基于免疫磁珠的喹乙醇磁酶联免疫吸附法的建立与应用

Establishment and Application of an Immunomagnetic Bead-based MELISA Method for Olaquindox

摘要:

【目的】建立基于免疫磁珠的喹乙醇（olaquindox，OLA）磁酶联免疫吸附法（magnetic bead enzyme-linked immunosorbent assay，MELISA）。【方法】首先合成喹乙醇酶标半抗原（OLA-HS-HRP），并制备免疫磁珠，随后利用偶联完成的免疫磁珠与OLA-HS-HRP，对MELISA的最佳反应条件进行筛选与优化。【结果】免疫磁珠与OLA-HS-HRP的最佳工作稀释倍数分别为100倍和8 000倍，抗体稀释液为含体积分数5%甲醇的PBS缓冲液（0.010 mol/L，pH 7.0）；在优化条件下建立的标准方程为y=13.593ln x+17.173（R²=0.994 3），其中IC₁₀值、IC₂₀值、IC₅₀值、IC₈₀值分别为0.59、1.23、11.19、101.70 ng/mL；该方法的回收率为93.17%~106.83%，变异系数（CV）<7%。【结论】该研究建立的MELISA灵敏度高且结果可靠，不仅为OLA残留检测提供了新方法，也为食品安全领域中其他小分子药物检测提供了理论依据与参考。

Abstract:

[Objective] This study aims to establish a magnetic bead enzyme-linked immunosorbent assay (MELISA) method for olaquindox (OLA) based on immunomagnetic beads.[Method] First, olaquindox-hapten conjugated with horseradish peroxidase (OLA-HS-HRP) and immunomagnetic beads were synthesized. Then, the conjugated immunomagnetic beads and OLA-HS-HRP were used to screen and optimize the reaction conditions of MELISA.[Result] The optimal working dilution factors for the immunomagnetic beads and OLA-HS-HRP were 100 times and 8 000 times, respectively, with the antibody dilution buffer being PBS solution (0.010 mol/L, pH 7.0) containing methanol at a volume fraction of 5%. Under the optimized conditions, the established standard equation was y=13.593ln x+17.173 (R²=0.994 3), with IC₁₀, IC₂₀, IC₅₀, and IC₈₀ values of 0.59, 1.23, 11.19, and 101.70 ng/mL, respectively. The recovery rate of this method was 93.17%~106.83%, and the coefficient of variation (CV) was <7%.[Conclusion]The MELISA established in this study is highly sensitive and reliable, providing a new method for the detection of OLA residues and offering a theoretical basis and reference for the detection of other small molecule drugs in food safety.