Currently, atherosclerosis accounts for the majority of cardiovascular morbidity and mortality worldwide, and predicting the stability of atherosclerotic plaque is the main method to prevent atherosclerotic death. This study aims to establish a dual-label time-resolved fluorescence immunoassay (TRFIA) of matrix metalloprotein-9 (MMP-9) and lipoprotein-associated phospholipaseA2 (Lp-PLA2) to predict atherosclerotic plaque stability. A dual-label TRFIA was introduced for the simultaneous quantification of MMP-9 and Lp-PLA2 using fluorescent lanthanide (Eu³⁺ and Sm³⁺) chelates. The performance (sensitivity, specificity, accuracy, precision and reference intervals in different subjects) of this TRFIA was evaluated and compared with commercial kit. The sensitivity of the TRFIA for MMP-9 was 0.85 ng/mL and for Lp-PLA2 was 0.68 ng/mL with high affinity and specificity. The average recoveries were 94.58% to 109.82%, and 104.32% to 109.26%, respectively. All intra- and inter-assay CVs ranged from 3.10% to 5.46%. For the normal subjects, the cutoff value was 160.70 ng/mL for MMP-9 and 183.73 ng/mL for LP-PLA2; for the subjects with stable plaque, the cutoff value was 181.98~309.22 ng/mL for MMP-9 and 194.73~337.89 ng/mL for LP-PLA2; for the subjects with unstable plaque, the cutoff value was 330.43 ng/mL for MMP-9 and 343.23 ng/mL for LP-PLA2. This TRFIA detection results agreed well with the results of commercial kit (R²=0.9567 and R²=0.9771, respectively) in clinical serum samples. The TRFIA developed has a wide detection range and good sensitivity for the high-throughput simultaneous detection of MMP-9 and Lp-PLA2 in serum, which provides a new method for predicting the stability of atherosclerotic plaque.

目前，动脉粥样硬化占全球心血管发病率和死亡率的大部分，预测动脉粥样硬化斑块的稳定性是预防动脉粥样硬化死亡的主要方法。本研究旨在建立一种基质金属蛋白 9 (MMP-9) 和脂蛋白相关磷脂酶 A2 (Lp-PLA2) 的双标记时间分辨荧光免疫测定法 (TRFIA) 来预测动脉粥样硬化斑块的稳定性。引入了双标记 TRFIA，用于使用荧光镧系元素（Eu ³⁺和 Sm ^{3+ ）同时定量 MMP-9 和 Lp-PLA2}) 螯合物。评估了该 TRFIA 的性能（灵敏度、特异性、准确度、精密度和不同受试者的参考区间），并与商业试剂盒进行了比较。TRFIA 对 MMP-9 的灵敏度为 0.85 ng/mL，对 Lp-PLA2 的灵敏度为 0.68 ng/mL，具有高亲和力和特异性。平均回收率分别为 94.58% 至 109.82% 和 104.32% 至 109.26%。所有测定内和测定间CV范围从 3.10% 到 5.46%。对于正常受试者，MMP-9 的临界值为 160.70 ng/mL，LP-PLA2 的临界值为 183.73 ng/mL；对于斑块稳定的受试者，MMP-9 的临界值为 181.98~309.22 ng/mL，LP-PLA2 的临界值为 194.73~337.89 ng/mL；对于斑块不稳定的受试者，MMP-9 的临界值为 330.43 ng/mL，LP-PLA2 的临界值为 343.23 ng/mL。该TRFIA检测结果与临床血清样本中商业试剂盒的结果（分别为R ² =0.9567和R ² =0.9771）吻合良好。开发的TRFIA检测范围广，灵敏度高，可用于血清中MMP-9和Lp-PLA2的高通量同时检测，为预测动脉粥样硬化斑块的稳定性提供了新的方法。