人25羥基維生素D3(25-OH-D3)說明書-1

1、人 25 羥基維生素D3(25-OH-D3)酶聯(lián)免疫分析(ELISA)試劑盒使用說明書本試劑僅供研究使用液體樣本中25 羥基維生素D3(25-OH-D3)的含量。實(shí)驗(yàn)原理：本試劑盒應(yīng)用雙抗體夾心法測定標(biāo)本中人25 羥基維生素D3(25-OH-D3) 水平。 用純化的抗 D3(25-OH-D3) 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人25羥基維生素 D3(25-OH-D3)，再與 HRP 標(biāo)記的 D3(25-OH-D3)抗體結(jié)合，形成抗體-抗原-酶標(biāo)抗體復(fù)合物，經(jīng)過徹底洗滌后加底物TMB 顯色。 TMB 在 HRP 酶的催化下轉(zhuǎn)化成藍(lán)色，并在酸的作用下轉(zhuǎn)化成最終的黃色。顏色的

2、深淺和樣品中的25 羥基維生素D3(25-OH-D3) 呈正相關(guān)。用酶標(biāo)儀在450nm 波長下測定吸光度( OD 值) ， 通過標(biāo)準(zhǔn)曲線計(jì)算樣品中人25 羥基維生素D3(25-OH-D3) 濃度。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( 48)2 片(96)密封袋1個(gè)1個(gè)酶標(biāo)包被板1 × 481× 962-8保存標(biāo)準(zhǔn)品：27 g/L0.5ml× 1 瓶0.5ml× 1 瓶2-8保存標(biāo)準(zhǔn)品稀釋液1.5ml × 1 瓶1.5ml × 1 瓶2-8保存酶標(biāo)試劑3 ml× 1 瓶6 ml

3、5; 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3ml× 1 瓶6ml× 1 瓶2-8保存濃縮洗滌液（ 20ml × 20 倍）×1 瓶（ 20ml × 30 倍）×1 瓶2-8保存樣本處理及要求：1. 血清： 室溫血液自然凝固10-20 分鐘， 離心 20 分鐘左右( 2000-3000 轉(zhuǎn) /分)。仔細(xì)收集上清，保存過程中

4、如出現(xiàn)沉淀，應(yīng)再次離心。2. 血漿：應(yīng)根據(jù)標(biāo)本的要求選擇EDTA 或檸檬酸鈉作為抗凝劑，混合10-20 分鐘后，離心20 分鐘左右(2000-3000 轉(zhuǎn) /分)。仔細(xì)收集上清，保存過程中如有沉淀形成，應(yīng)該再次離心。3. 尿液：用無菌管收集，離心20 分鐘左右(2000-3000 轉(zhuǎn) /分)。仔細(xì)收集上清，保存過程中如有沉淀形成，應(yīng)再次離心。胸腹水、腦脊液參照實(shí)行。4. 細(xì)胞培養(yǎng)上清：檢測分泌性的成份時(shí)，用無菌管收集。離心20 分鐘左右(2000-3000 轉(zhuǎn) /分） 。仔細(xì)收集上清。檢測細(xì)胞內(nèi)的成份時(shí)，用PBS（ PH7.2-7.4）稀釋細(xì)胞懸液，細(xì)胞濃度達(dá)到100 萬 /ml 左右。通過反

5、復(fù)凍融，以使細(xì)胞破壞并放出細(xì)胞內(nèi)成份。離心20 分鐘左右（2000-3000 轉(zhuǎn) /分）。仔細(xì)收集上清。保存過程中如有沉淀形成，應(yīng)再次離心。5. 組織標(biāo)本：切割標(biāo)本后，稱取重量。加入一定量的PBS， PH7.4。用液氮迅速冷凍保存?zhèn)溆?。?biāo)本融化后仍然保持2-8的溫度。加入一定量的PBS（ PH7.4） ，用手工或勻漿器將標(biāo)本勻漿充分。離心20 分鐘左右（2000-3000 轉(zhuǎn) /分）。仔細(xì)收集上清。分裝后一份待檢測，其余冷凍備用。6. 標(biāo)本采集后盡早進(jìn)行提取，提取按相關(guān)文獻(xiàn)進(jìn)行，提取后應(yīng)盡快進(jìn)行實(shí)驗(yàn)。若不能馬上進(jìn)行試驗(yàn)，可將標(biāo)本放于-20保存，但應(yīng)避免反復(fù)凍融.7. 不能檢測含NaN3的樣品，

6、因NaN3 抑制辣根過氧化物酶的（HRP）活性。操作步驟1. 標(biāo)準(zhǔn)品的稀釋與加樣：在酶標(biāo)包被板上設(shè)標(biāo)準(zhǔn)品孔10 孔，在第一、第二孔中分別加標(biāo)準(zhǔn)品 100 l，然后在第一、第二孔中加標(biāo)準(zhǔn)品稀釋液50l，混勻；然后從第一孔、第二孔中各取100 l 分別加到第三孔和第四孔，再在第三、第四孔分別加標(biāo)準(zhǔn)品稀釋液50 l，混勻；然后在第三孔和第四孔中先各取50l 棄掉，再各取50l 分別加到第五、第六孔中，再在第五、第六孔中分別加標(biāo)準(zhǔn)品稀釋液50ul，混勻；混勻后從第五、第六孔中各取 50l 分別加到第七、第八孔中，再在第七、第八孔中分別加標(biāo)準(zhǔn)品稀釋液50 l，混勻后從第七、第八孔中分別取50l 加到第九

7、、第十孔中，再在第九第十孔分別加標(biāo)準(zhǔn)品稀釋液50l，混勻后從第九第十孔中各取50l 棄掉。 （稀釋后各孔加樣量都為50 l，濃度分別為18g /L ， 12g /L， 6g /L， 3g /L，1.5g /L） 。2. 加樣：分別設(shè)空白孔（空白對照孔不加樣品及酶標(biāo)試劑，其余各步操作相同）、待測樣品孔。 在酶標(biāo)包被板上待測樣品孔中先加樣品稀釋液40 l， 然后再加待測樣品10 l（ 樣品最終稀釋度為5 倍） 。加樣將樣品加于酶標(biāo)板孔底部，盡量不觸及孔壁，輕輕晃動混勻。3. 溫育：用封板膜封板后置37溫育 30 分鐘。4. 配液：將30（ 48T 的 20 倍）倍濃縮洗滌液用蒸餾水30（ 48T

8、的 20 倍）倍稀釋后備用。5. 洗滌：小心揭掉封板膜，棄去液體，甩干，每孔加滿洗滌液，靜置30 秒后棄去，如此重復(fù) 5 次，拍干。6. 加酶：每孔加入酶標(biāo)試劑50l，空白孔除外。7. 溫育：操作同3。8. 洗滌：操作同5。9. 顯色：每孔先加入顯色劑A50l，再加入顯色劑B50 l，輕輕震蕩混勻，37避光顯色15 分鐘 .10. 終止：每孔加終止液50l ，終止反應(yīng)（此時(shí)藍(lán)色立轉(zhuǎn)黃色）。11. 測定：以空白空調(diào)零，450nm 波長依序測量各孔的吸光度（OD 值） 。 測定應(yīng)在加終止液后 15 分鐘以內(nèi)進(jìn)行。注意事項(xiàng)：1 試劑盒從冷藏環(huán)境中取出應(yīng)在室溫平衡15-30 分鐘后方可使用，酶標(biāo)包被板

9、開封后如未用完，板條應(yīng)裝入密封袋中保存。2 濃洗滌液可能會有結(jié)晶析出，稀釋時(shí)可在水浴中加溫助溶，洗滌時(shí)不影響結(jié)果。3 各步加樣均應(yīng)使用加樣器，并經(jīng)常校對其準(zhǔn)確性，以避免試驗(yàn)誤差。一次加樣時(shí)間最好控制在 5 分鐘內(nèi)，如標(biāo)本數(shù)量多，推薦使用排槍加樣。4 請每次測定的同時(shí)做標(biāo)準(zhǔn)曲線，最好做復(fù)孔。如標(biāo)本中待測物質(zhì)含量過高（樣本OD 值大于標(biāo)準(zhǔn)品孔第一孔的OD 值） ，請先用樣品稀釋液稀釋一定倍數(shù)（n 倍）后再測定，計(jì)11算時(shí)請最后乘以總稀釋倍數(shù)（×n × 5） 。5 封板膜只限一次性使用，以避免交叉污染。6 底物請避光保存。7 嚴(yán)格按照說明書的操作進(jìn)行，試驗(yàn)結(jié)果判定必須以酶標(biāo)儀讀數(shù)

10、為準(zhǔn)8 所有樣品，洗滌液和各種廢棄物都應(yīng)按傳染物處理。9 本試劑不同批號組分不得混用。10 如與英文說明書有異，以英文說明書為準(zhǔn)。計(jì)算：以標(biāo)準(zhǔn)物的濃度為橫坐標(biāo)，OD 值為縱坐標(biāo)，在坐標(biāo)紙上繪出標(biāo)準(zhǔn)曲線，根據(jù)樣品的OD值由標(biāo)準(zhǔn)曲線查出相應(yīng)的濃度；再乘以稀釋倍數(shù)；或用標(biāo)準(zhǔn)物的濃度與OD 值計(jì)算出標(biāo)準(zhǔn)曲線的直線回歸方程式，將樣品的OD 值代入方程式，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。試劑盒性能：1 .樣品線性回歸與預(yù)期濃度相關(guān)系數(shù)R 值為 0.92 以上。2 .批內(nèi)與批間應(yīng)分別小于9%和15%檢測范圍：0.6g /L 22g /L保存條件及有效期：1.試劑盒保存：； 2-8。2有效

11、期：6 個(gè)月FOR RESEARCH USE ONLYHuman 25- hydroxy vitamin D3Drug NamesGeneric Nam：e Human 25-hydroxy vitamin D3 (25-OH-D3) ELISA Kit.PurposeThis kit allows for the determination of 25-OH-D3 concentrations in Human serum, blood plasma, and other biological fluids.Principle of the assayThe kit assay Human2

12、 5-OH-D3l evel in the sample， use Purified Human 25-OH-D3 antibody to coat microtiter plate wells, make solid-phase antibody, then add 25-OH-D3 to wells, Combined 25-OH-D3 antibody which With HRP labeled , become antibody - antigen - enzyme-antibodyc omplex, after washing Completely,A dd TMB substra

13、te solution,TMB substrate becomes blue color At HRP enzyme-catalyzed, reaction is terminated by the addition of a sulphurica cid solution and the color change is measureds pectrophotometricalalyt a wavelength of 450 nm. The concentration of 25-OH-D3 in the samples is then determined by comparing the

14、 O.D. of the samples to the standard curve.Materials provided with the kitMaterials provided with the kit48determinations96 determinationsStorageUser manual11Closure plate membrane22Sealed bags11Microelisa stripplate112-8Standard： 27g /L0.5ml 1× bottle0.5ml 1× bottle2-8Standard diluent1.5m

15、l 1× bottle1.5ml 1× bottle2-8HRP-Conjugate reagent3ml ×1 bottle6ml ×1 bottle2-8Sample diluent3ml ×1 bottle6ml ×1 bottle2-8Chromogen Solution A3ml ×1 bottle6ml ×1 bottle2-8Chromogen Solution B3ml ×1 bottle6ml ×1 bottle2-8Stop Solution3ml ×1 bottl

16、e6ml ×1 bottle2-8wash solution( 20ml × 20 fold) × 1bottle( 20ml × 30 fold) × 1bottle2-8Specimen requirements1. serum- coagulation at room temperature 10-20， mciennstrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant, If precipitation appeared, Centrifugal

17、again.2. plasma-use suited EDTA or citrate plasma as an anticoagulant,mix 10-20 mins ,centrifugation2 0-min at the speed of 2000-3000r .p.m. remove supernatant,I f precipitation appeared, Centrifugal again.3. Urine-collect sue a sterile container, centrifugation 20-min at the speed of 2000-3000 r.p.

18、m. remove supernatant,I f precipitationa ppeared, Centrifugal again. The Operation of Hydrothorax and cerebrospinal fluid Reference to it.4. cell culture supernatant-detect secretory components,c ollect sue a sterile container, centrifugation2 0-min at the speed of 2000-3000r .p.m. remove supernatan

19、t,detectth e compositiono f cells, Dilut cell suspensionw ith PBS( PH7.2-7.)4 , Cell concentration reached 1 million / ml, repeated freeze-thawc ycles, damage cells and release of intracellular components, centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant, If precipitation ap

20、peared, Centrifugal again.5. Tissue samples- After cutting samples, check the weight,add( PPBHS7.2-7.4) , Rapidly frozen with liquid nitrogen, maintain samples at 2- a8fter melting,add PB(S PH7.4) ,Homogenized by hand or Grinders, centrifugation 20-min at the speed of 2000-3000 r.p.m. remove superna

21、tant.6. extract as soon as possible after Specimen collection,anda ccording to the relevant literature,a nd shouldb e experimenta s soon as possiblea fter the extraction.I f it can t,specimen can be kept in -20 to preserve, Avoid repeated freeze-thaw cycles.7. Can t detect the sample which coNnataNi

22、n3 , because NaN3 inhibits HRP active.Assay procedure1 .Dilute and add sample to Standard: set 10 Standard wells on the ELISA plates coated, addStandard 100 l to the first and the second well, then add Stand5a0rd dli ltuot itohne first andthe second well, mix; take out 100l fotrhme tsheec ofinrsdtwa

23、nedlldll then add it to the thirdand the forth well separately.t hen add Standard dilution 50 tl o the third and the forthwell ,mix ; then take out 50 l from the third and the forth well discard, add 50the sixth well ,then add Standard dilu5ti0o n l to the fifth and the sixth well, mix ; take out 50

24、from the fifth and the sixth well and add to the seventh and the eighth well, then add Standard dilution5 0 l to the seventh and the eighth well ,mix ; take out 50 th and the l from the seven eighth well and add to the ninth and the tenth well, add Standard 5d0ilutilotno the ninth andthe tenth well,

25、 mix , take out 50 l from the ninth and the tenth well discard(add Sample 5each well after Diluting ,(density: 18,12 g,/6L g/,3Lgg/L/,1L .5 g/L)2 .add sample： Set blank wells separately( blank comparisonw ells don atd d sample andHRP-Conjugate reagent, other each step operation is same). testing sam

26、ple well. add Sample dilution 40 tlo testing samplew ell, then add testing sample 10 (ls amplef inal dilution is5-fold), add sample to welldso,n t touch the well wall as far as poasnsidb lGe,e ntly mix.1 .Incubate: After closing plate with Closure plate membrane ,incubate for 30 m. in at 374.Configu

27、rate liquid: 30-fold (or 20-fold)wash solution diluted 30-fold (or 20-fold) with distilled water and reserve.5 .washin：g 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 enzym： e Add HRP-Conjug

28、ate reage5n0t l to each well, excepbtlank well.7.incubat：e Operation with 3.8.washin：g Operation with 5.9.colo：r Add Chromogen Solution A 50ul and Chromogen Solution B to each well, evade the light preservation for 15 min at 3710.Stopt he reaction： Add Stop Solution50l to each well, Stop the reactio

29、n(theb lue colorchange to yellow color).11.assa：y take blank well as zero , Read absorbance at 450nm after Adding Stop Solution andwithin 15min.Important notes1. 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 Crystallizations eparation,it can be heated the water helps dissolve whe