纳米金棒标记技术应用于旋毛虫感染诊断的研究

doi:10.12140/j.issn.1000-7423.2021.05.013

摘要/Abstract

摘要：

目的用聚合纳米金棒标记的巯基化旋毛虫排泄分泌抗原构建旋毛虫感染早期敏感的诊断方法。方法制备旋毛虫成虫、新生幼虫、囊包幼虫的粗抗原和纯化抗原,以及肌幼虫排泄分泌抗原。优化金晶种子生长法中的十六烷基三甲基溴化铵（CTAB）、抗坏血酸（AA）和硝酸银（AgNO₃）的用量,制备出长径比稳定的纳米金棒,与不同浓度（10、20、30、40、50 μg/ml）巯基化的旋毛虫排泄分泌抗原、虫体粗抗原以及虫体纯化抗原结合进行纳米金棒的功能化,通过观察紫外分光扫描表面等离子共振吸收峰的变化,筛选标记的最适浓度和诊断抗原。用纳米金棒标记最适诊断抗原,分别检测不同囊包幼虫感染度（轻、中、重度分别感染50、100、300条/鼠）小鼠感染后5、8、11、17、23 d的血清抗体以及重度感染的小鼠不同血清稀释度（1 ∶ 300、1 ∶ 400、1 ∶ 500、1 ∶ 600、1 ∶ 700、1 ∶ 800）的血清抗体情况,并与包被最适诊断抗原的ELISA法检测结果进行评价性比较,判定其检测早期低虫荷时的敏感性。结果 11.875 ml 0.2 mol/L CTAB、160 μl 100 mmol/L AA和150 μl 10 mmol/L AgNO₃制备出的纳米金棒径长比最长,溶液最稳定。用纳米金棒标记的不同浓度旋毛虫粗抗原、纯化抗原以及排泄分泌抗原,紫外分光光度计光谱显示表面等离子共振吸收峰位移最大值为87 nm,最适包被抗原为排泄分泌抗原。用纳米金棒标记的旋毛虫肌幼虫排泄分泌抗原能检出轻度、中度、重度感染囊包幼虫后5 d小鼠的血清抗体以及稀释度为1 ∶ 800的阳性血清抗体。ELISA法能检出轻度和中度感染囊包幼虫后11 d小鼠的血清抗体和重度感染囊包幼虫后8 d小鼠的血清抗体以及稀释度为1 ∶ 600的阳性血清抗体。结论纳米金棒能够有效标记旋毛虫抗原,排泄分泌抗原功能化的纳米金棒对旋毛虫早期感染及低虫荷的诊断具有明显优势。

关键词: 旋毛虫, 旋毛虫虫体抗原, 纳米金棒, 标记技术, 诊断

Abstract:

Objective To establish sensitive method for early diagnosis of Trichinella spiralis infection, polymeric gold nanorods were used to label the excretion and secretion antigens of T. spiralis. Methods Crude and purified antigens were extracted from adult T. spiralis, newborn larvae, and encapsulated larvae, and excretory and secreted antigens were prepared from larvae in muscle tissue of infected mice. We optimized the amount of cetyltrimethylammonium bromide (CTAB), ascorbic acid (AA), and silver nitrate (AgNO₃) in the procedure of gold crystal seed growth to yield of gold nanorods with stable length-diameter ratio. The gold nanorods were functionalized by coupling with different concentrations (10, 20, 30, 40, and 50 μg/ml) of mercaptosylated excreted/secreted antigens, worm crude antigen and worm purified antigen. Screening for optimum labeling concentrations and diagnostic target antigens was based on observation of the changes of surface plasma resonance absorption peak of labeled antigens by ultraviolet spectroscopic scanning. The serum antibody of mice with different infection intensity of encysted larvae (light: 50/mouse, medium: 100/mouse, severe: 300/mouse) was examined using optimal nanoro-labeled diagnostic antigen on 5, 8, 11, 17, and 23 d post-infection. Meantime, the serum antibody of severely infected mice were detected at different serum dilutions (1 ∶ 300, 1 ∶ 400, 1 ∶ 500, 1 ∶ 600, 1 ∶ 700, 1 ∶ 800); the results were compared with those assayed by ELISA with optimum coating antigen to evaluate the sensitivity in detecting early infection with low worm burden. Results Using 11.875 ml 0.2 mol/L CTAB, 160 μl 100 mmol/L AA, and 150 μl 10 mmol/L AgNO₃ in the growth media produced high length-diameter ratio and most stable solution of nanorods. The spectrophotometric spectrum of different concentrations of gold nanorod-labeled worm crude, purified, and excreted/secreted antigens showed a highest plasma resonance absorption peak of 87 nm, indicating the optimum antigen for coating gold-nanorod surface being excreted/secreted antigens. Using gold nanorod-labeled excreted/secreted antigens, serum antibody in mice with light, medium, and severe infection of encysted larvae could be detected on 5 d post-infection at 1 ∶ 800 dilution. By contrast, ELISA could detecte serum antibodies of mice with light and medium infection rate of encysted larvae on 11 d post-infection, while the serum antibody of mice with severe infection could be detected on 8 d post-infection, at 1 ∶ 600 dilution. Conclusion Gold nanorods can effectively label T. antigens, and the excreted/secreted antigen-functionalized gold nanorods show significant superiority in diagnosis of early infection and low worm burden of T. spiralis.

Key words: Trichinella spiralis, Trichinella spiralis worm antigens, Gold nanorod, Labeling technology, Diagnosis

中图分类号:

R532.14

蔡子涵, 曹颖, 朱逢龙, 李倩, 和艳红, 杨毅梅. 纳米金棒标记技术应用于旋毛虫感染诊断的研究[J]. 中国寄生虫学与寄生虫病杂志, 2021, 39(5): 652-658.

CAI Zi-han, CAO Ying, ZHU Feng-long, LI Qian, HE Yan-hong, YANG Yi-mei. Application of gold nanorod labeling in diagnosis of Trichinella spiralis infection[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2021, 39(5): 652-658.

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感染程度 The degree of infection	感染后时间/d Time after infection/d	A₂₈₀值 A₂₈₀ value	结果 Result
轻度（50条/只）Light (50 encysted larvae/mouse)	5	0.154	-
	8	0.201	-
	11	0.247	+
中度（100条/只）Medium (100 encysted larvae/mouse)	5	0.175	-
	8	0.211	-
	11	0.256	+
重度（300条/只）Severe (300 encysted larvae/mouse)	5	0.194	-
	8	0.232	+
	11	0.274	+

稀释度 Dilution degrees	A₂₈₀值 A₂₈₀ value	结果 Result
1 ∶ 300	0.231	+
1 ∶ 400	0.228	+
1 ∶ 500	0.225	+
1 ∶ 600	0.223	+
1 ∶ 700	0.216	-
1 ∶ 800	0.209	-