基于微流控技术的磁免疫荧光法在EB病毒检测中的应用

doi:10.3724/SP.J.1123.2021.09005

摘要/Abstract

摘要：

EB病毒(Epstein-Barr virus, EBV)的早期诊断能够降低患者发生重大疾病的风险。临床上常用的EBV抗体的检测方法存在耗时长、试剂消耗大和效率低等缺点。相比于传统的检测方法,微流控(microfluidics)技术具有高通量、试剂消耗少,污染少和自动化程度高等优点,磁免疫荧光技术具有检测效率高、信号强等优点,将两者的优势结合,能够弥补传统方法的不足。鉴于此,采用聚甲基丙烯酸甲酯(PMMA)作为芯片原材料,经过激光切割及真空热压加工工艺能够快速获得芯片。将包被抗原的磁珠及包被抗人抗体的荧光微球经过冷冻干燥工艺快速冻干成小球并嵌入芯片内,经过孵育和清洗后,进行检测。通过图像分析快速得到检测结果。通过精密度、特异性、剂量-反应曲线及检出限测试,进行性能验证。通过与化学发光免疫分析法(CLIA)检测的临床样本比对,进行方法学与临床应用评价。结果显示相对标准偏差(RSD)均小于10%。与多种常见的病原体抗体均无交叉反应。EB病毒衣壳抗原(Epstein-Barr viral capsid antigen, EB VCA)IgG项目的检出限为1.92 U/mL,线性范围为1.92~200 U/mL,阳性符合率为95.77%(68/71),阴性符合率为86%(43/50); EB VCA IgA项目的检出限为2.79 U/mL,线性范围为2.79~200 U/mL,阳性符合率为92%(46/50),阴性符合率为92.96%(66/71); EB病毒核心抗原1(Epstein-Barr nuclear antigen 1, EB NA1)IgG项目的检出限为3.13 U/mL,线性范围为3.13~200 U/mL,阳性符合率为92.96%(66/71),阴性符合率为92%(46/50); EB NA1 IgA项目的检出限为1.53 U/mL,线性范围为1.53~200 U/mL,阳性符合率为90%(45/50),阴性符合率为91.55%(65/71)。4个项目能在20 min内快速完成检测,且与临床上使用CLIA方法测试的结果具有良好的相关性,可以为临床提供一种快速、灵敏、简便、自动化程度高和易于基层推广的检测方法。

关键词: 微流控, 磁免疫荧光, EB病毒, 快速检测

Abstract:

Early diagnosis of Epstein-Barr virus (EBV) can reduce the risk of major illnesses. Disadvantages of EBV antibody detection methods that are commonly used clinically include lengthy assay time, need for a lot of reagent, and low efficiency. Compared with traditional detection methods, microfluidics technology offers high throughput, low reagent consumption, less bio-contamination, and a higher degree of automation. Advantages of magnetic immunofluorescence technology include high detection efficiency and a strong signal. The combined advantages of the two methods can compensate for the shortcomings of traditional methods. In the present study, polymethyl methacrylate (PMMA) as the raw material was subjected to laser cutting and vacuum hot pressing to quickly obtain chips. Magnetic beads labeled with antigen and fluorescent microspheres labeled with anti-human antibody were then rapidly lyophilized into microspheres by freeze-drying and embedded into the chips. After incubation and cleaning, the last step was detection. Image J software was used to analyze the mean fluorescence intensity and obtain negative or positive test results. To determine the precision of the chip, high- and low-value samples of each item were retested 10 times. The mean values were calculated to obtain the relative standard deviation (RSD) for several common pathogens. Furthermore, the coincidence rate of clinical samples was tested using a chemiluminescence immunoassay (CLIA) to determine the potential clinical application value. The RSD of the precision test for each item was <10%, indicating good precision. The precision of the accelerated stability test was not verified. Specificity test results revealed no cross-reaction with some common pathogen antibodies, indicating good specificity. It remains to be verified whether the antibodies detected by this method cross-react with other herpes simplex viruses, such as types 1 and 2, Kaposi’s sarcoma-associated virus, and human herpes virus type 6 and 7. Of the 121 clinical samples tested, statistical analysis of the data indicated good agreement with the chemiluminescence immunoassay in clinical trials. EB viral capsid antigen (EB VCA) IgG positive coincidence rate was 95.77% (68/71), the negative coincidence rate was 86% (43/50) (Kappa=0.828, P<0.05), the limit of detection (LOD) was 1.92 U/mL, and the linear range was 1.92 to 200 U/mL. The EB VCA IgA positive coincidence rate was 92% (46/50), negative coincidence rate was 92.96% (66/71) (Kappa=0.847, P<0.05), LOD was 2.79 U/mL, and the linear range was 2.79 to 200 U/mL. The positive coincidence rate of EB nuclear antigen 1 (EB NA1) IgG was 92.96% (66/71), the negative coincidence rate was 92% (46/50) (Kappa=0.847, P<0.05), the LOD was 3.13 U/mL, and the linear range was 3.13 to 200 U/mL. The positive coincidence rate of EB NA1 IgA was 90% (45/50), the negative coincidence rate was 91.55% (65/71) (Kappa=0.813, P<0.05), the LOD was 1.53 U/mL, and the linear range was 1.53 to 200 U/mL. Compared with the traditional enzyme-linked immunosorbent assay, the novel method featured a shorter detection time, reduced use of reagent, high degree of automation, and less bio-contamination. Compared with CLIA, advantages of the novel method include multi-item combined detection, long luminescence time, and simple use as a basic health service. Compared with silicon and ceramic microfluidic chips, advantages of the selected PMMA material include low processing cost, short processing time, simple processing technology, and easy industrialization. A refinement that can still be made include the use of molding instead of laser cutting technology, which can further shorten the chip processing time. In summary, a microfluidic detection platform was initially built to provide a rapid, sensitive, simple, highly automated, and easy to be used by basic health service for the quantitative combined detection of EBV VCA and EB NA1 IgG and IgA.

Key words: microfluidic, magnetic immunofluorescence, Epstein-Barr virus (EBV), rapid detection

中图分类号:

O658

李俊豪, 韩冠华, 林晓涛, 吴力强, 钱纯亘, 徐军发. 基于微流控技术的磁免疫荧光法在EB病毒检测中的应用[J]. 色谱, 2022, 40(4): 372-383.

LI Junhao, HAN Guanhua, LIN Xiaotao, WU Liqiang, QIAN Chungen, XU Junfa. Application of magnetic immunofluorescence assay based on microfluidic technology to detection of Epstein-Barr virus[J]. Chinese Journal of Chromatography, 2022, 40(4): 372-383.

图/表 16

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Stage	Number in Fig. 4	Rotational speed/(r/min)	t/s	Control	Specification
First incubation	1	-	-	-	sampling
	2	500	10	valve close	sample guiding
	3	800	10	valve close	sample aliquoting
	4	500	300	valve close	reacting (bi-directional running with 120°)
	5	800	10	magnetic beads accumulation; valve open	draining
Second incubation	6	-	-	magnetic beads re-suspension; valve close	adding deionized water (DW)
	7	300	10	valve close	DW guiding
	8	500	10	valve close	DW guiding
	9	800	10	valve close	DW aliquoting
	10	500	300	valve close	reacting (bi-directional running with 120°)
	11	800	10	magnetic beads accumulation; valve open	draining
Cleaning	12	-	-	magnetic beads re-suspension; valve close	adding cleaning buffer (CB)
	13	300	10	valve close	CB guiding
	14	500	10	valve close	CB guiding
	15	800	10	valve close	CB aliquoting
	16	500	300	valve close	cleaning (bi-directional running with 120°)
	17	800	10	magnetic beads accumulation; valve open	draining
Detection	18	-	-	-	signal collecting

Stage	Number in Fig. 4	Rotational speed/(r/min)	t/s	Control	Specification
First incubation	1	-	-	-	sampling
	2	500	10	valve close	sample guiding
	3	800	10	valve close	sample aliquoting
	4	500	300	valve close	reacting (bi-directional running with 120°)
	5	800	10	magnetic beads accumulation; valve open	draining
Second incubation	6	-	-	magnetic beads re-suspension; valve close	adding deionized water (DW)
	7	300	10	valve close	DW guiding
	8	500	10	valve close	DW guiding
	9	800	10	valve close	DW aliquoting
	10	500	300	valve close	reacting (bi-directional running with 120°)
	11	800	10	magnetic beads accumulation; valve open	draining
Cleaning	12	-	-	magnetic beads re-suspension; valve close	adding cleaning buffer (CB)
	13	300	10	valve close	CB guiding
	14	500	10	valve close	CB guiding
	15	800	10	valve close	CB aliquoting
	16	500	300	valve close	cleaning (bi-directional running with 120°)
	17	800	10	magnetic beads accumulation; valve open	draining
Detection	18	-	-	-	signal collecting

Channel location	Maximum channel depth/μm	Channel cross-sectional area/μm²
Between sampling pool and liquid separation pool	93.51±2.94	15935.2±712.5
Between fluorescent bead pool and liquid separation pool	96.44±3.28	16718.7±654.6
Between left fluid sac pool and fluorescent bead pool	92.43±3.66	15502.3±857.4
Between detection pool and waste liquid pool	96.93±2.78	16614.1±811.3

Channel location	Maximum channel depth/μm	Channel cross-sectional area/μm²
Between sampling pool and liquid separation pool	93.51±2.94	15935.2±712.5
Between fluorescent bead pool and liquid separation pool	96.44±3.28	16718.7±654.6
Between left fluid sac pool and fluorescent bead pool	92.43±3.66	15502.3±857.4
Between detection pool and waste liquid pool	96.93±2.78	16614.1±811.3

Item	Normal serum sample number	Positive serum sample number	OOP/(U/mL)	Sensitivity/%	Specificity/%	AUC
EB VCA IgG	46	75	9.97	93.3	91.3	0.9788
EB VCA IgA	70	51	9.95	92.2	92.9	0.9784
EB NA1 IgG	51	70	9.97	94.3	90.2	0.9672
EB NA1 IgA	52	69	10.05	90.4	92.8	0.9727