基于碱性磷酸酶-金刚烷体系的化学发光酶免疫分析法对磺胺对甲氧嘧啶的测定

Determination of Sulfameter by Chemiluminescence Enzyme Immunoassay Based on Alkaline Phosphatase-Adamantane

目的用碱性磷酸酶（ALP）-金刚烷（AMPPD）体系,建立化学发光酶免疫分析定量检测磺胺对甲氧嘧啶（SMD）的方法。方法利用重氮化偶联法标记ALP,在优化的实验条件下抗体包被浓度为5.0μg.mL-1,酶标记物工作液稀释度为1∶10,每孔加待测样品或标准品50μL和酶标记物50μL,在37℃下反应l h,洗涤后每孔加发光底物100μL,避光反应25 min后在发光检测仪下检测发光计数。结果该方法检测限为28.0 pg.mL-1,批内变异系数为13.5%~14.6%,批间变异系数为16.1%~17.5%。标准曲线回归方程为Y=-0.021 5X＋5.172 6（r=0.996 1）,线性范围为50~10 000 pg.mL-1。结论首次运用ALP-AMPPD发光体系的化学发光酶免疫分析法实现了对SMD残留的快速检测。

OBJECTIVE To establish a chemiluminescence enzyme immunoassay detection method for the quantitative assay of sulfameter, using alkaline phosphatase （ALP）-adamantane （AMPPD） system. METHODS The diazotization-coupling method was used to combine horseradish peroxidase with sulfameter. The optimal conditions were as follows. The concentration of antibody-coated solid-phase was 5.0 μg.mL-1. The ALP-sulfameter was diluted to l∶ 10. Every well was added by standards or samples （50 μL） and ALP-sulfameter （50 μL）, then plates were placed at 37 ℃ for 1 h. After washed, each well was added by 100 μL AMPPD. The result was obtained by luminometer after adding substrate for 25 min. RESULTS The limit of detection was 28.0 pg.mL-1, the relative standard deviations of intra-assay and inter-assay of ten samples were 13.5%-14.6% and 16.1%-17.5%, respectively. The regression equation was Y=-0.021 5X＋5.172 6 （r=0.996 1）, the linear range for the method was 50-10 000 pg.mL-1. CONCLUSION ALP-AMPPD chemiluminescence enzyme immunoassay can be used to determine the residues of veterinary drugs of sulfameter.