间接酶联免疫吸附法测定大鲵凝集素
An Indirect Non-compe titive Enzyme-linked Immunosorbent Asas y for Determin ation of Andrias davidianus Lectin
王晓龙 1佟长青 1李伟1
作者信息
- 1. 大连海洋大学食品科学与工程学院，辽宁大连116023
- 折叠
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∶16000，多克隆抗体血清的最佳稀释度为1∶3000，辣根过氧化物酶（HRP）标记的羊抗兔二抗最佳稀释度为1∶30000，抗原浓度在31．25～1000．00 ng／mL范围内呈良好的线性关系，相关系数 R2＞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。
Abstract
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%.[Conclu-sion] 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.
关键词
大鲵/凝集素/酶联免疫/多克隆抗体Key words
Andrias davidianus/Lectin/Enzyme-linked immunosorbent assay/Polyclonal antibody引用本文复制引用
基金项目
国家自然科学基金(31571916)
大连海洋大学引进人才及在职培养博士启动项目(100914262)
出版年
2016
国家科技期刊平台
NSTL
维普
万方数据