基于抗分析物\抗吖啶酯双特异性抗体的化学发光标记免疫分析方法研究

Immunoassay is an important method in food safety detection. Nowadays, its sensitivity is confronted with more demanding requirments. Chemiluminescent immunoassay (CLIA) is a sensitive immunoassay by conjugating a antibody or a antigen with acridinium esters. But the average quantity of acridinium esters conjugated to the antibody can not be more than 2.8. Otherwise, the antibody is apt to be denatured. Furthermore, the conjugating reaction is complicated and lacking repeatability. Based on the acquired monoclonal antibodies against two model analytes Sudan Red and BSA, this project is to develop a high affinity monoclonal antibodies against the acridinium ester, and further develop two bispecific monoclonal antibody against the model analyte (Sudan RedⅠor BSA) and the acridinium ester by hybridoma techniques. Meanwhile, a polymerized acridinium ester (n>10) will be synthesized to improve the chemiluminescent efficiency. Then, two model CLIA methods (competitive CLIA and sandwich CLIA) will be established on the two bispecific antibodies and the polymerized acridinium esters (such research has not been reported by now). The new methods aimed at improving the sensitivity of CLIA. In this project, systematic analysis work will be done on sensitivity, accuracy, and precision of the two CLIA methods. Those methods will also be compared with traditional CLIA methods. The target of this project is to exploring sensitive, stable, and efficient CLIA methods meeting the demands of modern food safety detection.

免疫分析在食品安全检测中发挥着重要作用，其灵敏度面临着越来越高的要求。化学发光标记免疫分析（chemiluminescent immunoassay，CLIA）是用化学发光剂（吖啶酯）偶联标记抗原或抗体的免疫分析方法，具有较高的灵敏度。但在CLIA中，抗体上偶联标记的吖啶酯数量不能太高（n<2.8），否则易造成抗体失活；且标记反应操作繁琐、重复性不好。本研究拟在已获苏丹红Ⅰ和BSA二种分析物单抗的基础上，制备抗吖啶酯的高亲和力单抗，再通过四瘤杂交法，研制二种抗分析物/抗吖啶酯的双特异性抗体，研究建立无需偶联标记抗体的竞争CLIA方法和夹心CLIA方法；同时，通过合成多聚吖啶酯（n>10）增加发光强度，以提高CLIA方法的灵敏度(该研究未见报道)。本课题将对这二种方法的检测限、精密度、准确度和稳定性进行系统分析，并与传统CLIA方法进行比较研究，探索灵敏度高、稳定性强的免疫分析新方法。