利用精制的N-月桂酰肌氨酸不溶性蛋白开发用于猫抓病血清学诊断的高度特异性抗汉赛巴尔通体IgM酶联免疫吸附测定法。
Development of a Highly Specific IgM Enzyme-Linked Immunosorbent Assay for Bartonella henselae Using Refined N-Lauroyl-Sarcosine-Insoluble Proteins for Serodiagnosis of Cat Scratch Disease.
作者信息
Otsuyama Ken-Ichiro, Tsuneoka Hidehiro, Kondou Kaori, Yanagihara Masashi, Tokuda Nobuko, Shirasawa Bungo, Ichihara Kiyoshi
机构信息
Department of Medical Education, Graduate School of Medicine, Yamaguchi University, Ube, Japan.
Department of Clinical Laboratory Science, Faculty of Health Science, Graduate School of Medicine, Yamaguchi University, Ube, Japan
出版信息
J Clin Microbiol. 2016 Apr;54(4):1058-64. doi: 10.1128/JCM.03009-15. Epub 2016 Feb 10.
The conventional anti-Bartonella henselaeIgM enzyme-linked immunosorbent assay (IgM-ELISA) methods for diagnosing cat scratch disease (CSD) remain poor in both sensitivity and specificity. We sought to develop an IgM-ELISA with improved accuracy in the serodiagnosis of CSD by exploring the antigens that are most suitable for an ELISA. We prepared 5 different protein antigens: antigen I (sonicatedB. henselaewhole-cell antigen), antigen II (N-lauroyl-sarcosine-insoluble antigen), antigen III (processed sarcosine-soluble antigen), and antigen IV and antigen V (sarcosine-insoluble and sarcosine-soluble antigens refined by DEAE-Sepharose Fast Flow ion-exchange chromatography). The IgM antibodies in the sera of 47 patients with clinically suspected CSD (24 definite, 23 suspected) and of 85 healthy individuals were examined by ELISAs using the 5 antigens, and the results were compared with those of an IgM indirect fluorescent antibody assay (IgM-IFA). In a reference panel, which consisted of 5 positive and 5 negative sera, antigen I and antigen III failed to distinguish between the two statuses, whereas the other three antigens succeeded in distinguishing between them. When the cutoff value was set at the 98th percentile of the ELISA index for healthy individuals, the sensitivity of IgM-IFA for the 24 cases of definite CSD was 54%, whereas the sensitivities of the IgM-ELISAs with antigen II, IV, and V were 75%, 83%, and 75%, respectively. The sensitivities of these three IgM-ELISAs for all 47 of the clinically suspected cases were 49%, 64%, and 51%, respectively. In contrast, the sensitivity of IgM-IFA was 28%. These results indicate that the refined sarcosine-insoluble proteins (antigen IV), which possessed the highest specificity among the 5 antigens, are the most appropriate for developing an IgM-ELISA for the highly specific serodiagnosis of CSD.
用于诊断猫抓病（CSD）的传统抗汉赛巴尔通体IgM酶联免疫吸附测定（IgM-ELISA）方法在敏感性和特异性方面仍然较差。我们试图通过探索最适合ELISA的抗原，开发一种在CSD血清诊断中准确性更高的IgM-ELISA。我们制备了5种不同的蛋白质抗原：抗原I（超声处理的汉赛巴尔通体全细胞抗原）、抗原II（N-月桂酰肌氨酸不溶性抗原）、抗原III（处理后的肌氨酸可溶性抗原）以及抗原IV和抗原V（通过DEAE-琼脂糖快速流动离子交换色谱法精制的肌氨酸不溶性和肌氨酸可溶性抗原）。使用这5种抗原，通过ELISA检测了47例临床疑似CSD患者（24例确诊，23例疑似）和85名健康个体血清中的IgM抗体，并将结果与IgM间接荧光抗体测定（IgM-IFA）的结果进行比较。在一个由5份阳性血清和5份阴性血清组成的参考组中，抗原I和抗原III无法区分两种状态，而其他三种抗原成功区分了它们。当将临界值设定为健康个体ELISA指数的第98百分位数时，IgM-IFA对24例确诊CSD病例的敏感性为54%，而使用抗原II、IV和V的IgM-ELISA的敏感性分别为75%、83%和75%。这三种IgM-ELISA对所有47例临床疑似病例的敏感性分别为49%、64%和51%。相比之下，IgM-IFA的敏感性为28%。这些结果表明，在这5种抗原中特异性最高的精制肌氨酸不溶性蛋白（抗原IV）最适合开发用于CSD高特异性血清诊断的IgM-ELISA。
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