Preparation and Characterization of Olaquindox Polyclonal Antibody
Preparation and Characterization of Olaquindox Polyclonal Antibody
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DOI：
10.1002/cjoc.200990170
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发表时间：
2009-05
影响因子：
5.4
通讯作者：
H. Che;Baoxian Shi;Jinlan Chen;Xue-yong Zhu;Jiguo He
中科院分区：
文献类型：
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作者：
H. Che;Baoxian Shi;Jinlan Chen;Xue-yong Zhu;Jiguo He
2-[N-（2-羟乙基）-氨甲酰基]-3-甲基-氧代喹喔啉-1，4-氧化物（奥拉）先与丁二酸酐发生单酯化反应，再经羧基化得到衍生物。合成产物经重结晶纯化，产率为52.47%。其化学结构经核磁共振谱、红外光谱和质谱确证。利用确定的结构合成了具有奥拉分子结构特征的奥拉半抗原。采用活化酯法将奥拉半抗原与牛血清白蛋白（BSA）偶联，采用混合酸酐法将半抗原与卵清蛋白（OVA）偶联。紫外扫描和红外光谱分析结果表明，半抗原和载体蛋白分别与BSA和OVA偶联成功，偶联比分别为3.8：1和5：1。以OLA-BSA为免疫原，免疫新西兰白色家兔4只，获得了较高效价的抗血清。以OLA-OVA为包被抗原，经间接ELISA测定，效价分别为1：6400、1：1600、1：12800和1：6400。奥拉在间接ELISA试验中的中间抑制浓度（IC 50）为743.3 ng/mL。最低检测限（IC 20）为5.71 ng/mL。通过饱和硫酸铵柱层析和离子交换柱层析，获得了高纯度的奥拉多克隆抗体。
2-[N-(2-Hydroxyethyl)-carbamoyl]-3-methyl-oxoquinoxaline-1,4-oxides (OLA) was first mono-esterificated by succinic acid anhydride, and was then turned into a derivative with carboxyl. The synthetic product was purified by recrystallization with a yield of 52.47%. Its chemical structure was determined by NMR, IR and MS spectra. The determined structure was used to synthese OLA hapten, which had the molecular structural characteristics of OLA. The OLA hapten and bovine serum albumin (BSA) were conjugated using the activated ester method, while the hapten and ovalbumin (OVA) were coupled by the mixed anhydride method. The UV scanning and infrared spectrum results showed that the hapten and the carrier protein were successfully coupled to BSA and OVA, with the combined ratios of 3.8:1 and 5:1, respectively. OLA-BSA was used as the immunogen to immunize four New Zealand white rabbits, and the high titer anti-serum produced relatively. The titers were 1:6400, 1:1600, 1:12800 and 1:6400, respectively, determined by indirect ELISA OLA-OVA as the coating antigen. The intermediate inhibition concentration (IC50) of OLA in the indirect ELISA test was 743.3 ng/mL. The lowest detection limit (IC20) was 5.71 ng/mL. High-purity OLA polyclonal antibody has been obtained by saturated ammonium sulfate and ion-exchange chromatography.