Establishment of cytometric bead assay combined detection on Cys C and NGAL and its application in CMV-infected patients after renal transplantation
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摘要:
目的 建立流式微球分析技术联合检测胱抑素(Cys) C和中性粒细胞明胶酶相关脂质运载蛋白(NGAL)的方法，并初步研究两者在肾移植术后巨细胞病毒(CMV)感染患者血清中的含量变化。 方法 实验方法的建立包括检测微球包被的效果和稳定性，优化Cys C和NGAL的检测含量范围，确定生物素化抗体浓度，确定PE-链霉亲和素浓度，绘制标准曲线图确定相关系数。采用该法检测对照组(10名健康人)、CMV阴性组(10例肾移植术后CMV-pp65阴性患者)和CMV阳性组(20例肾移植术后CMV-pp65阳性患者)的血清Cys C和NGAL含量。 结果 成功建立流式微球分析技术联合检测Cys C和NGAL的方法。对照组、CMV阴性组和CMV阳性组的血清NGAL含量分别为8 759(1 465)pg/ml、10 472(856)pg/ml、12 817(4 533)pg/ml，依次升高且差异有统计学意义(P < 0.001)。3组间Cys C比较差异无统计学意义(P > 0.05)。 结论 本实验建立流式微球分析技术联合检测Cys C和NGAL标准曲线的方法相关性和重复性良好，并初步验证NGAL含量在肾移植术后CMV感染中明显升高，提示肾移植术后CMV活动性感染可能对肾小管造成急性损伤。 Abstract:Objective To establish the method of cytometric bead assay in combined detection of cystatin C (Cys C) and neutrophil gelatinase-associated lipocalin (NGAL) and to preliminarily investigate the changes in the serum levels of Cys C and NGAL in cytomegalovirus (CMV)-infected patients after renal transplantation. Methods The experimental method was established as following. The effect and stability of microsphere capsulation were evaluated. The detection range of Cys C and NGAL concentration was improved. The concentrations of biotinylated antibody and PE-streptavidin were quantitatively measured. The correlation coefficient of the standard curve was calculated. The serum levels of Cys C and NGAL in the control (n=10, healthy person), negative CMV (n=10, CMV-pp65 negative patients after renal transplantation) and positive CMV groups (n=20, CMV-pp65 positive patients after renal transplantation) were detected using this method. Results The method of cytometric bead assay in combined detection of Cys C and NGAL was established successfully. In the control, negative and positive CMV groups, the serum levels of NGAL were 8 759(1 465) pg/ml, 10 472(856)pg/ml and 12 817(4 533)pg/ml with statistical significance (all P < 0.001). No statistical significance was noted in terms of Cys C concentration among three groups (all P > 0.05). Conclusions Combined application of cytometric bead assay detects the standard curve of Cys C and NGAL with high relevance and reproducibility. Preliminary results demonstrates that NGAL concentration is significantly elevated in CMV-infected patients following renal transplantation, hinting that CMV active infection probably leads to acute renal tubular injury. -
图 1 Cys C和NGAL标准曲线及各点MFI值
注：A图为Cys C标准曲线；B图为NGAL标准曲线
Figure 1. Standard curves of Cys C and NGAL and the MFI value of each point
表 1 包被后不同时间Cys C和NGAL的PE信号MFI值
Table 1. MFI value of PE signal of Cys C and NGAL at different time points after coating
检测管 包被后 0 d 7 d 1个月 2个月 3个月 4个月 Cys C 2 916.38 2 996.14 2 689.60 2 996.14 3 337.64 4 141.78 Cys C A4阴性对照 4.80 4.83 4.66 4.96 7.84 9.31 NGAL 3 367.78 3 854.23 3 459.89 3 718.03 4 293.51 5 327.98 NGAL B9阴性对照 2.23 2.37 2.11 2.30 3.11 3.82 
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