Jankovicova Barbora, Rosnerova Sarka, Slovakova Marcela, Zverinova Zuzana, Hubalek Martin, Hernychova Lenka, Rehulka Pavel, Viovy Jean-Louis, Bilkova Zuzana
Department of Biological and Biochemical Sciences, University of Pardubice, Pardubice, Czech Republic.
J Chromatogr A. 2008 Oct 3;1206(1):64-71. doi: 10.1016/j.chroma.2008.07.062. Epub 2008 Jul 26.
Specific allergen immunotherapy is frequently associated with adverse reactions. Several strategies are being developed to reduce the allergenicity while maintaining the therapeutic benefits. Peptide immunotherapy is one such approach. Methods for the simple and rapid identification of immunogenic epitopes of allergens (i.e. allergenic epitopes) are ongoing and could potentially lead to peptide-based vaccines. An epitope extraction technique, based on biofunctionalized magnetic microspheres self-organized under a magnetic field in a channel of a simple microfluidic device fabricated from polydimethylsiloxane, was applied in the isolation and identification of prospective allergenic epitopes. Similarly to chromatographic column separations, the easily replaceable plug of self-organized beads in the channel benefits especially from an even larger surface-to-volume ratio and an enhanced interaction of the surfaces with passing samples. Ovalbumin, the major protein of egg white and a typical representative of food allergens, was selected as the model molecule. Highly resistant ovalbumin was at first efficiently digested by a magnetic proteolytic reactor with trypsin treated with l-1-tosylamido-2-phenylethyl chloromethyl ketone and the second step, i.e. capture of allergenic epitopes from the mixture of peptides, was performed by a magnetic immunoaffinity carrier with orientedly immobilized rabbit anti-ovalbumin IgG molecules. Captured peptides were released with 0.05% trifluoroacetic acid. The elution fractions were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The peptide fragment of ovalbumin HIATNAVLFFGR (m/z: 1345.75, position: 371-382) was identified as a relevant allergenic epitope in this way. Such a microfluidic magnetic force-based epitope extraction technique applied in the epitope mapping of ovalbumin has the potential to be a significant step towards developing safe and cost-effective epitope-based vaccines.
特异性变应原免疫疗法常与不良反应相关。目前正在研发多种策略以降低变应原性，同时保持治疗效果。肽免疫疗法就是其中一种方法。用于简单快速鉴定变应原免疫原性表位（即变应原表位）的方法正在不断发展，有望开发出基于肽的疫苗。一种基于生物功能化磁性微球的表位提取技术被应用于分离和鉴定潜在的变应原表位，该技术是在由聚二甲基硅氧烷制成的简单微流控装置通道中，在磁场作用下自组装而成。与色谱柱分离类似，通道中易于更换的自组装磁珠塞尤其受益于更大的表面积与体积比以及表面与通过样品之间增强的相互作用。蛋清中的主要蛋白质卵清蛋白作为食物变应原的典型代表，被选作模型分子。首先用经l-1-甲苯磺酰氨基-2-苯乙基氯甲基酮处理的胰蛋白酶，通过磁性蛋白水解反应器高效消化高抗性卵清蛋白，第二步，即从肽混合物中捕获变应原表位，由定向固定有兔抗卵清蛋白IgG分子的磁性免疫亲和载体完成。捕获的肽用0.05%三氟乙酸洗脱。洗脱级分通过基质辅助激光解吸/电离飞行时间质谱分析。通过这种方式，卵清蛋白的肽片段HIATNAVLFFGR（m/z：1345.75，位置：371 - 382）被鉴定为相关变应原表位。这种基于微流控磁力的表位提取技术应用于卵清蛋白的表位图谱分析，有可能成为朝着开发安全且经济高效的基于表位的疫苗迈出的重要一步。
