Postgraduate Program in Health Sciences, State University of Maringá, Maringá, Paraná, Brazil.
Postgraduate Program in Biosciences Applied to Pharmacy, State University of Maringá, Paraná, Brazil.
Diagn Microbiol Infect Dis. 2014 Apr;78(4):411-7. doi: 10.1016/j.diagmicrobio.2013.08.020. Epub 2014 Jan 28.
This study evaluated the reactivity of membrane antigens of Leishmania (Viannia) braziliensis for the diagnosis of ACL by enzyme immunoassay (EIA). Promastigotes of L. (V.) braziliensis were grown in medium 199 and lysed in a sonicator. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting showed that specific proteins of L. (V.) braziliensis (apparent molecular weights 36 kDa and 48-56 kDa) were recognized by sera from ACL patients. These proteins were eluted from the SDS-PAGE and tested in EIA-IgG with sera from ACL patients, healthy individuals, patients with toxoplasmosis, paracoccidioidomycosis, syphilis, tuberculosis, leprosy, and Chagas disease. The EIA-IgG with membrane antigens allowed us to distinguish patients with ACL from healthy individuals and patients with other diseases (P < 0.0001), and showed a sensitivity of 93.3% and specificity of 90.8%, not including Chagas disease patients. 2D-SDS-PAGE followed by Western blotting was performed to improve the characterization of the antigens, and showed a component with isoelectric points near the acid pH side and apparent molecular weights of 48-56 kDa. The results showed good sensitivity and specificity of EIA-IgG with membrane antigens, indicating their potential use for diagnosis of ACL, as well as seroepidemiological surveys and follow-up of clinically cured patients.
本研究通过酶联免疫吸附试验（EIA）评估了巴西利什曼原虫（Viannia）膜抗原的反应性，用于 ACL 的诊断。巴西利什曼原虫的前鞭毛体在 199 培养基中生长，并在超声仪中裂解。十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）和 Western blot 显示，巴西利什曼原虫的特定蛋白（表观分子量 36 kDa 和 48-56 kDa）被 ACL 患者的血清识别。这些蛋白从 SDS-PAGE 洗脱后，在 EIA-IgG 中与 ACL 患者、健康个体、弓形虫病、副球孢子菌病、梅毒、结核病、麻风病和恰加斯病患者的血清进行了测试。膜抗原的 EIA-IgG 使我们能够将 ACL 患者与健康个体和其他疾病患者区分开来（P < 0.0001），其敏感性为 93.3%，特异性为 90.8%，不包括恰加斯病患者。进行了 2D-SDS-PAGE 后 Western blot 以改善抗原的表征，并显示出等电点在酸性 pH 侧附近且表观分子量为 48-56 kDa 的组分。结果表明，膜抗原的 EIA-IgG 具有良好的敏感性和特异性，表明其在 ACL 的诊断、血清流行病学调查和临床治愈患者的随访中具有潜在用途。
