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小豆蔻酰化蛋白-3 作为重组抗原和含有其线性 B 细胞表位的合成肽在犬内脏利什曼病和人体皮肤利什曼病血清学诊断中的潜在应用。

Potential application of small myristoylated protein-3 evaluated as recombinant antigen and a synthetic peptide containing its linear B-cell epitope for the serodiagnosis of canine visceral and human tegumentary leishmaniasis.

机构信息

Programa de Pós-Graduação em Ciências da Saúde: Infectologia e Medicina Tropical, Faculdade de Medicina, Universidade Federal de Minas Gerais, Belo Horizonte, 30130-100, Minas Gerais, Brazil.

Programa de Pós-Graduação em Ciências da Saúde, Universidade do Extremo Sul Catarinense, Criciúma, 88806-000, Santa Catarina, Brazil.

出版信息

Immunobiology. 2019 Jan;224(1):163-171. doi: 10.1016/j.imbio.2018.09.003. Epub 2018 Sep 21.

Abstract

Serological tests are important tools for the diagnosis of Leishmania infection. However, they are not effective markers to diagnose asymptomatic cases of visceral leishmaniasis (VL) and patients developing tegumentary leishmaniasis (TL), since antileishmanial antibodies can be encountered in low levels resulting in false-negative results in the serological trials. In this context, antigens able to be recognized by antibodies in sera from both VL and TL patients will be desirable to be employed in a more sensitivity and specific diagnosis of disease. In the present study, a conserved Leishmania protein, small myristoylated protein-3 (SMP-3), which was showed to be conserved in different Leishmania species and an effective vaccine candidate against Leishmania infantum infection in a murine model, was cloned and the recombinant protein was evaluated as a serological marker for the diagnosis of human TL and canine VL. In addition, a linear B cell-specific epitope (MQKDEESGEFKCEL) was identified, synthetized and also investigated as a serological marker. As antigen controls, rA2 protein and antigenic Leishmania extracts (SLA) were used. Results showed that ELISA-rSMP-3 and ELISA-Peptide presented sensitivity and specificity values higher than 90% in both diseases in humans and canids, having identified all asymptomatic cases and did not present cross-reaction with cross-reactivity diseases in both mammalian hosts. On the other hand, sensitivity and specificity values were worst when rA2 or SLA were used as antigens in humans and dogs. In conclusion, results showed the efficacy and Leishmania SMP-3 protein, employed as a recombinant antigen or a B cell epitope, for the improvement of the serodiagnosis of human TL and canine VL. This candidate can be tested in other diagnostic platforms, such as rapid immunochromatographic dipstick tests, aiming its use in epidemiological studies in remote areas where laboratories are not readily accessible for conventional assays.

摘要

血清学检测是诊断利什曼病感染的重要工具。然而,它们并不是诊断无症状内脏利什曼病(VL)和发生皮肤利什曼病(TL)患者的有效标志物,因为抗利什曼原虫抗体的水平可能较低,导致血清学检测出现假阴性结果。在这种情况下,能够被 VL 和 TL 患者血清中的抗体识别的抗原将是更敏感和特异的疾病诊断所需要的。在本研究中,我们克隆了一种保守的利什曼原虫蛋白,小豆蔻酰化蛋白-3(SMP-3),该蛋白在不同的利什曼原虫种中具有保守性,并且在小鼠模型中是有效的利什曼原虫婴儿感染疫苗候选物,然后评估了重组蛋白作为人类 TL 和犬 VL 的血清学诊断标志物。此外,还鉴定、合成了一个线性 B 细胞特异性表位(MQKDEESGEFKCEL),并对其进行了研究。作为抗原对照,使用了 rA2 蛋白和抗原性利什曼原虫提取物(SLA)。结果表明,ELISA-rSMP-3 和 ELISA-Peptide 在人类和犬科动物的两种疾病中均具有高于 90%的敏感性和特异性,能够识别所有无症状病例,并且与两种哺乳动物宿主的交叉反应性疾病无交叉反应。另一方面,当 rA2 或 SLA 用作人类和犬科动物的抗原时,敏感性和特异性值最差。总之,结果表明,SMP-3 蛋白或其 B 细胞表位作为重组抗原,可提高人类 TL 和犬科 VL 的血清学诊断效果。该候选物可以在其他诊断平台(如快速免疫层析试纸条检测)中进行测试,旨在用于实验室难以获得常规检测的偏远地区的流行病学研究。

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