zhong)左右(2000-3000轉/分(fen)(fen))。仔細收(shou)集上(shang)清。保存過程中如(ru)有(you)沉淀(dian)形(xing)成(cheng),應再次離(li)心(xin)。
5. 組織(zhi)標(biao)本(ben):切割標(biao)本(ben)后,稱取(qu)重量。加(jia)入一(yi)定(ding)量的(de)PBS,PH7.4。用液氮迅速冷凍(dong)保存備用。標(biao)本(ben)融化(hua)后仍(reng)然保持2-8℃的(de)溫度。加(jia)入一(yi)定(ding)量的(de)PBS(PH7.4),用手工(gong)或勻漿器(qi)將標(biao)本(ben)勻漿充分。離(li)心20分鐘(zhong)左(zuo)右(you)(2000-3000轉/分)。仔細(xi)收集上清。分裝后一(yi)份待(dai)檢測,其余冷凍(dong)備用。
6. 標本采集后盡早進行提取,提取按相(xiang)關文獻進行,提取后應盡快(kuai)進行實驗(yan)。若不(bu)能馬上進行試驗(yan),可(ke)將(jiang)標本放于(yu)-20℃保存,但應避(bi)免反復凍融.
7. 不能檢測含NaN3的(de)(de)樣品,因NaN3抑制辣根過(guo)氧化物酶的(de)(de)(HRP)活性(xing)。
操作步驟
1. 標(biao)(biao)準品(pin)的加樣(yang):設置(zhi)標(biao)(biao)準品(pin)孔和樣(yang)本孔,標(biao)(biao)準品(pin)孔各加不同濃度的標(biao)(biao)準品(pin)50μL;。
2. 加(jia)(jia)樣(yang)(yang):分別設空白(bai)(bai)孔(kong)(kong)(空白(bai)(bai)對照(zhao)孔(kong)(kong)不加(jia)(jia)樣(yang)(yang)品(pin)(pin)及酶標(biao)試(shi)劑,其余各(ge)步操作相同)、待測(ce)(ce)樣(yang)(yang)品(pin)(pin)孔(kong)(kong)。在酶標(biao)包(bao)被板(ban)上(shang)待測(ce)(ce)樣(yang)(yang)品(pin)(pin)孔(kong)(kong)中先加(jia)(jia)樣(yang)(yang)品(pin)(pin)稀釋(shi)液40μl,然(ran)后再加(jia)(jia)待測(ce)(ce)樣(yang)(yang)品(pin)(pin)10μl(樣(yang)(yang)品(pin)(pin)最終稀釋(shi)度(du)為5倍)。加(jia)(jia)樣(yang)(yang)將(jiang)樣(yang)(yang)品(pin)(pin)加(jia)(jia)于酶標(biao)板(ban)孔(kong)(kong)底部,盡量不觸及孔(kong)(kong)壁,輕輕晃(huang)動混勻。
3. 加酶:每孔(kong)加入(ru)酶標(biao)試劑100μl,空白孔(kong)除外。
4. 溫育(yu):用(yong)封(feng)板膜封(feng)板后(hou)置37℃溫育(yu)60分鐘。
5. 配液:將20倍濃縮洗滌(di)液用(yong)蒸餾(liu)水20倍稀釋后備用(yong)。
6. 洗(xi)滌:小(xiao)心揭掉封板膜,棄(qi)去液(ye)體,甩干(gan),每(mei)孔加滿(man)洗(xi)滌液(ye),靜(jing)置30秒后棄(qi)去,如此(ci)重復5次,拍(pai)干(gan)。
7. 顯(xian)色(se)(se)(se):每孔先(xian)加入(ru)顯(xian)色(se)(se)(se)劑(ji)A50μl,再加入(ru)顯(xian)色(se)(se)(se)劑(ji)B50μl,輕(qing)輕(qing)震(zhen)蕩混勻,37℃避光顯(xian)色(se)(se)(se)15分鐘.
8. 終止:每孔加終止液50μl,終止反應(此時藍色(se)(se)立轉(zhuan)黃(huang)色(se)(se))。
9. 測(ce)定(ding)(ding):以空白孔調零(ling),450nm波長依序測(ce)量各孔的吸光度(OD值(zhi))。 測(ce)定(ding)(ding)應在加終(zhong)止液后15分鐘以內進行。
注意事項:
1. 試(shi)劑盒從冷(leng)藏(zang)環(huan)境中取(qu)出應(ying)在(zai)室(shi)溫平衡15-30分鐘(zhong)后(hou)(hou)方可(ke)使用(yong),酶標包被板開封后(hou)(hou)如未用(yong)完(wan),板條應(ying)裝入密(mi)封袋中保存。樣本在(zai)使用(yong)前也要在(zai)室(shi)溫平衡60分鐘(zhong)。
2. 濃洗滌液(ye)可(ke)(ke)能會(hui)有結晶析出,稀釋(shi)時可(ke)(ke)在水浴中加溫助溶,洗滌時不影響(xiang)結果。
3. 各步加(jia)樣(yang)均應使(shi)用加(jia)樣(yang)器,并經常(chang)校對其準確性(xing),以避免試驗誤(wu)差。一次加(jia)樣(yang)時間最好控制在5分鐘(zhong)內,如標本數量多,推薦使(shi)用排(pai)槍(qiang)加(jia)樣(yang)。
4. 請每次測(ce)(ce)定的同時做標準曲線(xian),最好做復(fu)孔(kong)。如標本(ben)中待(dai)測(ce)(ce)物質含量過高(樣(yang)(yang)本(ben)OD值大于標準品(pin)(pin)孔(kong)第一孔(kong)的OD值),請先(xian)用樣(yang)(yang)品(pin)(pin)稀(xi)釋(shi)液稀(xi)釋(shi)一定倍數(n倍)后(hou)再測(ce)(ce)定,計算時請最后(hou)乘以總稀(xi)釋(shi)倍數(×n×5)。
5. 封(feng)板(ban)膜(mo)只限一次性使用(yong),以避免(mian)交叉污染。
6. 底物請避光(guang)保存。
7. 嚴格按照說(shuo)明(ming)書的(de)操作進行(xing),試驗結果判定(ding)必須(xu)以酶標儀讀數為(wei)準(zhun).
8. 所有樣品,洗滌液和(he)各種廢棄物都應按(an)傳染物處理。
9. 本(ben)試(shi)劑不同批號(hao)組(zu)分不得(de)混用。
10. 如與(yu)英文說(shuo)明書有(you)異,以英文說(shuo)明書為準(zhun)。
計算:
以標(biao)準物(wu)的濃度為橫坐(zuo)標(biao),OD值為縱坐(zuo)標(biao),
在坐標(biao)紙上繪出標(biao)準曲線(xian),根(gen)據樣品的(de)OD
值由標準曲線查出相應的濃度;再(zai)乘以稀釋
倍(bei)數;或(huo)用標準物的濃度與OD值計算(suan)出標
準曲線(xian)的直(zhi)線(xian)回(hui)歸方(fang)程式,將(jiang)樣品的OD值
代入(ru)方程式,計算(suan)出(chu)樣(yang)品濃(nong)度,再(zai)乘以稀釋
倍數(shu),即為樣品的實際濃度。
(此圖僅供參考)
試劑盒性能:
1.樣品(pin)線性(xing)回歸(gui)與預(yu)期濃度相關系數R值為0.95以上(shang)。
2.批內變異(yi)系數(shu)與批間變異(yi)系數(shu)應分別小于10%和(he)15% 。
檢測范圍:
6.25 ng/mL - 200 ng/mL
靈敏(min)度(du):
檢測(ce)濃度小于1.0 ng/mL
保存條件及有效期:
1.試(shi)劑盒保存: 2-8℃。
2.有(you)效期: 6個(ge)月
Rat Cluster of differentiation 31
FOR RESEARCH USE ONLY |
Drug Names
Generic Name:Rat Cluster of differentiation 31 (CD31) ELISA Kit.
Purpose
This kit allows for the determination of CD31 concentrations in Rat serum, plasma, tissue homogenates and other biological fluids.
Principle of the assay
The kit assay Rat CD31 level in the sample, use Purified Rat CD31 antibody to coat microtiter plate wells, make solid-phase antibody, then add CD31 to the wells, Combined antibody which With HRP labeled, become antibody-antigen-enzyme-antibody complex, after washing Completely, 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 CD31 in the samples is then determined by comparing the O.D. of the samples to the standard curve.
Materials provided with the kit
Materials provided with the kit | 48determinations | 96 determinations | Storage |
User manual | 1 | 1 | |
Closure plate membrane | 2 | 2 | |
Sealed bags | 1 | 1 | |
Microelisa stripplate | 1 | 1 | 2-8℃ |
Standard | 0.3ml×6 bottle | 0.3ml×6 bottle | 2-8℃ |
HRP-Conjugate reagent | 5ml×1 bottle | 10ml×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℃ |
20×Wash solution | 15ml×1 bottle | 25ml×1 bottle | 2-8℃ |
Note: Standard concentration was followed by:
200、100、50、25、12.5、0 ng/mL.
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. Add standard: Set Standard wells, testing sample wells. Add standard 50μl to standard well.
2.add sample:Set blank wells separately (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.add enzyme:Add HRP-Conjugate reagent 100μl to each well, except blank well.
4.Incubate: After closing plate with Closure plate membrane ,incubate for 60 min at 37℃.
5.Configurate liquid: 20-fold wash solution diluted 20-fold with distilled water and reserve.
6.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.
7.color:Add Chromogen Solution A 50ul and Chromogen Solution B to each well, evade the light preservation for 15 min at 37℃
8.Stop the reaction:Add Stop Solution 50μl to each well, Stop the reaction(the blue color change to yellow color).
9.assay:take blank well as zero , Read absorbance at 450nm after Adding Stop Solution and within 15min.