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1、大鼠极大鼠极低密度脂蛋白低密度脂蛋白（VLDL）酶联免疫分析）酶联免疫分析试剂盒使用说明书试剂盒使用说明书本试剂盒仅供研究使用。检测范围：检测范围： 96T3g/ml -120g/ml使用目的：使用目的：本试剂盒用于测定大鼠血清、血浆及相关液体样本中极低密度脂蛋白（VLDL）含量。实验原理实验原理本试剂盒应用双抗体夹心法测定标本中大鼠极低密度脂蛋白（VLDL）水平。用纯化的大鼠极低密度脂蛋白（VLDL）抗体包被微孔板，制成固相抗体，往包被单抗的微孔中依次加入极低密度脂蛋白（VLDL），再与 HRP 标记的极低密度脂蛋白（VLDL）抗体结合，形成抗体-抗原-酶标抗体复合物，经过彻底洗涤后加底物
2、TMB 显色。TMB 在 HRP 酶的催化下转化成蓝色，并在酸的作用下转化成最终的黄色。颜色的深浅和样品中的极低密度脂蛋白（VLDL）呈正相关。用酶标仪在 450nm 波长下测定吸光度（OD 值），通过标准曲线计算样品中大鼠极低密度脂蛋白（VLDL）浓度。试剂盒组成试剂盒组成 以标准物的浓度为横坐标，OD 值为纵坐标，在坐标纸上绘出标准曲线，根据样品的 OD 值由标准曲线查出相应的浓度；再乘以稀释倍数；或用标准物的浓度与 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，即为样品的实际浓度。注意事项注意事项1试剂盒从冷藏环境中取出应在室温平衡
3、 15-30 分钟后方可使用，酶标包被板开封后如未用完，板条应装入密封袋中保存。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，洗涤时不影响结果。3各步加样均应使用加样器，并经常校对其准确性，以避免试验误差。一次加样时间最好控制在 5 分钟内，如标本数量多，推荐使用排枪加样。4 请每次测定的同时做标准曲线，最好做复孔。如标本中待测物质含量过高（样本 OD 值大于标准品孔第一孔的 OD 值），请先用样品稀释液稀释一定倍数（n 倍）后再测定，计算时请最后乘以总稀释倍数（n5）。5 封板膜只限一次性使用，以避免交叉污染。6底物请避光保存。7严格按照说明书的操作进行，试验结果判定必须以酶标仪读数
4、为准.8所有样品，洗涤液和各种废弃物都应按传染物处理。9本试剂不同批号组分不得混用。10. 如与英文说明书有异，以英文说明书为准。保存条件及有效期保存条件及有效期1试剂盒保存：；2-8。2有效期：6 个月RDRat VLDLFOR RESEARCH USE ONLYAssay range：3g/mL 120g/mL 96determinationsPurposeThis kit allows for the determination ofVLDL concentrations in Ratserum, cellculture supernates and other biological f
5、luidsPrinciple of the assayThe kit assay RatVLDLlevel in the sample，use Purified RatVLDLantibody to coat microtiter plate wells, make solid-phase antibody, then addVLDLto wells,CombinedVLDL antibody which With HRP labeled,become antibody - antigen - enzyme-antibody complex, after washing Completely,
6、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 RatVLDLin the samples is then determined by
7、comparing the O.D. of the samples to the standard curve.Materials provided with the kit1wash solution20ml1bottle7Stopp Solution6ml1 bottle2HRP-Conjugate reagent6ml1 bottle8Standard（240g/ml）0.5ml1 bottle3Microelisa stripplate12well8strips9Standard diluent1.5ml1bottle4Sample diluent6ml1 bottle10Instru
8、ction15Chromogen Solution A6ml1 bottle11Closure plate membrane26Chromogen Solution B6ml1 bottle12Sealed bags1Specimen requirements1. extractas 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
9、 cant, specimen can be kept in -20 to preserve, Avoid repeated freeze-thaw cycles.2. Cant detect the sample which contain NaN3, because NaN3 inhibits HRP active.Assay procedure1. Dilute and add sample:Dilute Original density Standard as follow table:120g/ml5 Standard150l Original density Standard+15
10、0l Standard diluent80g/mL4 Standard150l 5 Standard+150l Standard diluent40g/mL3 Standard150l 4 Standard+150l Standard diluent20g/mL2 Standard150l 3 Standard +150l Standard diluent10g/mL1 Standard150l 2 Standard +150l Standard diluent2.add sample：Set blank wells separately (blank comparison wells don
11、t add sample and HRP-Conjugate reagent, other each step operation is same). testing sample well. add Sample dilution 40l to testing sample well, then add testing sample 10l (sample final dilution is 5-fold), add sample to wells , dont touch the well wall as far as possible, and Gently mix.3.Incubate
12、: 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, discardLiquid, dry by swing, add washing buffer to every well,
13、still for 30s then drain,repeat 5 times, dry by pat.6.add enzyme：Add HRP-Conjugate reagent 50l 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
14、at 3710.Stop the reaction：Add Stop Solution50l 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.Steps descriptionStandard, Sample diluentAddStandard, Sample diluent, incubate
15、 for 30 min at 37.Wash 5 time,AddHRP-Conjugate reagent, incubate for 30 min at 37.Wash 5 times,Add Chromogen Solution A and B, incubate for 30 min at 37.AddStopp SolutionRead absorbance at 450nm within 15 mincalculateCalculateTake the standard density as the horizontal, the OD value for the vertical
16、 ,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
