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人血管内皮生长因子(human vascular endothelial growth factor,hVEGF)是一种具有高度生物活性的二聚体阳离子糖蛋白[1],它特异性地作用于血管内皮细胞,使其增殖、迁移,并能促进血管形成和增加毛细血管通透性。肿瘤新生血管的生成是在hVEGF的调控下进行的。肿瘤新生血管的活跃程度是影响肿瘤细胞增殖的重要因素,而hVEGF是促进肿瘤新生血管生成最重要的生长因子之一,故其可反映肿瘤的发生、发展和转移[2-4]。测定血清中hVEGF的含量,对肿瘤的早期筛查、治疗疗效观察、复发监测及相关抗肿瘤药物疗效评价等都具有一定的参考价值[5-6]。目前hVEGF的检测方法主要有酶联免疫吸附法和荧光免疫层析法等[7-8]。但酶联免疫吸附法的灵敏度相对较低,荧光免疫层析法在定量检测中的批间变异性较大,不适宜在临床检测中推广。化学发光免疫测定(chemiluminescence immunoassay,CLIA)法是目前最先进的免疫分析技术之一,其灵敏度高,批内和批间变异性小,更适合在临床检测中使用[9-12]。本研究旨在研制以磁微粒为载体的hVEGF放射免疫测定(radioimmunoassay,RIA)和CLIA试剂盒,评价其分析灵敏度、回收率和精密度等技术指标,并通过对临床血清样本的检测探讨2种试剂盒对早期肿瘤的诊断价值 。
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由RIA和CLIA试剂盒测定hVEGF抗原标准品的结果可知,hVEGF抗原抗体结合较好,非特异性结合低,各标准品浓度的发光值梯度明显,2种试剂盒测定的标准曲线线性良好(图1、图2)。因此,磁微粒与包被抗体的最佳比例为1 ml磁微粒∶1 mg 抗体;125I与标记抗体的最佳比例为55.5 MBq 125I∶0.1 mg抗体,吖啶酯与标记抗体的最佳比例为25 μg吖啶酯∶0.2 mg抗体。
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RIA试剂盒检测20管零标准品,计算得到的每分钟放射性计数
$\bar x $'/>'/>'/>'/>'/>'/>'/>'/>'/>'/>'/>'/>'/>'/>'/> =844.35、SD=147.63、$\bar x $'/>'/>'/>'/>'/>'/>'/>'/>'/>'/>'/>'/>'/>'/>'/> +2SD=1139.61;CLIA试剂盒检测20管零标准品,计算得到的发光值$\bar x $'/>'/>'/>'/>'/>'/>'/>'/>'/>'/>'/>'/>'/>'/>'/> =8109.20、SD=1142.87、$\bar x $'/>'/>'/>'/>'/>'/>'/>'/>'/>'/>'/>'/>'/>'/>'/> +2SD=10394.95。将$\bar x $'/>'/>'/>'/>'/>'/>'/>'/>'/>'/>'/>'/>'/>'/>'/> +2SD结果取整后带入标准曲线,得到RIA和CLIA试剂盒的分析灵敏度分别为17.6和9.2 pg/ml。 -
RIA试剂盒的批内变异性分别为5.95%和4.19%,批间变异性分别为7.85%和7.06%;CLIA试剂盒的批内变异性分别为6.12%和4.35%,批间变异性分别为7.96%和6.37%。
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RIA和CLIA 2种试剂盒对低、中、高3个血清样本进行测定的平均回收率分别为103.28%和101.85%,说明CLIA试剂盒检测的准确率更高。
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RIA试剂盒测定正常人血清样本中hVEGF浓度,计算得到
$\bar x $'/>'/>'/>'/>'/>'/>'/> =99.82 pg/ml、SD=20.53、$\bar x $'/>'/>'/>'/>'/>'/>'/> +2SD=140.88;CLIA试剂盒测定正常人血清样本中hVEGF浓度,计算得到$\bar x $'/>'/>'/>'/>'/>'/>'/> =102.35 pg/ml、SD=19.59、$\bar x $'/>'/>'/>'/>'/>'/>'/> +2SD=141.53。因此将RIA和CLIA试剂盒测定hVEGF的正常参考值统一确定为<142 pg/ml。 -
由表1可知,每种试剂盒检测肺癌患者和结直肠癌患者分别与正常人血清样本hVEGF测定值均值相比,差异均有统计学意义(t=−16.695~−14.920,均P<0.01)。RIA试剂盒和CLIA试剂盒检测出肺癌、结直肠癌灵敏度分别为90.38%(47/52)和92.31%(48/52)、92.86%(52/53)和91.07%(51/56);RIA试剂盒和CLIA试剂盒检测出正常人血清样本的特异度分别为98.11%(104/106)和99.06%(105/106)。
血清样本来源 试剂盒 测定值>正
常参考值
样本数(例)hVEGF
浓度均值
(pg/ml)灵敏度
(%)特异度
(%)肺癌患者(n=52) RIA 47 378.65a 90.38 − CLIA 48 389.52a 92.31 − 结直肠癌患者(n=56) RIA 52 458.39a 92.86 − CLIA 51 469.18a 91.07 − 正常人(n=106) RIA 2 99.82 − 98.11 CLIA 1 102.35 − 99.06 注:a表示与正常人血清样本hVEGF浓度均值相比,差异均有统计学意义(t=−14.929、−14.920、−16.695、−16.415,均P<0.01);−表示无此项数据。RIA为放射免疫测定;CLIA为化学发光免疫测定 表 1 RIA和CLIA 2种试剂盒测定临床血清样本中人血管内皮生长因子的结果分析
Table 1. Analysis of the results of determination of human vascular endothelial growth factor in clinical serum samples by RIA and CLIA kits
两种人血管内皮生长因子免疫测定试剂盒的研制及其对早期肿瘤的诊断价值探讨
Development of two kinds of human vascular endothelial growth factor immunoassay kits and their diagnostic value in early tumor
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摘要:
目的 研制人血管内皮生长因子(hVEGF)放射免疫测定(RIA)试剂盒和化学发光免疫测定(CLIA)试剂盒,探讨其在早期肺癌和结直肠癌诊断中的价值。 方法 以2种试剂盒的技术指标为参考依据建立检测方法,确定磁微粒包被抗体的工艺方法及125I和吖啶酯标记抗体的工艺方法,并评价2种试剂盒的分析灵敏度、精密度、回收率。通过检测临床癌症(早期肺癌和结直肠癌)患者和正常人血清样本中的hVEGF,评价2种试剂盒对早期肿瘤诊断的灵敏度和特异度。2组间的比较采用两独立样本t检验。 结果 磁微粒与包被抗体的最佳比例为1 ml磁微粒∶1 mg 抗体;125I与标记抗体的最佳比例为55.5 MBq 125I∶0.1 mg抗体,吖啶酯与标记抗体的最佳比例为25 μg吖啶酯∶0.2 mg抗体。RIA和CLIA试剂盒的分析灵敏度分别为17.6、9.2 pg/ml。在精密度方面,CLIA试剂盒的批内变异性略高于RIA试剂盒,而批间变异性略低于RIA试剂盒。RIA和CLIA试剂盒的平均回收率分别为103.28%和101.85%,说明CLIA试剂盒检测的准确率更高。在临床样本检测方面,2种试剂盒对早期肺癌和结直肠癌的临床诊断灵敏度和特异度均可达90%以上。RIA试剂盒和CLIA试剂盒检测出正常人血清样本的特异度分别为98.11%(104/106)和99.06%(105/106)。癌症患者与正常人hVEGF测定值相比,差异均有统计学意义(t=−16.695~−14.920,均P<0.01)。 结论 2种试剂盒各项技术指标较好,其中CLIA试剂盒的分析灵敏度和特异度更高,在早期肺癌和结直肠癌的诊断中具有一定的临床价值。 Abstract:Objective To prepare human vascular endothelial growth factor (hVEGF) radioimmunoassay (RIA) and chemiluminescence immunoassay (CLIA) kits and evaluate their clinical value in diagnosis of early lung cancer and colorectal cancer. Methods A detection method was established according to the technical indices of the kits. The techniques for coating magnetic particles and 125iodine and acridine ester labeling with antibody were evaluated. Sensitivity, precision, and recovery of the kits were determined. The sensitivity and specificity of the kits in the diagnosis of early cancers (lung and colorectal cancers) were evaluated by testing cancer and normal serum samples. Independent sample t-test was used for inter-group comparison. Results The optimal ratios were as follows: 1 mL of magnetic particles to 1 mg of the antibody, 55.5 MBq 125iodine to 0.1 mg of the antibody, and 25 μg acridine ester to 0.2 mg of the antibody. The detection sensitivities of RIA and CLIA kits were 17.6 and 9.2 pg/mL respectively. For precision, the CLIA kit had slightly higher intra-batch variability and lower inter-batch variability than the RIA kit. The mean recovery levels of the RIA and CLIA kits were 103.28% and 101.85% respectively, and the latter had higher detection accuracy. The clinical sensitivity and specificity in the diagnosis of lung cancer and colorectal cancer were all above 90%. RIA kit and CLIA kit showed that the specificity of hVEGF in normal serum samples was 98.11% (104/106) and 99.06% (105/106), respectively.. There were significant differences in hVEGF between cancer patients and normal subjects (t=−16.695–−14.920, all P<0.01). Conclusions The technical indices of the hVEGF RIA and CLIA kits were good. The CLIA kit had higher sensitivity and specificity than the RIA kit and has clinical value in screening and auxiliary diagnosis of early lung and colorectal cancers. -
表 1 RIA和CLIA 2种试剂盒测定临床血清样本中人血管内皮生长因子的结果分析
Table 1. Analysis of the results of determination of human vascular endothelial growth factor in clinical serum samples by RIA and CLIA kits
血清样本来源 试剂盒 测定值>正
常参考值
样本数(例)hVEGF
浓度均值
(pg/ml)灵敏度
(%)特异度
(%)肺癌患者(n=52) RIA 47 378.65a 90.38 − CLIA 48 389.52a 92.31 − 结直肠癌患者(n=56) RIA 52 458.39a 92.86 − CLIA 51 469.18a 91.07 − 正常人(n=106) RIA 2 99.82 − 98.11 CLIA 1 102.35 − 99.06 注:a表示与正常人血清样本hVEGF浓度均值相比,差异均有统计学意义(t=−14.929、−14.920、−16.695、−16.415,均P<0.01);−表示无此项数据。RIA为放射免疫测定;CLIA为化学发光免疫测定 -
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