抗心型脂肪酸结合蛋白单克隆抗体制备、鉴定和在侧向免疫层析方法中的应用

目的:制备抗心型脂肪酸结合蛋白(H-FABP)单克隆抗体(mAb),并建立侧向免疫层析方法检测血浆中H-FABP。方法:用H-FABP蛋白免疫纯系Balb/c小鼠,采用杂交瘤技术建立能稳定分泌抗人H-FABP的单克隆杂交瘤细胞株。常规制备腹水,纯化后得到特异性抗H-FABP单克隆抗体,进行效价、特异性、亲和力的鉴定分析,并在ELISA平台进行抗体配对,用所筛选到的抗体对初步建立了检测H-FABP的侧向免疫层析方法。结果:成功获得12株稳定分泌抗体的阳性细胞株,并筛选出能相互配对,并应用于侧向免疫层析平台的抗体3D1和5F4,检测临床样品与对照试剂比较总符合率为100%。结论:筛选能稳定分泌抗体的细胞株,配对抗体应用于侧向免疫层析检测方法中,能快速、特异、灵敏的检测出临床样品中H-FABP,为临床应用快速检测H-FABP指标提供了方法和关键材料。

Development and Characterization of Monoclonal Antibodies Against Human Heart Type Fatty Acid Binding Protein and Its Application in Lateral Flow Immunoassay

AIM: To develop and characterize monoclonal antibodies (mAb) against human heart type fatty acid binding protein (H-FABP); To develop a rapid lateral flow immunoassay for detecting H-FABP in plasma using the identified mAbs. Methods: BALB/c mice were immunized with H-FABP. The hybridoma cells were generated by fusing spleen cells and myeloma cells SP2/0 followed by screening and clonally selecting. The mAbs were purified from ascites and characterized by indirect ELISA and Western Blot, and were used for developing a rapid lateral flow immunoassay.Results: Twelve hybridoma cell lines were found to secret anti-H-FABP with high titers, specificity and affinity. Among them, the antibody pair of 3D1 and 5F4 were used as conjugate and coating antibody, respectively, in lateral flow immunoassay. The newly developed assay was used to detect 203 patients plasma. A 100% consistence was observed when compared with the reference kit.Conclusion: Two mAbs against H-FABP with high titers, specificity and affinity were obtained. The rapid lateral flow immunoassay was successfully developed for sensitive and specific detection of H-FABP in human plasma by using these mAbs.