摘要
[目的]建立基于大鲵凝集素多克隆抗体酶联免疫定量分析的方法。[方法]以大鲵凝集素为抗原,免疫新西兰白兔获得多克隆抗体,建立间接非竞争酶联免疫检测方法,并对该方法的精密度及准确度进行了测试。[结果]获得的抗血清效价为1∶16 000,多克隆抗体血清的最佳稀释度为1∶3 000,辣根过氧化物酶(HRP)标记的羊抗兔二抗最佳稀释度为1∶30 000,抗原浓度在31.25~1 000.00 ng/m L范围内呈良好的线性关系,相关系数R^2>0.99,板内及板间变异系数范围分别为2.50%~9.88%和2.69%~11.59%,回收率为91.5%~109.5%。分布结果显示,大鲵凝集素在躯干部肌肉组织中的含量较高。[结论]成功建立了大鲵凝集素间接非竞争酶联免疫检测方法,该方法灵敏度高,重复性好,可用于实际检测大鲵肌肉组织中的凝集素含量。
[Objective] To prepare polyclonal antibody against the content of lectin in skin mucous of Andrias davidianus and develop an indirect non-competitive enzyme-linked immunosorbent assay （ELISA）.[Method] Andrias davidianus lectin was used as complete antigen.The New Zeal-and white rabbits were immunized according to the immunization procedure .Finally through the optimization of coating amount of lectin and anti-body dilution,an indirect non-competitive enzyme-linked immunosorbent assay was developed .[ Result] The titer of the antiserum determined by ELISA was up to 1∶16 000.Determined polyclonal antibody diluted was 1∶3 000.The best working dilution of horseradish peroxidase-conjugated affinipure goat anti rabbit IgG was 1∶30 000.The linear range of the method was 31.25-1 000.00 ng/mL （R2 〉0.99）.The variation coefficients of intra -assay and inter-assay were 2.50%-9.88% and 2.69%-11.59%,respectively.Recoveries ranged from 91.5% to 109.5%.[Conclusion] A rapid,sensitive and repeatable indirect non-competitive ELISA detection method for An drias davidianus lectin allergens was established successfully.This method is suitable for qualitative determination of Andrias davidianus lectin.
出处
《安徽农业科学》
CAS
2016年第33期74-76,102,共4页
Journal of Anhui Agricultural Sciences
基金
国家自然基金项目(31571916)
大连海洋大学引进人才及在职培养博士启动项目(100914262)
关键词
大鲵
凝集素
酶联免疫
多克隆抗体
Andrias davidianus
Lectin
Enzyme-linked immunosorbent assay
Polyclonal antibody
