阿莫西林人工抗原的合成及其单克隆抗体的制备

Synthesis of artificial antigens of amoxicillin and preparation of monoclonal antibody to amoxicillin

目的:合成阿莫西林(AMX)完全抗原,获得稳定、高效分泌抗AMX单克隆抗体(mAb)的杂交瘤细胞株。方法:采用N-羟基琥珀酰亚胺活性酯(NHS)法制备AMX-OVA(免疫原)AMX-BSA(检测抗原),紫外光谱分析检测偶联物克分子比,以AMX-OVA免疫BALB/c小鼠,用细胞融合技术筛选分泌抗AMX mAb的杂交瘤细胞,体内诱生法制备腹水,辛酸-硫酸胺法从腹水中纯化mAb,ELISA法测定mAb亚类。结果:紫外光谱显示偶联物表现出小分子与蛋白载体叠加的特征,具有良好的免疫原性和反应原性。获得了5株可稳定分泌特异性抗AMX mAb的杂交瘤细胞株,亚类鉴定均为IgG1。mAb对AMX最小检测极限(limit of detection,LOD)为0.25 ng/mL,但与同属β-内酰胺类抗生素的氨苄青霉素有一定程度的交叉反应。结论:该细胞株可满足建立免疫学检测方法的需要和开发应用的要求。

AIM：To synthesize two kinds of amoxicillin artificial antigen and to establish hybridoma cell lines secreting monoclonal antibody against amoxillin.METHODS：Amoxicillin were respectively coupled with ovalbumin（OVA） and bovine serum albumin（BSA） by using N-hydroxysuc-cinimide active ester（NHS） method to prepared the immunogen and detection antigen.Molar ratio of conjugates were detected by ultraviolet spectrum analysis.BALB/c mice was immunized with AMX-OVA.The hybridomas were obtained by fusing mouse myeloma cells Sp2/0 with splenocytes from the mice immunized with AMX-OVA.The aseites with mAb were purified by octanoic acid-saturated ammonium sulphate method.Isotype was determinated by Mouse Isotype kit（Bio-RadLabs.）.RESULTS：UV spectra shows overlay features between a small molecular compound and carrier protein.Five hybridoma cell strains secreting specific mAb against amoxicillin were obtained and the five Amb belong to IgG1 kappa isotype.The limit of detection of AMX was 0.25 ng/mL and but there is mild degree of cross-reactions with ampicillin with same structure of β-lactam ring.CONCLUSION：The anti-AMX Amb could be used to develop immunoassay for detection of residues.