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

关键词:  碱性磷酸酶  金刚烷  化学发光酶免疫分析  磺胺对甲氧嘧啶

Key words:  alkaline phosphatase  adamantane  chemiluminescence enzyme immunoassay  sulfameter

摘要:

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 5.0 μg·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50 μL和酶标记物50 μL，在37 ℃下反应l h，洗涤后每孔加发光底物100 μL，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。

Abstract:

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.