快速检测美洲钩虫特异抗体胶体金免疫层析试条方法的建立与效果评价

摘要/Abstract

摘要：

目的建立简便、快速检测美洲钩虫特异抗体胶体金免疫层析试条方法并评价其检测效能。方法将美洲钩虫成虫置液氮中研磨,通过膜蛋白表面活性剂浸泡、再经液氮反复冻融和硫酸铵沉淀提取成虫可溶性抗原。用制备的成虫可溶性抗原作为包被抗原,以胶体金标记G蛋白为检测探针,制备检测钩虫特异抗体的免疫层析试条。用该试条检测美洲钩虫、蛔虫、鞭虫、日本血吸虫和刚地弓形虫等寄生虫感染者的血清以及健康人血清,评价该试条检测的敏感性和特异性,同时用ELISA进行平行检测,以评价该试条的检测效能。结果制备了检测钩虫特异抗体的胶体金免疫层析试条。用该试条和ELISA法分别检测美洲钩虫、蛔虫、鞭虫、血吸虫、刚地弓形虫感染者血清95、10、11、10和10份,健康人血清74份。试条和ELISA检测美洲钩虫感染者血清的敏感性分别为88.4%（84/95）和90.5%（86/95）,检测74份健康人血清假阳性率分别为4.1%（3/74）和6.8%（5/74）;与蛔虫感染者血清的交叉反应分别为2/10和4/10,与鞭虫感染者血清的交叉反应分别为1/11和4/11,试条法与血吸虫和弓形虫感染者血清无交叉反应,ELISA法与血吸虫感染者的交叉反应为1/10,与弓形虫感染者血清无交叉反应。试条法与ELISA法的特异性分别为94.9%（109/115）和88.7%（102/115）,检测效能分别为91.9%和89.5%。试条法与ELISA法敏感性、特异性的差异均无统计学意义（P > 0.05）。结论以美洲钩虫成虫可溶性抗原制备的快速检测美洲钩虫特异抗体胶体金免疫层析试条检测美洲钩虫感染者血清的敏感性和特异性均较高。

关键词: 钩虫病, 美洲钩虫, 可溶性抗原, 免疫层析试条, 诊断

Abstract:

Objective To establish and evaluate colloid gold-labeled immunochromatographic test strip for rapid and convenient detection of antibody against Necator americanus. Methods Adult worms of N. americanus was ground in liquid nitrogen, immersed in membrane protein surfactant, followed by repeated freezing and thawing in liquid nitrogen and ammonium sulfate precipitation, to extract soluble antigen. The colloid gold-labeled immunochromatographic test strip was established using the soluble antigen as a coating antigen and the colloidal gold-labeled G protein as the detective probe. This test strip was used to examine sera from patients infected with N. americanus, Ascaris lumbricoides, Trichuris trichiura, Schistosoma japonicum and Toxoplasma gondii and sera from healthy participants, to evaluate the sensitivity and specificity of the assay, accompanied by ELISA as a parallel control to evaluate the detection efficacy. Results The colloid gold-labeled immunochromatographic test strip for detecting antibody against N. americanus was established. This test was performed to examine sera from patients infected with N. americanus (95 samples), A. lumbricoides (10 samples), T. trichiura (11 samples), S. japonicum (10 samples) and T. gondii (10 samples) and 74 serum samples from healthy participants, accompanied by ELISA as a parallel. The sensitivity of the colloid gold-labeled immunochromatographic strip test and ELISA in detecting N. americanus was 88.4% （84/95) and 90.5% （86/95), respectively, with false positive rates of 4.1% （3/74） and 6.8% （5/74） in 74 healthy serum samples, with rates of cross reaction with ascariasis being 2/10 and 4/10, rates of cross reaction with trichuriasis being 1/11 and 4/11. The strip test had no cross reaction with schistosomiasis or toxoplasmosis, while ELISA had a cross reaction rate of 1/10 with schistosomiasis but no cross reaction with toxoplasmosis. The specificity of the strip test and ELISA was 94.9% (109/115) and 88.7% (102/115), and the diagnostic efficacy was 91.9% and 89.5%, respectively. There was no significant difference in sensitivity （P > 0.05） or specificity （P > 0.05） between the strip test and ELISA. Conclusion The colloid gold-labeled immunochromatographic test strip shows relatively high sensitivity and specificity in diagnosis of ancylostomiasis.

Key words: Ancylostomiasis, Necator americanus, Soluble antigen, Immunochromatographic strip, Diagnosis

中图分类号:

R383.132

石锋, 杨益, 汪俊云, 杨玥涛, 高春花. 快速检测美洲钩虫特异抗体胶体金免疫层析试条方法的建立与效果评价[J]. 中国寄生虫学与寄生虫病杂志, 2018, 36(2): 119-123.

Feng SHI, Yi YANG, Jun-yun WANG, Yue-tao YANG, Chun-hua GAO. Establishment and evaluation of colloid gold-labeled immunochromatographic test strip for rapid detection of antibody against Necator americanus[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2018, 36(2): 119-123.

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血清 Serum	检测数 No. detected	阳性数No. positives
血清 Serum	检测数 No. detected	试条法 Immunochromatographic strip test	ELISA法 Enzyme-linked immunosorbent assay
钩虫感染 Infected with Necator americanus	95	84	86
蛔虫感染 Infected with Ascaris lumbricoides	10	2	4
鞭虫感染 Infected with Trichuris trichiura	11	1	4
日本血吸虫感染 Infected with Schistosoma japonicum	10	0	1
弓形虫感染 Infected with Toxoplasma gondii	10	0	0
健康者Healthy participants	74	3	4

试条法 Immunochromatographic strip test	ELISA法 Emzyme-linked immunosorbent assay		合计 Total
试条法 Immunochromatographic strip test	阳性Positive	阴性Negative	合计 Total
阳性Positive	84	0	84
阴性Negative	2	9	11
合计 Total	86	9	95