Jiacomini Isabella, Silva Sabrina K, Aubrey Nicolas, Muzard Julien, Chavez-Olortegui Carlos, De Moura Juliana, Billiald Philippe, Alvarenga Larissa M
Laboratório de Imunoquímica, Departamento de Patologia Básica, Universidade Federal do Paraná, Curitiba CEP 81531-980, PR, Brazil.
Université de Tours-INRA UMR 1282, Infectiologie et Santé Publique, 37200 Tours, France.
Immunol Lett. 2016 May;173:1-6. doi: 10.1016/j.imlet.2016.03.001. Epub 2016 Mar 10.
Bites by spiders from Loxosceles genus often lead to a wide variance in envenomation profile of patients and diagnosis is difficult due to the number of diseases that mimic loxoscelism. In such a context, it is of interest to consider the design of standardized recombinant colorimetric antibodies for diagnosis and specific detection of individual circulating toxins in biological fluids of envenomed patients. We have previously prepared a monoclonal murine IgG (LiMab7) that reacts with Loxosceles intermedia venom components of 32-35kDa and neutralizes the dermonecrotic activity of the venom. Here, we re-engineered LiMab7 into a colorimetric bifunctional protein consisting in the corresponding single-chain antibody fragment (scFv) fused to alkaline phosphatase (AP) of Escherichia coli. The immune tracer was tested in two different types of immunoassays and it proved to be efficient in both. Thus, this recombinant fusion protein (scFv-LiMab7/AP) can be used for rapid and specific immunotitration of L. intermedia venom with a linear range of 39-20000ng/mL and a detection limit of 39ng/mL without any cross-reaction.
巴西游走蛛属蜘蛛的叮咬常常导致患者的中毒情况差异很大，并且由于有多种疾病会模仿游走蛛中毒，所以诊断很困难。在这种情况下，考虑设计标准化的重组比色抗体用于中毒患者生物体液中个体循环毒素的诊断和特异性检测是很有意义的。我们之前制备了一种单克隆鼠IgG（LiMab7），它能与中间巴西游走蛛毒液中32 - 35kDa的成分发生反应，并中和毒液的皮肤坏死活性。在此，我们将LiMab7重新设计成一种比色双功能蛋白，该蛋白由与大肠杆菌碱性磷酸酶（AP）融合的相应单链抗体片段（scFv）组成。这种免疫示踪剂在两种不同类型的免疫分析中进行了测试，结果证明在两种分析中都很有效。因此，这种重组融合蛋白（scFv - LiMab7/AP）可用于快速、特异性地免疫滴定中间巴西游走蛛毒液，线性范围为39 - 20000ng/mL，检测限为39ng/mL，且无任何交叉反应。
