小鼠C反應(yīng)蛋白 (CRP)-ELISA試劑盒說(shuō)明書(shū)

1、小鼠C反應(yīng)蛋白 (CRP酶聯(lián)免疫吸附測(cè)定試劑盒使用說(shuō)明書(shū)產(chǎn)品編號(hào):E-EL-M0053(本試劑盒僅供體外研究使用、不用于臨床診斷!聲明:尊敬的客戶,感謝您選用本公司的產(chǎn)品。本產(chǎn)品選用世界著名生產(chǎn)廠家的原料,采用專業(yè)ELISA kit生產(chǎn)技術(shù)制造。適用于體外定量檢測(cè)小鼠血清、血漿、組織勻漿或細(xì)胞培養(yǎng)上清液中天然和重組CRP濃度。使用前請(qǐng)仔細(xì)閱讀說(shuō)明書(shū)并檢查試劑組分!如有疑問(wèn),請(qǐng)及時(shí)聯(lián)系伊萊瑞特生物科技有限公司。 *: 96T/48T(打開(kāi)包裝后請(qǐng)及時(shí)檢查所有物品是否齊全完整檢測(cè)原理:本試劑盒采用雙抗體夾心ELISA法。用抗小鼠CRP抗體包被于酶標(biāo)板上,實(shí)驗(yàn)時(shí)標(biāo)本或標(biāo)準(zhǔn)品中的CRP會(huì)與包被抗體結(jié)合

2、,游離的成分被洗去。依次加入生物素化的抗小鼠CRP抗體和辣根過(guò)氧化物酶標(biāo)記的親和素。抗小鼠CRP抗體與結(jié)合在包被抗體上的小鼠CRP結(jié)合、生物素與親和素特異性結(jié)合而形成免疫復(fù)合物,游離的成分被洗去。加入顯色底物(TMB,TMB在辣根過(guò)氧化物酶的催化下現(xiàn)藍(lán)色,加終止液后變黃。用酶標(biāo)儀在450nm波長(zhǎng)處測(cè)OD值,CRP濃度與OD450值之間呈正比,通過(guò)繪制標(biāo)準(zhǔn)曲線求出標(biāo)本中CRP的濃度。標(biāo)本收集:1.血清:全血標(biāo)本于室溫放置2小時(shí)或4過(guò)夜后于1000×g離心20分鐘,取上清即可檢測(cè),收集血液的試管應(yīng)為一次性的無(wú)熱原,無(wú)內(nèi)毒素試管。2.血漿:抗凝劑推薦使用EDTA.Na2,標(biāo)本采集后30分鐘

3、內(nèi)于1000×g離心15分鐘,取上清即可檢測(cè)。避免使用溶血,高血脂標(biāo)本。3.組織勻漿:用預(yù)冷的PBS (0.01M, pH=7.4沖洗組織,以去除殘留血液(勻漿中裂解的紅細(xì)胞會(huì)影響測(cè)量結(jié)果,稱重后將組織剪碎。將剪碎的組織與對(duì)應(yīng)體積的PBS(一般按1:9的重量體積比,比如1g的組織樣本對(duì)應(yīng)9mL的PBS,具體體積可根據(jù)實(shí)驗(yàn)需要適當(dāng)調(diào)整,并做好記錄。推薦在PBS中加入蛋白酶抑制劑加入玻璃勻漿器中,于冰上充分研磨。為了進(jìn)一步裂解組織細(xì)胞,可以對(duì)勻漿液進(jìn)行超聲破碎,或反復(fù)凍融。最后將勻漿液于5000×g 離心510分鐘,取上清檢測(cè)。4.細(xì)胞培養(yǎng)上清:取細(xì)胞培養(yǎng)上清于1000

4、5;g離心20分鐘,除去雜質(zhì)及細(xì)胞碎片。取上清檢測(cè)。5.其它生物標(biāo)本:1000×g離心20分鐘,取上清即可檢測(cè)6.標(biāo)本應(yīng)清澈透明,懸浮物應(yīng)離心去除。7.標(biāo)本收集后若不及時(shí)檢測(cè),請(qǐng)按一次使用量分裝,凍存于-20/-80冰箱內(nèi),避免反復(fù)凍融,1-6月內(nèi)檢測(cè),4保存的應(yīng)在1周內(nèi)進(jìn)行檢測(cè)。8.如果您的樣本中檢測(cè)物濃度高于標(biāo)準(zhǔn)品最高值,請(qǐng)根據(jù)實(shí)際情況,做適當(dāng)倍數(shù)稀釋(建議先做預(yù)實(shí)驗(yàn),以確定稀釋倍數(shù)。試驗(yàn)所需自備物品:1.酶標(biāo)儀(450nm波長(zhǎng)濾光片2.高精度移液器,EP管及一次性吸頭:0.5-10L, 2-20L, 20-200L, 200-1000L3.37恒溫箱, 雙蒸水或去離子水檢測(cè)前準(zhǔn)

5、備工作:1.請(qǐng)?zhí)崆?0分鐘從冰箱中取出試劑盒,平衡至室溫。2.將濃縮洗滌液用雙蒸水稀釋(1:25。未用完的放回4。從冰箱中取出的濃縮洗滌液可能有結(jié)晶,屬于正?，F(xiàn)象,可用40水浴微加熱使結(jié)晶完全溶解后再配制洗滌液(加熱溫度不要超過(guò)50,使用時(shí)洗滌液應(yīng)為室溫。3.標(biāo)準(zhǔn)品: 加入標(biāo)準(zhǔn)品&樣品稀釋液1.0mL至凍干標(biāo)準(zhǔn)品中,靜置10分鐘,待其充分溶解后,輕輕混勻(濃度為25ng/mL。然后根據(jù)需要進(jìn)行倍比稀釋(注:不要直接在反應(yīng)孔中進(jìn)行倍比稀釋。建議配制成以下濃度:25、12.5、6.25、3.13、1.56、0.78、0.39、0 ng/mL ,樣品稀釋液直接作為空白孔0ng/mL。如配制1

6、2.5ng/mL標(biāo)準(zhǔn)品:取0.5mL 25ng/mL的上述標(biāo)準(zhǔn)品加入含有0.5mL樣品稀釋液的EP管中,混勻即可,其余濃度依此類推。4.生物素化抗體工作液:實(shí)驗(yàn)前計(jì)算當(dāng)次實(shí)驗(yàn)所需用量(以100L/孔計(jì),實(shí)際配制時(shí)應(yīng)多配制100-200L。使用前15分鐘,以生物素化抗體稀釋液稀釋濃縮生物素化抗體(1:100成工作濃度。當(dāng)日使用。5.酶結(jié)合物工作液:實(shí)驗(yàn)前計(jì)算當(dāng)次實(shí)驗(yàn)所需用量(以100L/孔計(jì),實(shí)際配制時(shí)應(yīng)多配制100-200L。使用前15分鐘,以酶結(jié)合物稀釋液稀釋濃縮HRP酶結(jié)合物(1:100成工作濃度。當(dāng)日使用。標(biāo)準(zhǔn)品稀釋方法圖例:(以500L/管為例,也可根據(jù)實(shí)際用量來(lái)稀釋,如200L/管

7、25 12.5 6.25 3.13 1.56 0.78 0.39 0 ng/mL洗滌方法:1.自動(dòng)洗板機(jī):每孔加入洗滌液350L,注入與吸出間隔60秒。2.手工洗板:甩盡孔內(nèi)液體,在潔凈的吸水紙上拍干,每孔加洗滌液350L,浸泡1-2分鐘,吸去(不可觸及板壁或甩掉酶標(biāo)板內(nèi)的液體,在厚的吸水紙上拍干。操作步驟:實(shí)驗(yàn)開(kāi)始前,各試劑均應(yīng)平衡至室溫;試劑或樣品配制時(shí),均需充分混勻,并盡量避免起泡。1.加樣:分別設(shè)空白孔、標(biāo)準(zhǔn)孔、待測(cè)樣品孔?？瞻卓准訕悠废♂屢?00L,余孔分別加標(biāo)準(zhǔn)品或待測(cè)樣品100L,注意不要有氣泡,加樣時(shí)將樣品加于酶標(biāo)板底部,盡量不觸及孔壁,輕輕晃動(dòng)混勻。給酶標(biāo)板覆膜,37孵育90

8、分鐘。為保證實(shí)驗(yàn)結(jié)果有效性,每次實(shí)驗(yàn)請(qǐng)使用新的標(biāo)準(zhǔn)品溶液。2.棄去液體,甩干,不用洗滌。每個(gè)孔中加入生物素化抗體工作液100L(在使用前15分鐘內(nèi)配制,酶標(biāo)板加上覆膜,37溫育1小時(shí)。3.棄去孔內(nèi)液體,甩干,洗板3次,每次浸泡1-2分鐘,大約350L/每孔,甩干并在吸水紙上輕拍將孔內(nèi)液體拍干。4.每孔加酶結(jié)合物工作液(臨用前15分鐘內(nèi)配制100L,加上覆膜,37溫育30分鐘。5.棄去孔內(nèi)液體,甩干,洗板5次,方法同步驟3。6.每孔加底物溶液(TMB100L,酶標(biāo)板加上覆膜37避光孵育15分鐘左右(根據(jù)實(shí)際顯色情況酌情縮短或延長(zhǎng),但不可超過(guò)30分鐘。當(dāng)標(biāo)準(zhǔn)孔出現(xiàn)明顯梯度時(shí),即可終止。7.每孔加終

9、止液50L,終止反應(yīng),此時(shí)藍(lán)色立轉(zhuǎn)黃色。終止液的加入順序應(yīng)盡量與底物溶液的加入順序相同。8.立即用酶標(biāo)儀在450nm波長(zhǎng)測(cè)量各孔的光密度(OD值。應(yīng)提前打開(kāi)酶標(biāo)儀電源,預(yù)熱儀器,設(shè)置好檢測(cè)程序。9.實(shí)驗(yàn)完畢后將未用完的試劑按規(guī)定的保存溫度放回冰箱保存。注意事項(xiàng):1.保存:試劑盒中各試劑請(qǐng)按說(shuō)明書(shū)提示合理存放。在儲(chǔ)存及溫育過(guò)程中避免將試劑暴露在強(qiáng)光中。所有試劑瓶蓋須旋緊以防止蒸發(fā)和微生物的污染,否則可能會(huì)出現(xiàn)錯(cuò)誤的結(jié)果。2.酶標(biāo)板:剛開(kāi)啟的酶標(biāo)板孔中可能會(huì)有少許水樣物質(zhì),此為正?，F(xiàn)象,不會(huì)對(duì)實(shí)驗(yàn)結(jié)果造成任何影響。3.加樣:加樣或加試劑時(shí),第一個(gè)孔與最后一個(gè)孔的加樣時(shí)間間隔如果太大,將會(huì)導(dǎo)致不同的

10、“預(yù)溫育”時(shí)間,從而明顯地影響到測(cè)量值的準(zhǔn)確性及重復(fù)性。每次的加樣時(shí)間最好控制在10分鐘內(nèi)。推薦設(shè)置復(fù)孔。4.溫育:為防止樣品蒸發(fā),實(shí)驗(yàn)時(shí)必須給酶標(biāo)板覆膜;洗板后應(yīng)盡快進(jìn)行下步操作,避免酶標(biāo)板處于干燥狀態(tài);嚴(yán)格遵守給定的溫育時(shí)間和溫度。5.洗滌:洗滌過(guò)程中反應(yīng)孔中殘留的洗滌液應(yīng)在吸水紙上拍干,勿將濾紙直接放入反應(yīng)孔中吸水。在讀數(shù)前要注意清除底部殘留的液體和手指印,以免影響酶標(biāo)儀讀數(shù)。6.試劑配制:Concentrated Biotinylated Detection Ab及Concentrated HRP Conjugate體積較小,運(yùn)輸過(guò)程會(huì)使液體沾到管壁或瓶蓋,因此使用前1000轉(zhuǎn)/分離心

11、1min,以使附著管壁或瓶蓋的液體沉積到管底。取用前,請(qǐng)用移液器小心吹打4-5次使溶液混勻。標(biāo)準(zhǔn)品、生物素化抗體工作液、酶結(jié)合物工作液請(qǐng)根據(jù)所需用量配制,并使用相應(yīng)的稀釋液配制,不能混淆。請(qǐng)精確配制標(biāo)準(zhǔn)品及工作液,盡量不要微量配制(如吸取Concentrated Biotinylated Detection Ab時(shí),一次不要小于10L,以避免由于不準(zhǔn)確稀釋而造成濃度誤差;請(qǐng)勿重復(fù)使用已稀釋過(guò)的標(biāo)準(zhǔn)品、生物素化抗體工作液、酶結(jié)合物工作液。若需要分次使用標(biāo)準(zhǔn)品應(yīng)按照每一次用量分裝,將其放在-20-80貯存。避免反復(fù)凍融。7.顯色時(shí)間的控制:加入底物后請(qǐng)定時(shí)觀察反應(yīng)孔的顏色變化(比如每隔5分鐘,如梯

12、度已很明顯,請(qǐng)?zhí)崆凹尤虢K止液終止反應(yīng),避免顏色過(guò)深影響酶標(biāo)儀讀數(shù)。8.底物:底物請(qǐng)避光保存,在儲(chǔ)存和溫育時(shí)避免強(qiáng)光直接照射。9.混勻:充分輕微混勻?qū)Ψ磻?yīng)結(jié)果尤為重要,最好使用微量振蕩器(使用最低頻率,如無(wú)微量振蕩器,可在反應(yīng)前手工輕輕敲擊酶標(biāo)板框混勻。10.安全:試驗(yàn)中請(qǐng)穿著實(shí)驗(yàn)服并帶乳膠手套做好防護(hù)工作。特別是檢測(cè)血液或者其他體液標(biāo)本時(shí),請(qǐng)按國(guó)家生物試驗(yàn)室安全防護(hù)條例執(zhí)行。11.不同批號(hào)的試劑盒組份不能混用(洗滌液和反應(yīng)終止液除外12.試驗(yàn)中所用的EP管和吸頭均為一次性使用,嚴(yán)禁混用,否則將影響試驗(yàn)結(jié)果!結(jié)果判斷:1.每個(gè)標(biāo)準(zhǔn)品的OD值減去空白孔的OD值后作圖,如設(shè)置復(fù)孔,則應(yīng)取其平均值計(jì)算

13、。以標(biāo)準(zhǔn)品的濃度為橫坐標(biāo),OD值為縱坐標(biāo),繪出標(biāo)準(zhǔn)曲線。亦可以O(shè)D值為橫坐標(biāo),標(biāo)準(zhǔn)品的濃度為縱坐標(biāo),繪出標(biāo)準(zhǔn)曲線。2.推薦使用專業(yè)的曲線制作軟件,如curve expert 1.3,在軟件界面既可根據(jù)樣品OD值,由標(biāo)準(zhǔn)曲線查出相應(yīng)的濃度,乘以稀釋倍數(shù);亦可將樣品的OD值代入標(biāo)準(zhǔn)曲線的擬合方程式,計(jì)算出樣品濃度,再乘以稀釋倍數(shù),即為樣品的實(shí)際濃度。3.若標(biāo)本OD值高于標(biāo)準(zhǔn)曲線上限,應(yīng)適當(dāng)稀釋后重測(cè),計(jì)算濃度時(shí)應(yīng)乘以稀釋倍數(shù)。靈敏度、檢測(cè)范圍、特異性和重復(fù)性: 靈敏度:最小可測(cè)0.23ng/mL。檢測(cè)范圍:0.39 25ng/mL。 特異性:可檢測(cè)重組或天然的小鼠CRP,且與其它相關(guān)蛋白無(wú)交叉反應(yīng)

14、。 重復(fù)性:板內(nèi),板間變異系數(shù)均<10%。 說(shuō)明1.限于現(xiàn)有條件及科學(xué)技術(shù)水平,尚不能對(duì)所有原料進(jìn)行全面的鑒定分析,本產(chǎn)品可能存在一定的質(zhì)量技術(shù)風(fēng)險(xiǎn)。2.最終的實(shí)驗(yàn)結(jié)果與試劑的有效性、實(shí)驗(yàn)者的相關(guān)操作以及當(dāng)時(shí)的實(shí)驗(yàn)環(huán)境密切相關(guān),請(qǐng)務(wù)必準(zhǔn)備充足的待測(cè)樣品。3.只有全部使用Elab TM試劑才能保證檢測(cè)效果,不能混用其他制造商的產(chǎn)品。只有嚴(yán)格遵守Elab TM試劑的實(shí)驗(yàn)說(shuō)明才會(huì)得到最佳的檢測(cè)結(jié)果。4.有效期:6個(gè)月。5.本操作說(shuō)明同樣適用于48T試劑盒。Mouse CRP (C-Reactive Protein ELISA KitProduct DescriptionCatalog No:

15、E-EL-M0053(FOR RESEARCH USE ONLY. DO NOT USE IT IN CLINICAL DIAGONOSIS !Dear customer, Thank you for choosing our products. This product is produced using raw materials from world-renowned manufacturer, and professional manufacturing technology of ELISA kits. Please read the instructions carefully b

16、efore use and check all the reagent compositions! If in doubt, please contact Elabscience Biotechnology Co., Ltd.Intended useThis immunoassay kit allows for the in vitro quantitative determination of Mouse CRP concentrations in serum, plasma and other biological fluids. Test principleThis ELISA kit

17、uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to CRP Standards or samples are then added to the appropriate micro ELISA plate wells and combined to the specific antibody. Then a biotinylated detection antibody specific for

18、 CRP and Avidin-Horseradish Peroxidase (HRP conjugate is added to each micro plate well and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain CRP, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color.

19、 The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color turn yellow. The optical density (OD is measured spectrophotometrically at a wavelength of 450 nm ±2 nm. The OD value is proportional to the concentration of CRP. You can calculate the concen

20、tration of CRP in the samples by comparing the O.D. of the samples to the standard curve.Sample collection and storageSerum- Allow samples to clot for 2 hours at room temperature or overnight at 4°C before centrifugation for 20 minutes at approximately 1000×g. Collect the supernatant and c

21、arry out the assay immediately. Blood collection tubes should be disposable, non-pyrogenic, and non-endotoxin.Plasma- Collect plasma using EDTA.Na2 or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000×g at 2 - 8°C within 30 minutes of collection. Collect the supernatan

22、t and carry out the assay immediately. Avoid hemolysis, high cholesterol samples.Tissue homogenates:For general information, hemolysis blood may affect the result, so you should rinse the tissues with ice-cold PBS (0.01M, pH=7.4 to remove excess blood thoroughly. Tissue pieces should be weighed and

23、then minced to small pieces which will be homogenized in PBS (the volume depends on the weight of the tissue. 9mL PBS would be appropriate to 1 gram tissue pieces.Some protease inhibitor is recommended to add into the PBS. with a glass homogenizer on ice. To further break the cells, you can sonicate

24、 the suspension with an ultrasonic cell disrupter or subject it to freeze-thaw cycles. The homogenates are then centrifugated for 5minutes at 5000×g to get the supernate.Cell culture supernateCentrifuge supernate for 20 minutes to remove insoluble impurity and cell debris at 1000×g at 2 -

25、8°C. Collect the clear supernate and carry out the assay immediately.Sample preparation Samples should be clear and transparent and be centrifuged to remove suspended solids.Note: Serum and plasma to be used within 7 days when stored at 2-8°C, otherwise samples must be divided and stored a

26、t -20°C (1 month or -80°C (6 months to avoid loss of bioactivity andcontamination. Avoid freeze-thaw cycles. When performing the assay slowly bring samples to room temperature. If the sample concentration is higher than the maximum standard value, please dilute it with appropriate factor a

27、ccording to the actual situation. (A pre-test is recommended to determine the dilute factorOther supplies requiredMicroplate reader with 450nm wavelength filterHigh-precision transferpettor, EP tubes and disposable pipette tips37°C Incubator, Deionized or distilled water.Absorbent paperReagent

28、preparationBring all reagents to room temperature before use.Wash Buffer - Dilute 30mL of Concentrated Wash Buffer into 750 mL of Wash Buffer with deionized or distilled water. Put unused solution back at 4°C. If crystals have formed in the concentrate, you can warm it with 40°C water bath

29、 (Heating temperature should not exceed 50°C and mix it gently until the crystals have completely dissolved. The solution should be cooled to room temperature before use. Standard - Reconstitute the Standard with 1.0mL of Sample Diluent, let it stand for 10minutes until it dissolved fully. This

30、 reconstitution produces a stock solution of 25ng/mL. Then make serial dilutions as needed (Making serial dilution in the wells directly is not permitted. The recommended concentrations are as follows: 25、12.5、6.25、3.13、1.56、0.78、0.39、0ng/mL . As if you want to make standard solution at the concentr

31、ation of 12.5ng/mL, you can take 0.5mL the standard at 25ng/mL, add it to an EP tube with 0.5mL sample dilution, and mix it. The procedures of making the remaining concentrations are all the same. The undiluted standard serves as the highest standard (25ng/mL. The Sample Diluent serves as the zero (

32、0ng/mL.(500L/tube,for example. Can also be diluted according to the actual amount,such as 200L/tube 25 12.5 6.25 3.13 1.56 0.78 0.39 0 ng/mLBiotinylated Detection Ab Calculate the required amount before experiment (100L /well. In actual preparation you should prepare 100200L more. Dilute the concent

33、rated Biotinylated Detection Ab to the working concentration using Diluent for Biotinylated Detection Ab (1:100.Concentrated HRP Conjugate Calculate the required amount before experiment (100L/well. In actual preparation you should prepare 100200L more. Dilute the Concentrated HRP Conjugate to the w

34、orking concentration using Diluent for Concentrated HRP Conjugate (1:100.Washing Procedure:1. Automated washer: add 350L wash buffer into each well, the interval between injection andsuction should be set about 60s.2. Manual wash: add 350L wash buffer into each well, soak it for 12minutes, suck(no i

35、nside walltouching or get rid of liquid within the micro ELISA plate and pat it dry on thick clean absorbent paper.Assay procedureAllow all reagents to reach room temperature All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming.1.Add Sample: Add 100L of Stan

36、dard, Blank, or Sample per well. The blank well is added withsample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming to the best of your ability. Mix it gently. Cover the plate with sealer we provided. Incubate for 90 minutes at 37°C.2

37、.Biotinylated Detection Ab: Remove the liquid o f each well, dont wash. Immediately add 100Lof Biotinylated Detection Ab working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C.3.Wash: Aspirate each well and wash, r

38、epeating the process three times. Wash by filling each wellwith Wash Buffer (approximately 350L using a squirt bottle, multi-channel pipette, manifold dispenser or automated washer. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wa

39、sh Buffer by aspirating or decanting.Invert the plate and pat it against thick clean absorbent paper.4. HRP Conjugate: Add 100L of HRP Conjugate working solution to each well. Cover with thePlate sealer. Incubate for 30 minutes at 37°C.5. Wash: Repeat the wash process for five times as conducte

40、d in step 3.6. Substrate:Add 100L of Substrate Solution to each well. Cover with a new Plate sealer. Incubatefor about 15 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but not more than 30minutes. When apparen

41、t gradient appeared in standard wells, you can terminate the reaction.7. Stop: Add 50L of Stop Solution to each well. Color turn to yellow immediately. The adding order of stop solution should be as the same as the substrate solution. 8. OD Measurement: Determine the optical density (OD value of eac

42、h well at once, using a microplate reader set to 450nm. You should open the microplate reader ahead, preheat the instrument, and set the testing parameters. 9. After experiment, put all the unused reagents back into the refrigerator according to the specified storage temperature respectively until t

43、heir expiry. Important Note: 1. Storage: All the reagents in the kit should be stored following the instructions. Exposure of reagents to strong light should be avoided in the process of incubation and storage. All the taps of reagents should be tightened to prevent evaporation and microbial contami

44、nation, or erroneous results may occur. 2. ELISA Plate: Little water-like substance may appear in the ELISA Plate just opened, this is normal and will not have any impact on the experiment results. 3. Add Sample: The interval of sample adding between the first well and the last well should not be to

45、o long, otherwise will cause different pre-incubation time, which will significantly affect the experiments accuracy and repeatability. The interval controlled within 10minutes is good. Parallel measurement is recommended. 4. Incubation: To prevent evaporation, proper adhesion of plate sealers durin

46、g incubation steps is necessary. Do not allow wells to sit uncovered for extended periods between incubation steps. Do not let the strips dry at any time during the assay. Strict compliance with the given incubation time and temperature. 5. Washing: The wash procedure is critical. Insufficient washi

47、ng will result in poor precision and falsely elevated absorbance readings Residual liquid in the reaction wells should be pat dry against absorbent paper in the washing process. But dont put absorbent paper into reaction wells directly. Note that clear the residual liquid and fingerprint in the bott

48、om before measurement, so as not to affect the microtiter plate reader. 6. Reagent Preparation: As the volume of Concentrated Biotinylated Detection Ab and Concentrated HRP Conjugate is very small, liquid may adhere to the tube wall or tube cap when being transported. You better hand-throw it or cen

49、trifugal it for 1 minute at 1000rpm. Please pipette the solution for 4-5 times before pippeting. Please carefully reconstitute Standards, working solutions of Biotinylated Detection Ab and HRP Conjugate according to the instructions. To minimize imprecision caused by pipetting, ensure that pipettors

50、 are calibrated. It is recommended to suck more than 10L for once pipetting. Do not reuse standard solution, working solution of Biotinylated Detection Ab and HRP Conjugate, which have been diluted. If you need to use standard repeatedly, you can divide the standard into small pack according to the

51、amount of each assay, keep them at -20-80° and avoid repeated freezing and thawing. C 7. 8. 9. Reaction Time Control: Please control reaction time strictly following this product description! Substrate: Substrate Solution is easily contaminated. Please protect it from light. Mixing: Youd better

52、 use microoscillator at the lowest frequency, as sufficient and gentle mixing is particularly important to reaction result. If there is no microsocillator available, you can knock the ELISA plate frame gently with your finger before reaction. 10. Security: Please wear lab coats and latex gloves for

53、protection. Especially detecting samples of blood or other body fluid, please perform following the national security columns of biological laboratories. 11. Do not use component from different batches of kit(washing buffer and stop solution can be an exception 12. To avoid cross-contamination, chan

54、ge pipette tips between adding of each standard level, between sample adding, and between reagent adding. Also, use separate reservoirs for each reagent. Otherwise, the results will be inaccurate! Calculation of results Average the duplicate readings for each standard and samples and subtract the av

55、erage zero standard optical density. Create a standard curve by plotting the mean OD value for each standard on the y-axis or x-axis against the concentration on the x-axis or y-axis and draw a best fit curve through the points on the graph. It is recommended to use some professional software to do

56、this calculation, such as curve expert 1.3. In the software interface, a best fitting equation of standard curve will be calculated using OD values and concentrations of standard sample. The software will calculate the concentration of samples after entering the OD value of samples. Also, you can en

57、ter the corresponding fitting equation and OD value of samples into Excel to get the concentration of samples. If samples have been diluted, the concentration calculated from the standard curve must be multiplied by the dilution factor. If the OD of the sample surpasses the upper limit of the standa

58、rd curve, you should re-test it after appropriate dilution. The actual concentration is the calculated concentration multiplied dilution factor. Sensitivity The minimum detectable dose of Mouse CRP is 0.23ng/mL (The sensitivity of this assay, or Lower Limit of Detection (LLD was defined as the lowest protein concentration that could be differentiated from zero. Detection Range 0.39 25 ng/mL. Specificity This kit recognizes recombinant and natural Mouse CRP. No significant cross-reactivity or interference was observed. Repeatability: Coefficient of variation were&