Deng Qi, Qiu Mei, Wang Yaling, Lv Pengli, Wu Chaojin, Sun Lijun, Ye Riying, Xu Defeng, Liu Ying, Gooneratne Ravi
College of Food Science and Technology, Guangdong Ocean University, Guangdong Provincial Key Laboratory of Aquatic Product Processing and Safety, Key Laboratory of Advanced Processing of Aquatic Products of Guangdong Higher Education Institution, Zhanjiang 524088, China.
College of Food Science and Technology, Guangdong Ocean University, Guangdong Provincial Key Laboratory of Aquatic Product Processing and Safety, Key Laboratory of Advanced Processing of Aquatic Products of Guangdong Higher Education Institution, Zhanjiang 524088, China; National Marine Products Quality Supervision and Inspection Centre, Zhanjiang 524096, China.
Ecotoxicol Environ Saf. 2017 Aug;142:441-447. doi: 10.1016/j.ecoenv.2017.04.037.
Accurate analyses of total T-2 (free and modified) in aquatic organisms including shrimp are important as the hazard caused by T-2 has been caught increasing attention. Therefore, acurate analysis of free T-2 especially of modified T-2 in shrimp tissues is important. A rapid, sensitive, and validated method for quantitative determination of free T-2 and modified T-2 toxin was developed using immunomagnetic-bead based enzyme-linked immunosorbent assay (IMB-ELISA). Super paramagnetic particles with a carboxyl group activated by an ester method coupled with envelope antigen 3- acetylneosolaniol- hemisuccinate - ovalbumin (3-Ac-NEOS-HS-OVA) was used to form immunomagnetic beads which could bind to T-2 skeletal structure antibodies. The conditions for magnetic bead coating of T-2 skeletal structure antibodies, and the concentrations of the polyclonal antibody and HRP-labeled goat anti-rabbit antibody were optimized. A good linear relationship with T-2 concentrations ranging from 5-75ng/mL (R =0.9965) was observed. The detection limit of different shrimp tissues of the IMB-ELISA ranged from 2.53 to 3.20ng/mL. And the IC was 63ng/mL. The recovery varied from 86% to 99% with a standard deviation of 2.8-5.8%. The application of this method to study the distribution in tissues showed that the total T-2 concentration in hepatopancreas was 26.7µg/kg > blood > head > muscle in the highest dose group of 12.2mg/kg. Our research showed a combination of ELISA and immunomagnetic bead technology provide a new, convenient approach to significantly improve the accuracy and sensitivity of total T-2 measurement in shrimp tissues.
准确分析包括虾在内的水生生物中的总T-2（游离和修饰型）非常重要，因为T-2所造成的危害已日益受到关注。因此，准确分析虾组织中的游离T-2，尤其是修饰型T-2至关重要。利用基于免疫磁珠的酶联免疫吸附测定法（IMB-ELISA）开发了一种快速、灵敏且经过验证的游离T-2和修饰型T-2毒素定量测定方法。使用通过酯法活化的带有羧基的超顺磁性颗粒与包膜抗原3-乙酰新茄病醇-半琥珀酸酯-卵清蛋白（3-Ac-NEOS-HS-OVA）结合，形成可与T-2骨架结构抗体结合的免疫磁珠。优化了T-2骨架结构抗体的磁珠包被条件以及多克隆抗体和辣根过氧化物酶标记的山羊抗兔抗体的浓度。观察到在5-75ng/mL的T-2浓度范围内具有良好的线性关系（R = 0.9965）。IMB-ELISA对不同虾组织的检测限为2.53至3.20ng/mL。半数抑制浓度为63ng/mL。回收率在86%至99%之间，标准偏差为2.8-5.8%。将该方法应用于组织分布研究表明，在最高剂量组12.2mg/kg中，肝胰腺中的总T-2浓度为26.7µg/kg＞血液＞头部＞肌肉。我们的研究表明，酶联免疫吸附测定法和免疫磁珠技术相结合提供了一种新的便捷方法，可显著提高虾组织中总T-2测量准确性和灵敏度。
