新霉素免疫亲和柱的制备及性能鉴定

Preparation and characterization of an immunoaffinity column for the selective extraction of neomycin

中文关键词:  新霉素  免疫亲和柱  高效液相色谱法

英文关键词:Neomycin  Immunoaffinity Column  High Performance Liquid Chromatography

中文摘要:

      目的 制备新霉素多克隆抗体免疫亲和柱并鉴定其性能。方法 将经过蛋白A柱纯化的新霉素多克隆抗体, 非定向偶联于sepharose 4B琼脂糖凝胶, 间接竞争ELISA方法检测抗体特异性、亲和常数及纯化前后效价; 通过考马斯亮蓝法测定偶联前后蛋白浓度计算偶联率, 高效液相色谱方法(HPLC)筛选亲和柱最优洗脱剂; 将免疫亲和萃取技术与HPLC以离线方式联用测定南美白对虾中的新霉素。结果 抗体纯化后效价提高5.67倍, 亲和常数为2.02×108 L/mol; 在4 ℃、6 h条件下可实现充分偶联; HPLC法筛选亲和柱最优洗脱剂为0.2 mol/L柠檬酸盐缓冲液(含0.5 mol/L NaCl, pH 2.3), 平均回收率为104.57%±0.03%(n=6); 重复使用5次, 柱容量为初始柱容量的85%以上; 储存于含有0.02%NaN3的PBS平衡缓冲液中, 110 d后800 ng加标回收率在75%以上; 分别按照500、5000、7500 μg /kg的浓度对南美白对虾进行加标, 加标回收率依次为63.29%±5.30%、80.15%±6.27%、83.87%±0.88%; 单个样本免疫萃取柱净化前处理时间较固相萃取缩短2~4 h。结论 制备所得的亲和柱准确性高、重现性好, 在食品安全检测中具有实际应用的潜力。

英文摘要:

      Objective To prepare and characterize an immune affinity column for the selective extraction of neomycin. Methods The immune affinity column of neomycin (NEO) was prepared by coupling CNBr-activated Sepharose-4B with the purified anti-NEO polyclonal antibody. An indirect competitive ELISA method was established to detect the specificity, affinity constant and titer of antibody. The changes of protein concentrations were determined by Coomassie brilliant blue method. HPLC method was used to explore the optimal elute condition. A rapid and reliable immune affinity column (IAC) clean-up based high performance liquid chromatography (HPLC) method was developed for the determination of neomycin (NEO) in Penaeus vannamei Boone products. Results The titer of the lgG was improved 5.67 times than before and the affinity constant (Ka) of lgG was about 2.02×108 L/mol. The optimal coupling conditions were at 4 ℃ coupling 6 h. The immunoaffinity column could be eluted with 5 mL 0.2 mol/L Citrate buffer (0.5 mol/L NaCl, pH 2.3). The average recoveries were 104.57%±0.03% (n=6) and the rate of recovery of neomycin was more than 85% after repeatedly for 5 times. After storage at 4 ℃ for 110 d, the rate of recovery to neomycin was more than 75% after loaded NEO for 800 ng. The average recoveries of NEO in spiked Penaeus vannamei Boone under levels of 500, 5000 and 7500 μg/kg ranged from 63.29% to 83.87% with the relative standard deviations of 0.88%~6.27%. Compared with solid phase extraction, immunoaffinity extraction saved 2~4 hours. Conclusion The preparation neomycin affinity column has the advantages of high accuracy and good reproducibility, which has the potential application value in food safety testing.