为了快速高效检测食品中胭脂红酸含量，本研究通过羟基二咪唑法制备了胭脂红酸人工抗原；通过免疫新西兰大耳白兔获得了胭脂红酸抗血清，经硫酸铵沉淀法分离纯化获得抗胭脂红酸多克隆抗体；利用棋盘滴定法检测了抗体效价，并探究了溶液pH值、离子强度和有机溶剂对ELISA反应的影响，优化出最佳ELISA反应条件；在最佳反应条件下，建立了胭脂红酸的间接竞争ELISA抑制曲线。紫外光谱扫描结果显示，免疫抗原（CA-BSA）和检测抗原(CA-OVA)均与载体蛋白成功偶联；经分离纯化获得的胭脂红酸多克隆抗体效价为1:16000；检测抗原最佳浓度为1 μg/mL、ELISA反应最适缓冲液为pH 7.4、含50 mmol/L Na+且无甲醇的磷酸盐缓冲液；间接竞争ELISA标准曲线的线性范围为7.8~1150 ng/mL，检测限为1 ng/mL，灵敏度IC50值为95.2 ng/mL。本研究为开发CA快速检测试剂盒对食品中胭脂红酸含量的大量快速测定奠定了基础。

In order to detect rapidly and efficiently the content of carminic acid (CA) in food, the artificial antigen of CA was prepared by hydroxydiimidazole method; A high-titer polyclonal antibody (Pab) against CA was obtained through immunizing New Zealand white rabbits to obtain antiserum (against CA) for further separation and purification by ammonium sulfate precipitation. The titer of the antibody was tested by checkerboard titration, and the effects of factors such as solution pH, ionic strength, and organic solvent were investigated to optimize the conditions of the enzyme-linked immunosorbent assay (ELISA) reaction. Under the optimal conditions, an indirect competitive ELISA inhibition curve for CA was established. Ultraviolet (UV) spectroscopy analysis showed that both the immunogenic antigen (CA-BSA) and coating antigen (CA-OVA) were successfully conjugated with carrier protein. The titer of the Anti-CA polyclonal antibody obtained after separation and purification was 1:16000; The optimal concentration of the detection antigen was 1 μg/mL, the optimum buffer for ELISA reaction was the phosphate buffer at pH 7.4 containing 50 mmol/L Na+ but no methanol. The linear range of the indirect competitive ELISA standard curve was 7.8~1150 ng/mL, with the limit of detection (LOD) as 1 ng/mL, and the sensitivity IC50 was 95.2 ng/mL. This study has laid the foundation for the development of a rapid CA rapid test kit for the determination of CA in food.

武汉市黄鹤英才计划（2014）