呋喃它酮代谢物人工抗原的合成及抗体的制备
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农业部兽药质量安全监管项目(农财发[2012]36号)


Synthesis of artificial antigens and preparation for specific antisera against 3-amino-2-oxazolidinone,a metabolite of furaltadone
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    【目的】建立高灵敏度的呋喃它酮酶联免疫检测方法.【方法】以戊二醛法将呋喃它酮代谢物5-甲基吗啉-3-氨基-2口恶唑烷基酮(AMOZ)与牛血清白蛋白(BSA)和卵清白蛋白(OVA)偶联,再由NaBH4 还原西佛碱得到结构稳定的免疫原AMOZ-BSA和AMOZ-OVA;用对醛基苯甲酸(CPA)对AMOZ进行衍生化,得到衍生物CPAMOZ,由质谱鉴定,经N-羟基琥珀酰亚胺活化酯法将CPAMOZ与BSA和OVA偶联得到免疫原CPAMOZ-BSA和包被原CPAMOZ-OVA.经紫外扫描鉴定表明人工抗原合成成功.免疫Balb/c小鼠,ic-ELISA方法测定鼠血清多克隆抗体对AMOZ和NPAMOZ的特异性.【结果和结论】结果显示CPAMOZ-BSA免疫制备的抗体对NPAMOZ有良好的特异性.抗体效价为1∶32×104,对NPAMOZ的半抑制质量浓度为4.53 ng·mL-1;抗血清与CPAMOZ、AMOZ及呋喃它酮的交叉反应率为78.6%、1.5%及4.6%;与其他3种硝基呋喃类原药及其代谢物对硝基苯甲醛、对醛基苯甲酸等结构相似物的交叉反应均小于0.01%.

    Abstract:

    【Objective】To establish a high sensitivity enzyme-linked immunosorbent assay for the detection of furaltadone.【Method】Furaltadone metabolite (3-amino-5-morpholinomethyl-2-oxazolidinone, AMOZ) was coupled to carrier proteins (bovine serum albumin or ovalbumin) and reduced with NaBH4 for the production of immunogen AMOZ-BSA and coating antigen AMOZ-OVA through glutaraldehyde method. With 4-carboxybenzaldehyde for derivatization reagent, AMOZ was made into CPAMOZ, which was successfully conjugated to carrier proteins according to the active ester method to form CPAMOZ-BSA, CPAMOZ-OVA. UV scanning showed that antigens were successfully linked to carrier proteins. After immunizing animal (Balb/c mice), polyclonal antibody against NPAMOZ was produced.【Result and conclusion】 Antibody was diluted at 1∶32×104 and the IC50 value was 4.53 ng·mL-1. The cross-reactivity (CR) of the antibody with CPAMOZ, AMOZ and furaltadone was 78.6%, 1.5% and 4.6% respectively. There is almost no CR with other three nitrofurans and their metabolites, which indicates a high selectivity of the antibody.

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束建花,郭红艳,王立琦,高 艳,曾振灵.呋喃它酮代谢物人工抗原的合成及抗体的制备[J].华南农业大学学报,2014,35(2):18-23

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  • 收稿日期:2013-05-31
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  • 在线发布日期: 2014-02-28