Laboratório de Tecnologia Diagnóstica, Instituto de Tecnologia em Imunobiológicos, Fundação Oswaldo Cruz, RJ, Brazil.
Parasite Immunol. 2012 Jan;34(1):1-7. doi: 10.1111/j.1365-3024.2011.01334.x.
The present work describes the isolation and purification of two Leishmania chagasi (= syn. Leishmania infantum) recombinant proteins, rLci2B and rLci1A, and their use in the development of an immunoassay for the diagnostic of canine leishmaniasis. After protein expression and cell disruption, rLci2B was purified by immobilized metal affinity chromatography followed by size exclusion chromatography, whereas rLci1A, expressed as an inclusion body, was treated with urea and purified by anion-exchange chromatography. Homogeneities were ascertained by denaturing gel electrophoresis (MW (rLci2B) = 46,370; MW(rLci1A) = 88,400), isoelectric focusing (pI (rLci2B) = 5·91; pI (rLci1A) = 6·01) and Western blot. An indirect ELISA was developed using the purified antigens rLci2B and rLci1A and a leishmaniasis canine serum panel (n = 256). The ELISA showed 100% sensitivity and 95% specificity for rLci2B and 96% sensitivity and 92% specificity for rLci1A. The purified proteins did not present cross-reactivity with sera from dogs infected with Trypanosoma caninum, Babesia canis and Ehrlichia canis. Cross-reaction was verified with sera from dogs infected with Leishmania brasiliensis (11·7% for rLci2B and 2·9% for rLci1A). Based on ELISA results, it is suggested the use of rLci2B and rLci1A as antigens in an alternative serological assay for diagnostic of canine leishmania.
本工作描述了两种莱什曼原虫（=同义名利什曼原虫婴儿）重组蛋白 rLci2B 和 rLci1A 的分离和纯化，以及它们在用于诊断犬利什曼病的免疫测定中的应用。在蛋白表达和细胞破碎后，rLci2B 通过固定金属亲和层析随后通过分子筛层析进行纯化，而作为包涵体表达的 rLci1A 则用尿素处理并通过阴离子交换层析进行纯化。变性凝胶电泳（MW（rLci2B）=46,370；MW（rLci1A）=88,400）、等电聚焦（pI（rLci2B）=5·91；pI（rLci1A）=6·01）和 Western blot 确定了均一性。使用纯化抗原 rLci2B 和 rLci1A 以及犬利什曼病血清面板（n=256）开发了间接 ELISA。ELISA 对 rLci2B 的敏感性为 100%，特异性为 95%，对 rLci1A 的敏感性为 96%，特异性为 92%。纯化的蛋白与感染了锥虫、犬巴贝斯虫和埃立克体的犬血清没有交叉反应。与感染了巴西利什曼原虫的犬血清的交叉反应得到了验证（rLci2B 为 11·7%，rLci1A 为 2·9%）。根据 ELISA 结果，建议使用 rLci2B 和 rLci1A 作为抗原，用于替代犬利什曼病的血清学诊断。
