呋喃西林代谢物单克隆抗体的制备及免疫学特性鉴定

Preparation and Immunological Characterization of Monoclonal Antibody against Furacillin Metabolite

为建立呋喃西林代谢物氨基脲(semicarbazide,SEM)的免疫学检测方法,采用碳化二亚胺法成功合成人工免疫抗原SEM-牛血清白蛋白(bovine serum albumen,BSA)(SEM-BSA),并用该抗原免疫BALB/c小鼠,采用聚乙二醇介导的细胞融合技术制备SEM的单克隆抗体(SEM mAb),采用辛酸-硫酸铵沉淀法纯化单抗,并对其效价、敏感性、特异性和亲和力等免疫学特性进行鉴定。结果表明:成功制备SEM单克隆抗体SEM-3D9,抗体效价达到1∶5×10^6,敏感性半抑制质量浓度为0.42μg/L,亚型为IgG1,亲和常数Ka=2.1×10^8 L/mol,与呋喃唑酮代谢物2-NP-3-氨基-2-恶唑酮、呋喃它酮代谢物2-NP-5-甲基吗啉-3-氨基-2-唑烷基酮类似物的交叉反应率(cross reaction,CR)均小于0.01%,与呋喃妥因代谢物2-NP-1-氨基-2-乙内酰的CR为0.0168%,特异性良好。

In order to establish an immunological method for the detection of the furacillin metabolite semicarbazide(SEM),a conjugate of SEM with bovine serum albumin(BSA)was synthesized by the carbodiimide method and was used as an immunogen to immunize BALB/c mice for the preparation of a monoclonal antibody against SEM(SEM mAb)by polyethylene glycol(PEG)-mediated cell fusion.SEM mAb was purified by caprylic acid-ammonium sulfate precipitation.Finally,the immunological characteristics of the monoclonal antibody including titer,sensitivity,specificity and affinity were identified.The results showed that the monoclonal antibody SEM-3D9 was successfully prepared.The serum titer of immunized mice was 1:5×10^6,the sensitivity IC50 was 0.42μg/L,and the antibody belonged to IgG1 subtype with an affinity constant Ka of 2.1×10^8 L/mol.Its cross-reaction rate with 2-NP-3-amino-2-oxazolidinone and 2-nitrophenyl-5-morpholine-methyl-3-amino-2-oxazolidinone analogues was less than 0.01%,and its cross-reaction rate with 2-nitrophenyl-1-aminohydantoin was 0.0168%,indicating good specificity.