Khabiri A R, Bagheri F, Alimohammadian M H, Assmar M, Nadaf S R
Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran.
Exp Parasitol. 2005 Dec;111(4):239-43. doi: 10.1016/j.exppara.2005.08.011. Epub 2005 Oct 4.
A series of hybridomas was produced by fusion of SP2/0 myeloma cells with spleen cells of mice immunized with Leishmania major (L. major). The reactivity of secreted monoclonal antibodies (mAbs) was evaluated against available leishmanin antigen by enzyme linked immunosorbent assay. Only one hybridoma designated as 7F9 secreted IgG1 mAb which was shown to be reactive with leishmanin. This mAb was further tested against four species of Leishmania (L. donovani, L. tropica, L. infantum, L. major) and a recombinant gp63. Among the four species tested it was shown to be only reactive with promastigotes of L. major. The antigen recognized by this mAb was purified and analyzed from both sonicated and supernatant cultures of L. major by immunoaffinity chromatography and reverse phase high performance liquid chromatography. The purified antigen, which gave a single band of 56kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis elicited a strong delayed-type hypersensitivity (DTH) reaction in guinea pigs sensitized with L. major. It was almost of the same degree as that produced by leishmanin. These results suggest that an L. major-specific antigen is an alternative as a specific diagnostic skin test reagent, which could lead to a better understanding of the mechanism of DTH in L. major.
通过将SP2/0骨髓瘤细胞与用硕大利什曼原虫（L. major）免疫的小鼠脾细胞融合，产生了一系列杂交瘤。通过酶联免疫吸附测定法评估分泌的单克隆抗体（mAb）对可用利什曼原虫素抗原的反应性。只有一个命名为7F9的杂交瘤分泌与利什曼原虫素反应的IgG1 mAb。进一步用四种利什曼原虫（杜氏利什曼原虫、热带利什曼原虫、婴儿利什曼原虫、硕大利什曼原虫）和一种重组gp63对该mAb进行测试。在所测试的四种利什曼原虫中，它仅与硕大利什曼原虫的前鞭毛体反应。通过免疫亲和色谱和反相高效液相色谱从硕大利什曼原虫的超声破碎物和上清液培养物中纯化并分析该mAb识别的抗原。纯化的抗原在十二烷基硫酸钠聚丙烯酰胺凝胶电泳上呈现一条56kDa的单条带，在用硕大利什曼原虫致敏的豚鼠中引发强烈的迟发型超敏反应（DTH）。其程度几乎与利什曼原虫素产生的反应相同。这些结果表明，一种硕大利什曼原虫特异性抗原可作为一种特异性诊断皮肤试验试剂，这可能有助于更好地理解硕大利什曼原虫中DTH的机制。
