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人Nestin酶联免疫阐发(ELISA)

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Nestin酶联免疫阐发(ELISA
试剂盒利用说明书
本试剂仅供研讨利用      目标:本试剂盒用于测定人血清,血浆及相干液体样本中Nestin的含量。
尝试道理:
 本试剂盒使用双抗体夹心法测定标本中人Nestin程度。用纯化的人Nestin抗体包被微孔板,制成固相抗体,往包被单抗的微孔中顺次参加Nestin,再与HRP标识表记标帜的Nestin抗体分离,构成抗体-抗原-酶标抗体复合物,颠末完全洗涤后加底物TMB显色。TMB在HRP酶的催化下转化成蓝色,并在酸的感化下转化成终极的黄色。色彩的深浅和样品中的Nestin呈正相干。用酶标仪在450nm波长下测定吸光度(OD值),经由过程尺度曲线计较样品中人Nestin含量。
老金沙网址
试剂盒构成
48孔设置
96孔设置
保留
说明书
1份
1份
 
封板膜
2片(48)
2片(96)
 
密封袋
1个
1个
 
酶标包被板
1×48
1×96
2-8保留
尺度品:135pg/mL
0.5ml×1瓶
0.5ml×1瓶
2-8保留
尺度品稀释液
1.5ml×1瓶
1.5ml×1瓶5wk金沙备用网址
2-8保留
酶标试剂
3 ml×1瓶
6 ml×1瓶澳门jin2015com
2-8
样品稀释液
3 ml×1瓶
6 ml×1瓶
2-8保留
显色剂A液
3 ml×1瓶
6 ml×1瓶
2-8保留
显色剂B液
3 ml×1瓶
6 ml×1瓶
2-8保留
停止液
3ml×1瓶
6ml×1瓶
2-8保留
稀释洗涤液
(20ml×20倍)×1瓶
(20ml×30倍)×1瓶
2-8保留
样本处置及要求
1. 血清:室温血液天然凝固10-20分钟,离心20分钟阁下(2000-3000转/分)。认真搜集上清,保留过程中如呈现沉淀,应再次离心。
2. 血浆:应按照标本的要求挑选EDTA或柠檬酸钠作为抗凝剂,混淆10-20分钟后,离心20分钟阁下(2000-3000转/分)。认真搜集上清,保留过程中如有沉淀构成,该当再次离心。
3. 尿液:用无菌管搜集,离心20分钟阁下(2000-3000转/分)。认真搜集上清,保留过程中如有沉淀构成,应再次离心。胸腹水、脑脊液参照实施。
4. 细胞培养上清:检测排泄性的成份时,用无菌管搜集。离心20分钟阁下(2000-3000转/分)。认真搜集上清。检测细胞内的成份时,用PBS(PH7.2-7.4)稀释细胞悬液,细胞浓度到达100万/ml阁下。经由过程重复冻融,以使细胞毁坏并放出细胞内成份。离心20分钟阁下(2000-3000转/分)。认真搜集上清。保留过程中如有沉淀构成,应再次离心。
5. 构造标本:切割标本后,称取重量。参加一定量的PBS,PH7.4。用液氮疾速冷冻保留备用。标本熔化后仍旧连结2-8的温度。参加一定量的PBS(PH7.4),用手工或匀浆器将标本匀浆充实。离心20分钟阁下(2000-3000转/分)。认真搜集上清。分装后一份待检测,其他冷冻备用。
6. 标本收罗后尽早停止提取,提取按相干文献停止,提取后应尽快停止尝试。若不能即刻停止实验,可将标本放于-20℃保留,但应制止重复冻融.
7. 不能检测含NaN3的样品,因NaN3抑止辣根过氧化物酶的(HRP)活性。
操纵步调
1.         尺度品的稀释与加样:在酶标包被板上设尺度品孔10孔,在第一、第二孔平分别加尺度品100μl,然后在第一、第二孔中加尺度品稀释液50μl,混匀;然后从第一孔、第二孔中各取100μl别离加到第三孔和第四孔,再在第三、第四孔别离加尺度品稀释液50μl,混匀;然后在第三孔和第四孔中先各取50μl弃掉,再各取50μl别离加到第五、第六孔中,再在第五、第六孔平分别加尺度品稀释液50ul,混匀;混匀后从第五、第六孔中各取50μl别离加到第七、第八孔中,再在第七、第八孔平分别加尺度品稀释液50μl,混匀后从第七、第八孔平分别取50μl加到第九、第十孔中,再在第九第十孔别离加尺度品稀释液50μl,混匀后从第九第十孔中各取50μl弃掉。(稀释后各孔加样量都为50μl,浓度别离为90pg/mL,60pg/mL ,30pg/mL,15pg/mL,7.5 pg/mL)。
2.         加样:别离设空缺孔(空缺比较孔不加样品及酶标试剂,其他各步操纵不异)、待测样品孔。在酶标包被板上待测样品孔中先加样品稀释液40μl,然后再加待测样品10μl(样品终极稀释度为5倍)。加样将样品加于酶标板孔底部,只管不触及孔壁,悄悄晃悠混匀。
3.         温育:用封板膜封板后置37℃温育30分钟。
4.         配液:将30(48T的20倍)倍稀释洗涤液用蒸馏水30(48T的20倍)倍稀释后备用。
5.         洗涤:当心揭掉封板膜,弃去液体,甩干,每孔加满洗涤液,静置30秒后弃去,云云反复5次,拍干。
6.         加酶:每孔参加酶标试剂50μl,空缺孔除外。
7.         温育:操纵同3。
8.         洗涤:操纵同5。
9.         显色:每孔先参加显色剂A50μl,再加入显色剂B50μl,悄悄震动混匀,37℃避光显色15分钟.
10.     停止:每孔加停止液50μl,停止反响(此时蓝色立转黄色)。
11.     测定:以空缺空调zero,450nm波长依序丈量各孔的吸光度(OD值)。 测定应在加停止液后15分钟之内停止。
注意事项:
1. 试剂盒从冷藏情况中掏出应在室温均衡15-30分钟前方可利用,酶标包被板开封后如未用完,板条应装入密封袋中保留。
2. 浓洗涤液可能会有结晶析出,稀释时可在水浴中加温助溶,洗涤时不影响成果。
3. 各步加样均应利用加样器,并常常校正其准确性,以制止实验偏差。一次加样工夫最好掌握在5分钟内,如标本数目多,推荐利用排枪加样。
4. 请每次测定的同时做尺度曲线,最好做复孔。如标本中待测物资含量太高(样本OD值大于尺度品孔第一孔的OD值),请先用样品稀释液稀释必然倍数(n倍)后再测定,计较时请最初乘以总稀释倍数(×n×5)。
5. 封板膜只限一次性利用,以制止穿插净化。
6. 底物请避光保留。
7. 严厉根据说明书的操纵停止,实验成果断定必需以酶标仪读数为准.
8. 所有样品,洗涤液和各类废弃物都应按感染物处置。
9. 本试剂差别批号组分不得混用。
10. 如与英文说明书有异,以英文说明书为准。老金沙网址
文本框:  老金沙网址
以尺度物的浓度为横坐标,OD值为纵坐标,   
在坐标纸上绘出尺度曲线,按照样品的OD     
值由尺度曲线查出响应的浓度;再乘以稀释      
倍数;或用尺度物的浓度与OD值计算出标      
准曲线的直线回归方程式,将样品的OD值      
代入方程式,计算出样品浓度,再乘以稀释      
倍数,即为样品的实践浓度。                  
                                             
(此图仅供参考)
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试剂盒机能:
1.样品线性回归与预期浓度相关系数R值为0.95以上。
2.批内与批间应分别小于9%和11%
 
检测范畴:                                             
5pg/mL -110pg/mL
                                       
                           
保留条件及有效期:
1.试剂盒保留:;2-8
2.有效期:6个月
 

RD
Human Nestin

FOR RESEARCH USE ONLY
 
Drug Names
Generic Name:Human Nestin ELISA Kit.
Purpose
This kit allows for the determination of Nestin concentrations in Human serum, blood plasma, and other biological fluids.
Principle of the assay
The kit assay Human Nestin level in the sample,use Purified Human Nestin antibody to coat microtiter plate wells, make solid-phase antibody, then add Nestin to wells, Combined Nestin 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 Nestin 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:135pg/mL
0.5ml×1 bottle
0.5ml×1 bottle
2-8
Standard diluent
1.5ml×1 bottle
1.5ml×1 bottle
2-8
HRP-Conjugate reagent
3ml×1 bottle
6ml×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
wash solution
(20ml×20 fold)
×1bottle
(20ml×30 fold)
×1bottle
2-8
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 2-8after 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 separately. 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: 90pg/mL,60pg/mL ,30pg/mL,15pg/mL,7.5 pg/mL)
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.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.
 

Take the standard density as the horizontal, the OD value for the vertical ,draw the standard curve on graph paper, Find out the corresponding density according to the sample OD value by the Sample curve, multiplied by the dilution multiple, or calculate the straight line regression equation of the standard curve with the standard density and the OD value ,with the sample OD value in the equation, calculate the sample density, multiplied by the dilution factor, the result is the sample actual density.
Calculate

This chartis for reference only
 

 

 
 
Assay range
5pg/mL -110pg/mL
 
Storage and validity
1.Storage: 2-8℃.
2.validity: six months.
 
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