首页> 中文期刊> 《华南农业大学学报》 >呋喃它酮代谢物人工抗原的合成及抗体的制备

呋喃它酮代谢物人工抗原的合成及抗体的制备

         

摘要

[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 NaBH 4 for the pro-duction 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 successful-ly 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 .%[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-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%.

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