De Lima Rivero A R, Farías Tamoy M N, Tortolero Leal E, Navarro Aguilera M C, Contreras Alvarez V T
Laboratorio Protozoología, Centro Biología Molecular de Parásitos, Facultad Ciencias de la Salud, Universidad de Carabobo, Bárbula, Valencia, Venezuela.
Acta Cient Venez. 2001;52(4):235-47.
Protein and glycoprotein fractions are used for Chagas' disease diagnosis, but they are degraded by parasite endogenous proteases. Therefore protease inhibitors are used for their conservation increasing the antigen cost. The possibility of using protease-resistant glycosidic fractions could solve this problem allowing to obtain stable antigens, with a high sensitivity at a lower cost. This proposal is reinforced by the existence of anti-galactosyl antibodies against T. cruzi oligosaccharic fractions in sera from chagasic patients. The aim of this work was to obtain T. cruzi glycosidic fractions and to evaluate their possible use for Chagas' disease serologic diagnosis. Total protein and glycoproteins from the four stages of T. cruzi were obtained. The glycosidic fractions were obtained by exhaustive proteolysis with Proteinase K. Protein, glycoprotein and glycosidic profiles were analysed by SDS-PAGE followed by staining proteins (Coomassie/silver) or glycoproteins and glycosidic fractions (APABGP). Antigenicity of the different fractions was determined by Western Blot and luminography, using control hyperimmune serum anti-epimastigote and a "pool" of chagasic human sera. Finally, the capacity of the glycosidic fractions to discriminate chagasic and non-chagasic human sera and the sensitivity of the fractions respect to control serum, were determined by ELISA. Main findings were: a) there are peptides, glycopeptides and glycosidic fractions that are common and specific to parasite stages; b) there are more antigenic glycoproteins in epimastigotes and metacyclics than in trypomastigotes and amastigotes; c) there is a correlation between the glycosidic fraction-pattern and the host type; d) glycosidic fractions can be used as antigens to discriminate, by ELISA, chagasic and non chagasic patients, but show lower titers with respect to total proteic and glycoprotein antigens. It is concluded that it is possible to develop a diagnostic kit for Chagas' disease employing glycosidic fractions, eliminating the disadvantages of the current kits.
蛋白质和糖蛋白组分可用于恰加斯病的诊断，但它们会被寄生虫内源性蛋白酶降解。因此，蛋白酶抑制剂被用于保存这些组分，这增加了抗原成本。使用抗蛋白酶的糖苷组分的可能性可以解决这个问题，从而获得稳定的抗原，以较低成本实现高灵敏度。恰加斯病患者血清中存在针对克氏锥虫寡糖组分的抗半乳糖基抗体，这进一步支持了这一提议。这项工作的目的是获得克氏锥虫糖苷组分，并评估其在恰加斯病血清学诊断中的潜在用途。我们获取了克氏锥虫四个阶段的总蛋白和糖蛋白。通过用蛋白酶K进行彻底的蛋白水解获得糖苷组分。通过SDS-PAGE分析蛋白质、糖蛋白和糖苷谱，随后对蛋白质（考马斯亮蓝/银染）、糖蛋白和糖苷组分（APABGP）进行染色。使用对照超免疫抗无鞭毛体血清和恰加斯病患者血清“混合样本”，通过蛋白质印迹法和发光成像测定不同组分的抗原性。最后，通过ELISA测定糖苷组分区分恰加斯病患者和非恰加斯病患者血清的能力以及这些组分相对于对照血清的灵敏度。主要发现如下：a）存在寄生虫不同阶段共有的和特定的肽、糖肽和糖苷组分；b）无鞭毛体和后循环体中的抗原性糖蛋白比锥鞭毛体和无鞭毛体中的更多；c）糖苷组分模式与宿主类型之间存在相关性；d）糖苷组分可用作抗原，通过ELISA区分恰加斯病患者和非恰加斯病患者，但相对于总蛋白质和糖蛋白抗原，其滴度较低。结论是，有可能开发一种使用糖苷组分的恰加斯病诊断试剂盒，消除现有试剂盒的缺点。
