流式微球分析技术联合检测Cys C、NGAL方法的建立及其在肾移植术后CMV感染患者中的应用

Establishment of cytometric bead assay combined detection on Cys C and NGAL and its application in CMV-infected patients after renal transplantation

  • 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.

     

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