间接竞争化学发光酶免疫法检测动物源食品中的 呋喃西林代谢物

Determination of nitrofurazone metabolite in animal-derived food by indirect competitive chemiluminescence enzyme immunoassay

中文关键词:  呋喃西林  氨基脲  化学发光酶免疫  硝基呋喃类药物

英文关键词:nitrofurazone  semicarbazide  chemiluminescence enzyme immunoassay  nitrofurans

中文摘要:

      目的 建立动物源食品中呋喃西林代谢物间接竞争化学发光酶免疫检测方法。方法 采用活化酯法将衍生后的呋喃西林代谢物半抗原与卵清蛋白(ovalbumin, OVA)偶联成为包被原; 其次, 对化学发光液体系、包被原和单克隆抗体的最优稀释倍数及其他反应条件进行优化; 最后对该法的灵敏度、特异性、精密度及准确度进行评价。结果 化学发光液A液为8 mmol/L对碘苯酚溶液和10 mmol/L鲁米诺溶液(1:1, V:V)混合, B液为每10 mL三(羟甲基)氨基甲烷(Tris)-盐酸缓冲液加5 μL 30% H2O2溶液, A液与B液临用前体积比1:1混匀; 最优反应条件是包被原和单抗稀释倍数均为800, 封闭液为1%脱脂乳, 竞争时间30 min, 酶标二抗孵育60 min; 该法的线性方程为Y=-0.4654X+0.3768(r2=0.993), 线性范围为0.123~2.398 ng/mL, IC50为0.544 ng/mL, 批内和批间变异系数分别为1.9%~4.1%和2.8%~5.3%, 空白鸡肉样品添加回收率为89.6%~98.0%。结论 该检测方法简单快速, 可用于实验室或现场动物源食品中呋喃西林代谢物的筛查。

英文摘要:

      Objective To establish a method for determination of nitrofurazone metabolite semicarbazide (SEM) in animal-derived food by indirect competitive chemiluminescence enzyme immunoassay (CLEIA). Methods Firstly, derived 3-(4-carboxyl benzyl)-semicarbazone (CPSEM) hapten was conjugated with ovalbumin (OVA) by using N-hydroxysuccinimide ester method to synthesize coating antigen. Secondly, chemiluminescence solution system, the best concentration of coating antigen and monoclonal antibody, and other reaction conditions were optimized. Finally, the sensitivity, specificity, precision and accuracy of the CLEIA method were evaluated. Results Chemiluminescence solution A was mixed by the same volume of 8 mmol/L iodine phenol solution and 10 mmol/L luminol solution, solution B was 10 mL Tris-HCl buffer solution including 5 μL 30%H2O2 solution, and solution A and solution B were mixed with the same volume before using. The optimized reaction conditions were as follows: fold dilution of monoclonal antibody and CPSEM-OVA were both 800, blocking solution was 1% skimmed milk, competitive reaction time was 30 min, and HRP-IgG incubation time was 60 min. The linear equation of CLEIA was Y=-0.4654X+0.3768(r2=0.993) with a linear detection range of 0.123~2.398 ng/mL, the 50% inhibitory concentration (IC50) was 0.544 ng/mL, the intra-assay and inter-assay coefficients of variation (CV) were respectively 1.9%~4.1% and 2.8%~5.3%, and the recoveries in negative chicken were 89.6%~98.0%. Conclusion The established detection method is simple and rapid, which can be applied in screening SEM from animal-derived food in laboratory or on-site.