呋喃妥因残留代谢物人工抗原的合成与鉴定

PREPARATION AND CHARACTERIZATION OF NITROFURANTOIN METABOLITE ANTIGEN COUPLED TO BOVINE SERUM ALBUMIN

利用对醛基苯甲酸对呋喃妥因代谢物1-氨基乙内酰脲(1-Aminohydantoin hydrochloride,AHD)进行衍生化,衍生物1-氨基乙内酰脲-4-羧苯基肟(CPAHD)通过混合酸酐法与牛血清蛋白(bovine serum albumin,BSA)偶联,用紫外扫描(UV)和变性凝胶电泳(SDS-PAGE)对偶联物进行验证。将偶联成功的人工全抗原(CPAHD-BSA)免疫BALB/c小鼠,通过间接酶联免疫吸附法(ELISA)测定小鼠血清抗体效价为1:32 000。用间接竞争ELISA鉴定抗体特性,结果显示:抗体对AHD与对硝基苯甲醛的衍生物(NPAHD)的灵敏度高,IC50为30.63μg/L,特异性好,与CPAHD有部分交叉反应外,与其他3类硝基呋喃类药物及代谢产物无交叉反应(交叉反应率<0.01%)。结果表明,用CPAHD-BSA作免疫原免疫小鼠可诱导产生针对AHD衍生物(NPAHD)的特异性抗体,为进一步制备AHD单克隆抗体和研制快速筛选检测试剂盒奠定了基础。

The metabolite l-aminohydantoin hydrochloride （AHD） of nitrofiarantoin was coupled to bovine serum albumin （BSA） with the presence of 4-formylbenzoic acid. Success of the coupled antigen （CPAHD-BSA） was confirmed via ultraviolet （UV） and degenerative gel electrophoresis （SDS-PAGE）. The CPAHD-BSA was injected to BALB/c mice for the preparation of polyclonal antibody. The mouse antiserum was titrated to be 1：32 000 in indirect ELISA. The mouse antibody was highly sensitive to p-nitrobenzaldehyde derivative ofAHD （NPAHD） and the IC50 of NPAHD was 30.63 txg/L. The specialty of mouse antiserum was determined in competitive ELISA. Except CPAHD, no cross reaction was found between mouse antiserum and other nitrofiaran drugs and their metabolites. The preparation of coupled antigen CPAHD-BSA and specific antibody to AHD derivative （NPAHD） was of importance for generation of monoclonal antibody used for developing AHD rapid testing kit.