呋喃妥因代谢物AHD单抗的制备及鉴定

Preparation and identification of monoclonal antibody to the derivative of 1-Amino-hydantoin( AHD)

将呋喃妥因代谢物1-氨基-乙内酰胺脲(AHD)的衍生物1-氨基乙内酰脲-4-羧苯基肟(CPAHD)分别与牛血清蛋白(BSA)和Jeffamine修饰的卵清蛋白(OVA)偶联,经紫外光谱扫描鉴定,成功合成了免疫抗原CPAHD-BSA和包被抗原CPAHD-Jeffamine-OVA。应用杂交瘤技术将免疫鼠脾细胞与骨髓瘤细胞SP2/0融合,获得了稳定分泌抗4-NPAHD单克隆抗体的杂交瘤细胞株,命名为B10E7。使用本细胞株制备的McAb对4-NPAHD的半数抑制质量浓度(IC50)为3.65 ng/mL,与同类抗生素及其代谢物未发现交叉反应,显示高度的灵敏性和特异性。本抗体的研制为建立间接竞争酶联免疫方法奠定了基础。

In this study,CPAHD,which was the derivative of nitrofurantoin metabolite 1- Amino- hydantoin（ AHD）,was conjugated with proteins BSA and chemically modified Jeffamine- OVA,respectively. Then the compounds were identified by UV spectra scanning methods. The results indicated that CPAHD was conjugated with BSA and Jeffamine- OVA successfully. The spleen cells and the myeloma cells were fused by hybridoma technology. One hybridoma which produced antibody steadily from the well of B10E7 was characterized by in- direct ELISA method. It was found that the IC 50 value of the monoclonal antibody for 4- NPAHD was 3. 65 ng / mL. The results of cross-reac- tivity showed that there was no cross reactivity（ CR 0. 01%） with other testing compounds except CPAHD and nitrofurantoin. The prepara- tion of the mo-noclonal antibody to 4- NPAHD will apply a basis for establishing the methods of indirect competitive ELISA.