呋喃妥因残留代谢物人工抗原的合成与鉴定

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。

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

The metabolite 1-aminohydantoin hydrochloride(AHD) of nitrofurantoin 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 of AHD(NPAHD) and the IC50 of NPAHD was 30.63 μg/L.The specialty of mouse antiserum was determined in competitive ELISA.Except CPAHD,no cross reaction was found between mouse antiserum and other nitrofuran 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.