摘要
目的制备能够识别多种β-内酰胺酶结构的广谱特异性抗体,并初步建立用于检测牛奶中违法添加β-内酰胺酶的间接竞争酶联免疫分析方法(ELISA)。方法以混合β-内酰胺酶作为全抗原,免疫动物获得β-内酰胺酶广谱特异性多克隆抗体,分离纯化后进行详细表征,确定最佳使用条件,并开发适用于牛奶样品中β-内酰胺酶检测的免疫分析方法。结果获得了能够识别多种β-内酰胺酶的多克隆抗体,血清中抗体浓度达到17.9～18.9mg/ml,抗体重链和轻链分子量分别为55和25kD,总分子量为160kD,亲和常数为3.6×108L/mol,以该抗体为基础建立了检测β-内酰胺酶的间接竞争ELISA法,方法检出限为0.2ng/ml,线性范围0.2～200ng/ml,平均回收率在80%～115%之间,相对标准偏差小于20%。结论获得了β-内酰胺酶多克隆抗体,并建立了间接竞争ELISA方法测定牛奶中β-内酰胺酶含量。
Objective To produce polyclonal antibody with affinity against several types of β-lactamase and develop an indirect competitive ELISA for detection of β-lactamase in milk.Methods A mixture of β-lactamase was used as immunogen for animal immunization to produce polyclonal antibody.After purification and characterization,the obtained antibody was employed to establish an ELISA method for β-lactamase determination.Results The polyclonal antibody was acquired,with the concentration of 17.9-18.9mg/ml in antiserum.The molecular weights were 55,25 and 160kD for heavy chain,light chain and the whole antibody,respectively.The developed ELISA method showed the LOD 0.2ng/ml and the linear range of 0.2-200ng/ml.The recoveries ranged from 80% to 115%,with RSD bellow 20%.Conclusion With the polyclonal antibody,the proposed indirect competitive ELISA method was successfully applied to determin β-lactamase in milk.
出处
《卫生研究》
CAS
CSCD
北大核心
2013年第3期386-391,共6页
Journal of Hygiene Research
基金
国家自然科学基金(No.81001248)
