4-甲基苯酚多克隆抗体的制备及应用

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%。

Preparation and application of polyclonal antibody against 4-methylphenol

Based on the structure of 4-methylphenol(4-MP) and the design principle of hapten,3-(4-Hydroxyphenyl)propionic acid(34-HPA) was selected as the hapten of 4-MP.34-HPA was coupled with bovine serum albumin(BSA) to prepare complete antigen 34-HPA-BSA and immunized 3~4 months old rabbits.The titer and identification of the polyclonal antibodies against 4-MP were characterized with indirect enzyme-linked immunosorbent assay(iELISA) which reached 1∶204 800.Indirect competitive ELISA(icELISA) was developed using 34-HPA coupled with ovalbmin(OVA) as the original coating.The results showed the best detection conditions for icELISA:the coating antigen 34-HPA-OVA concentration was 1.05 μg/mL,the antibodies 12 800 times diluted,0.1% gelatin solution blocking 1.5 h,competitive time was 1.0 h,horse radish peroxidase(HRP) labeled goat anti-rabbit IgG diluted 2 000 times and incubated 1.0 h.The linear range of icELISA to detect 4-MP was δ1.0×10-2~1.0×103 and the regression equation is y=-12.76x+66.66.The detection limit was δ0.02,the intra-assay and inter-assay coefficient of standard variation is 7.0% and 8.4%.