双抗夹心测蛋白实验的分析与改进

Analysis and Improvement of Protein Detection Test by Double Antibodies Sandwich

[目的]探索ELISA双抗夹心法的最优实验条件,充分发挥其高灵敏、强特异的优点。[方法]从操作的主要步骤出发,在标准品和溶液的配制以及温育等方面进行对比分析。[结果]合格标准品标准曲线的斜率在1.70～2.00,R2在0.99以上;样品稀释浓度的最佳临界值为60ng/ml。[结论]ELISA是个很敏感的试验,在操作过程中,标准品应在-80℃冷冻保存,配制时应避免偏高、偏低或没有梯度,加样量要仔细,应控制为100μl每孔。谨慎选择酶标板,确保吸附性能好、空白值低、孔底透明度高,各板之间和同一板各孔之间性能相近。洗涤浸泡时间确保为3min。

[Objective] To search the optimal conditions of Double antibodies sandwich ELISA and enhances its reliability and accuracy.[Methods] The preparation of standard substances and solutions and incubation et al.were analysed and compared.[Results] The slope of standard curve of qualified standard substances is 1.70-2.00,with R2 more than 0.99;the optimal critical value of sample diluted concentrationwas 60 ng/ml.[Conclusion] ELISA is a sensitive experiments test.During the process,the standard substance,whose concentrations should not be too high,too low or with no gradients,should be preserved at-80 ℃.The sampling volume should be controlled at 100 μl/hole.The ELISA plates must be selected carefully,to ensure good adsorption capacity,low blank value,high transparency and similar performance of plates and holes.The washing duration should be longer than 3 min.