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针对4-甲基苯酚(4-MP)的结构特点及半抗原设计原则,选择对羟基苯丙酸(34-HPA)作为半抗原,通过活化酯法与牛血清白蛋白(BSA)偶联合成人工免疫原,免疫3～4 m的雄性大白兔,制得多克隆抗体效价为1∶204 800。同时采用混合酸酐法合成半抗原-卵清蛋白(OVA)偶联物作为包被原,以4-MP为竞争的抗原,建立间接竞争ELISA检测方法。实验结果表明,理想工作条件为:包被原浓度1.05μg/mL,抗体稀释倍数1∶12 800,酶标二抗稀释倍数1∶1 000,0.1%明胶封闭1.5 h,竞争时间与酶标二抗反应时间均为1.0 h。4-甲基苯酚在δ1.0×10-2～1.0×103的范围内呈线性相关,得标准曲线方程y=-12.76x+66.66,最低检测限δ0.02,板内差异为7.0%,板间差异为8.4%。
According to the structural characteristics of 4-methylphenol (4-MP) and the design principle of hapten, p-hydroxyphenylpropionic acid (34-HPA) was selected as a hapten and coupled with bovine serum albumin (BSA) Artificial immunogen, immune 3 ~ 4 m of the male rabbits, polyclonal antibody titer 1:20 800. At the same time, the mixed anhydride method was used to synthesize the hapten-ovalbumin (OVA) conjugate as a coated antigen, and the indirect competitive ELISA was established by using 4-MP as antigen. The experimental results showed that the ideal working conditions were as follows: the original concentration of coating was 1.05μg / mL, the dilution of antibody was 1:12 800, the dilution of enzyme secondary antibody was 1: 1 000, 0.1% gelatin was blocked for 1.5 h, Anti-reaction time are 1.0 h. 4-methylphenol in the range of 1.0 × 10-2 ~ 1.0 × 103 linearly correlated with the standard curve equation y = -12.76x +66.66, the minimum detection limit δ0.02, the difference between the plate was 7.0% The difference between the plates was 8.4%.