采用免疫磁珠富集呋喃唑酮残留标示物的研究

Enrichment of the residual marker of furazolidone by immunomagnetic beads

将呋喃唑酮代谢物3-氨基-2-恶唑烷酮（AOZ）单克隆抗体偶联至经3-乙基碳二亚胺（EDC）和N-羟基琥珀酰亚胺（NHS）活化的磁珠上制备成免疫磁珠,建立快速、特异性富集鸡肉中残留标示物NPAOZ（3-[[（4-硝基苯基）-亚甲基]-氨基]-2-恶唑烷酮）的方法。纳米磁珠经EDC和NHS活化后与AOZ单克隆抗体偶联,得到抗体偶联量为50μg/mg的免疫磁珠。该免疫磁珠只对NPAOZ具有较高的特异性吸附,而对呋喃妥因代谢物AHD、呋喃西林代谢物SEM、呋喃它酮代谢物AMOZ的衍生物均无吸附作用。在样品前处理过程中利用免疫磁珠富集呋喃唑酮代谢物,当加标量为0.25~2 ng/g时回收率均在78%以上,变异系数为5.3%~10.9%,最低检测限为0.049μg/kg,远低于样品常规样品前处理法的最低检测限0.139μg/kg。结果显示基于免疫磁珠的样品前处理方法能有效富集鸡肉中NPAOZ,进一步有效提高ELISA检测方法的灵敏度。

Monoclonal antibody（ Mc Ab） against 3-amino-2-oxazolidinone（ AOZ）,the metabolite of furazolidone was coupled to actived immunomagnetic beads to develop a rapid and specific method for detection of the derivatives of AOZ（ NPAOZ） in chicken. Immunomagnetic beads were prepared by coupling 50 μg of anti-AOZ Mc Ab with 1 mg of magnetic beads that were activated by 1-ethyl-3-（ 3-dimethylaminopropyl） carbodiimide hydrochloride（ EDC） and N-Hydroxysuccinimide（ NHS）. The immunomagnetic beads could only specifically adsorb the derivatives of AOZ not the derivatives of 1-aminohydantoin（ AHD）,3-amino-5-morpholinomethyl-2-oxazolidone（ AMOZ） or semicarbazide（ SEM）. The sample pretreatment procedure was optimized after the immunomagnetic beads were obtained.The performance of immunomagnetic beads was evaluated by addition and recovery experiments. The recovery rates of AOZ at spiked levels from 0. 25 to 2 ng / g were more than 78%,and the coefficient of variation was between 5. 3% and 10. 9%. The limit of detection was lower than that of common sample pretreatment（ 0. 049 μg / kg vs 0. 139 μg / kg）. The results indicate that the immune-extraction method based on immunomagnetic beads with anti-AOZ Mc Ab could effectively enrich NPAOZ in chicken,and thus improve the sensitivity of ELISA method.