基于裸磁珠体系检测血清淀粉样蛋白A浓度方法的建立与效果评估

doi:10.19586/j.2095-2341.2021.0095

生物技术进展 ›› 2022, Vol. 12 ›› Issue (2): 281-287.DOI: 10.19586/j.2095-2341.2021.0095

基于裸磁珠体系检测血清淀粉样蛋白A浓度方法的建立与效果评估

高欣欣¹(), 马梦男¹, 刘春龙¹^,², 王梦凡¹^,³()

^1.天津大学化工学院，天津 300072
^2.侵袭性真菌病机制研究与精准诊断北京市重点实验室丹娜生物分中心，天津 300467
^3.天津大学生命科学院，天津 300072

收稿日期:2021-05-28 接受日期:2021-06-09 出版日期:2022-03-25 发布日期:2022-03-25
通讯作者: 王梦凡
作者简介:高欣欣E-mail：xinxingao163163@163.com；
基金资助:
天津市科技重大专项(20ZXGBSY00040)

Method Establishment and Performance Evaluation of Detecting Concentration of Serum Amyloid A Based on Bare Magnetic Particles Bead System

Xinxin GAO¹(), Mengnan MA¹, Chunlong LIU¹^,², Mengfan WANG¹^,³()

^1.School of Chemical Engineering and Technology，Tianjin University，Tianjin 300350，China
^2.Dynamiker Sub?center for Beijing Key Laboratory for Mechanisms Research and Precision Diagnosis of Invasive Fungal Disease，Tianjin 300467，China
^3.School of Life Sciences，Tianjin University，Tianjin 300072，China

Received:2021-05-28 Accepted:2021-06-09 Online:2022-03-25 Published:2022-03-25
Contact: Mengfan WANG

摘要/Abstract

摘要：

传统链霉亲和素磁珠体系及酶促发光体系中通常存在生物素干扰强、发光率低、特异性差的缺陷。基于此，以裸磁珠为载体偶联血清淀粉样蛋白A（serum amyloid A, SAA）抗体，以吖啶酯作为发光标记物，依据双抗夹心原理，建立了一种快速测定SAA的方法，并对该方法的检测性能（包括：空白限、检测限、线性范围、精密度验证、干扰试验、HOOK效应、方法学比较等）进行了评估。结果表明，SAA抗体与裸磁珠成功偶联；该方法空白限为0.6 mg·mL^-1，检出限为1.0 mg·mL^-1；线性范围为1~100 mg·mL^-1 （R²＞0.990）；在精密度方面，以CV表示的重复性、室内精密度均<10%；血红蛋白、胆红素、甘油三酯对临床样本的干扰检测结果相对偏差均<10%。Bland-Altman偏倚分析表明，该方法检测值与西门子试剂的测量值差异基本在95％CI的一致性界限内波动，回归分析结果显示有较好的相关性（R²=0.987）。上述结果表明，研究所建立的裸磁珠-吖啶酯发光检测体系性能参数可满足临床检测要求，适用于血清中SAA的快速检测。

关键词: 血清淀粉样蛋白A, 裸磁珠, 吖啶酯, 磁微粒化学发光法, 性能评价

Abstract:

Biotin interference， low luminescence yield and poor specificity are the main problems of traditional streptavidin-magnetic particles and enzymatic luminescence systems. In this study， based on the sandwich principle of antibody-antigen-antibody， a bare magnetic particles-acridinyl ester luminescence system was established for the rapid determination of serum amyloid A（SAA） by using bare magnetic particles as carriers to conjunct SAA antibodies and acridinium ester as luminescent marker. The detection performance of the method （including： blank limit， detection limit， linear range， precision verification， interference test， Hook effect， methodological comparison， etc.） was evaluated. The results showed that the SAA antibody was successfully coupled to the bare magnetic particles. The blank limit of this method was 0.6 mg·mL^-1， and the detection limit was 1.0 mg·mL^-1 with the linear range of 1~100 mg·mL^-1 （R²>0.990）. The repeatability and within-laboratory precision expressed by CV were both <10%. The interference test of hemoglobin， bilirubin， and triglycerides in clinical samples were all <10%. Bland-Altman analysis indicated that the difference fluctuated within the consistency limit of 95% CI between the established method and the commercial Siemens reagent and showed a good correlation （R²=0.987）. The results of this study indicated that the established bare magnetic particles-acridinyl ester luminescence detection system is promising for the rapid detection of SAA in serum.

Key words: serum amyloid A, bare magnetic particles, acridinium ester, magnetism particulate immuno chemistry luminescence method, performance evaluation

中图分类号:

R446.1

高欣欣, 马梦男, 刘春龙, 王梦凡. 基于裸磁珠体系检测血清淀粉样蛋白A浓度方法的建立与效果评估[J]. 生物技术进展, 2022, 12(2): 281-287.

Xinxin GAO, Mengnan MA, Chunlong LIU, Mengfan WANG. Method Establishment and Performance Evaluation of Detecting Concentration of Serum Amyloid A Based on Bare Magnetic Particles Bead System[J]. Current Biotechnology, 2022, 12(2): 281-287.

图/表 8

图1 血清淀粉样蛋白A抗体添加量与相对发光单位（RLU）的关系

Fig 1 Relationship between serum amyloid antibody addition and relative light escence unit (RLU)

图2 磁微粒添加量与相对发光单位（RLU）的关系

Fig 2 Relationship between magnetic particle addition and relative light emitting unit (RLU)

图3 线性范围的评估

Fig. 3 Evaluation of the learar range

表1 精密度检测结果

Table 1 Precision test results

样本编号	均值/（mg·L^-1）	重复性		室间精密度
样本编号	均值/（mg·L^-1）	SD	CV	SD	CV
1	5.11	0.165	3.2%	0.262	5.1%
2	19.90	0.587	3.0%	0.964	4.8%
3	2.02	0.104	5.1%	0.123	6.1%
4	14.74	0.467	3.2%	0.771	5.2%
5	30.25	1.160	3.8%	1.245	4.1%
6	59.21	1.512	2.6%	2.246	3.8%

表2 不同干扰物质添加量下样品检测浓度和理论浓度的相对偏差

Table 2 Relative deviation of test concentration and theoretical concentration of samples under the different add amount of interference materials

干扰物质添加量		低值样品检测浓度	相对偏差	高值样品检测浓度	相对偏差
血红蛋白/（mg·mL^-1）	0	5.37	—	49.44	—
	1	4.86	-6%	49.08	-3%
	3	5.21	1%	50.25	-1%
	5	5.07	-2%	48.38	-4%
	7	5.18	0%	52.87	4%
胆红素/（mg·L^-1）	0	4.98	—	52.27	—
	30	5.29	6%	48.53	-7%
	75	5.26	6%	50.60	-3%
	150	5.38	8%	50.56	-3%
	300	4.90	-2%	51.44	-2%
甘油三酯/（mmol·L^-1）	0	5.10	—	50.79	—
	1.5	5.34	5%	48.25	-5%
	2.5	4.85	-5%	51.85	2%
	5.0	4.89	-4%	49.69	-2%
	7.5	5.26	3%	51.87	2%

图4 HOOK效应检测结果

Fig 4 HOOK effect detection results

图5 测试结果的偏差分析

Fig 5 Deviation analysis of the test results

图6 回归分析

Fig 6 Regression analysis

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基于裸磁珠体系检测血清淀粉样蛋白A浓度方法的建立与效果评估

Method Establishment and Performance Evaluation of Detecting Concentration of Serum Amyloid A Based on Bare Magnetic Particles Bead System

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