Shelver Weilin L, Smith David J
U.S. Department of Agriculture, Agricultural Research Service, Biosciences Research Laboratory, Fargo, ND 58105-5674, USA.
J AOAC Int. 2002 Nov-Dec;85(6):1302-7.
A monoclonal antibody-based immunoaffinity column (RAC-IAC) was developed as a cleanup method for the determination of ractopamine and ractopamine glucuronides. [14C]Ractopamine (5 microg) and [14C]ractopamine glucuronides (5 microg) were fortified into 10 mL cattle urine, and loaded onto an RAC-IAC (5 mg IgG/mL) column. The column was washed and the bound analytes were eluted. In the initial loading and washing, 22% of the radioactivity was washed off and the subsequent elution step recovered 78%. A blank column prepared from nonspecific IgG retained <10% of the radioactivity. The RAC-IACs were damaged by high methanol concentrations, preventing reuse. Elution of the analytes with 50mM glycine buffer, pH 2.8, prevented damage, and the columns could be reused at least 20 times with no change in performance. They were stored >3 months in phosphate-buffered saline with 0.02% sodium azide at 4 degrees C. The method was used with fortified cattle muscle, liver, and kidney samples with recoveries of 82.1+/-7.6, 87.8+/-1.9, and 92.5+/-0.4%, respectively (n = 3). Similar studies with sheep muscle, liver, and kidney samples gave recoveries of 91.8+/-0.2, 91.7+/-0.3, and 92.3+/-0.3, respectively (n = 3). Liver and kidney samples were diluted to prevent column plugging, but all of the eluants were suitable for liquid chromatography analysis. This IAC is a selective, efficient, and economical cleanup method in a variety of matrixes for ractopamine determination.
开发了一种基于单克隆抗体的免疫亲和柱（RAC-IAC），作为测定莱克多巴胺及其葡萄糖醛酸苷的净化方法。将[14C]莱克多巴胺（5微克）和[14C]莱克多巴胺葡萄糖醛酸苷（5微克）添加到10毫升牛尿中，然后加载到RAC-IAC柱（5毫克免疫球蛋白G/毫升）上。对该柱进行清洗，然后洗脱结合的分析物。在初始加载和清洗过程中，22%的放射性被洗去，随后的洗脱步骤回收率为78%。由非特异性免疫球蛋白G制备的空白柱保留的放射性小于10%。高甲醇浓度会损坏RAC-IAC柱，使其无法重复使用。用pH值为2.8的50mM甘氨酸缓冲液洗脱分析物可防止柱损坏，并且这些柱可重复使用至少20次，性能无变化。它们在含有0.02%叠氮化钠的磷酸盐缓冲盐水中于4℃下保存超过3个月。该方法用于添加了目标物的牛肌肉、肝脏和肾脏样品，回收率分别为82.1±7.6%、87.8±1.9%和92.5±0.4%（n = 3）。对绵羊肌肉、肝脏和肾脏样品进行的类似研究，回收率分别为91.8±0.2%、91.7±0.3%和92.3±0.3%（n = 3）。肝脏和肾脏样品进行了稀释以防止柱堵塞，但所有洗脱液都适用于液相色谱分析。这种免疫亲和柱是一种在多种基质中测定莱克多巴胺的选择性高、效率高且经济的净化方法。
