Mendes Thais Melo, Roma Eric Henrique, Costal-Oliveira Fernanda, Dhom-Lemos Lucas de Carvalho, Toledo-Machado Cristina Monerat, Bruna-Romero Oscar, Bartholomeu Daniella Castanheiras, Fujiwara Ricardo Toshio, Chávez-Olórtegui Carlos
Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, UFMG, Belo Horizonte-Minas Gerais, Brazil.
Departamento de Parasitologia, Instituto de Ciências Biológicas, UFMG, Belo Horizonte-Minas Gerais, Brazil.
PLoS Negl Trop Dis. 2017 May 30;11(5):e0005562. doi: 10.1371/journal.pntd.0005562. eCollection 2017 May.
Leishmaniasis is one of the most important zoonotic diseases spread in Latin America. Since many species are involved in dog infection with different clinical manifestations, the development of specific diagnostic tests is mandatory for more accurate disease control and vaccine strategies.
METHODOLOGY/PRINCIPAL FINDINGS: Seventy-five 15-mer peptides covering the sequence of recombinant Leishmania donovani virulence factor A2 (recLdVFA2) protein were prepared by Spot synthesis. Membrane-bound peptides immunoreactivity with sera from dogs immunized with recLdVFA2 and with a specific anti-recLdVFA2 monoclonal antibody allowed mapping of continuous B-cell epitopes. Five epitopes corresponding to the N-terminal region of recLdVFA2 (MKIRSVRPLVVLLVC, RSVRPLVVLLVCVAA, RPLVVLLVCVAAVLA, VVLLVCVAAVLALSA and LVCVAAVLALSASAE, region 1-28) and one located within the repetitive units (PLSVGPQAVGLSVG, regions 67-81 and 122-135) were identified. A 34-mer recLdVFA2-derived bi-epitope containing the sequence MKIRSVRPLVVLLVC linked to PLSVGPQAVGLSVG by a Gly-Gly spacer was chemically synthesized in its soluble form. The synthetic bi-epitope was used as antigen to coat ELISA plates and assayed with dog sera for in vitro diagnosis of canine visceral leishmaniasis (CVL). The assay proved to be highly sensitive (98%) and specific (99%).
CONCLUSIONS/SIGNIFICANCE: Our work suggests that synthetic peptide-based ELISA strategy may be useful for the development of a sensitive and highly specific serodiagnosis for CVL or other parasitic diseases.
利什曼病是在拉丁美洲传播的最重要的人畜共患疾病之一。由于有许多物种参与犬类感染且临床表现各异，因此开发特定的诊断测试对于更准确地控制疾病和制定疫苗策略至关重要。
方法/主要发现：通过点合成制备了覆盖重组杜氏利什曼原虫毒力因子A2（recLdVFA2）蛋白序列的75个15肽。膜结合肽与用recLdVFA2免疫的犬血清以及特异性抗recLdVFA2单克隆抗体的免疫反应性，使得能够绘制连续B细胞表位图谱。鉴定出了5个对应于recLdVFA2 N端区域（MKIRSVRPLVVLLVC、RSVRPLVVLLVCVAA、RPLVVLLVCVAAVLA、VVLLVCVAAVLALSA和LVCVAAVLALSASAE，区域1 - 28）的表位以及1个位于重复单元内（PLSVGPQAVGLSVG，区域67 - 81和122 - 135）的表位。通过化学合成制备了一种34肽的recLdVFA2衍生双表位，其包含通过甘氨酸 - 甘氨酸间隔连接到PLSVGPQAVGLSVG的序列MKIRSVRPLVVLLVC的可溶形式。该合成双表位用作抗原包被酶联免疫吸附测定（ELISA）板，并与犬血清一起用于犬内脏利什曼病（CVL）的体外诊断。该测定法被证明具有高度敏感性（98%）和特异性（99%）。
结论/意义：我们的工作表明，基于合成肽的ELISA策略可能有助于开发用于CVL或其他寄生虫病的敏感且高度特异的血清学诊断方法。
