食物过敏血清sIgE抗体致病活性精准检测的研究

Accurate determination of the pathogenic activity of sIgE antibody in serum is of great value to the diagnosis and treatment of food allergy. So far, only by using the basophil activation test can we achieve it. The method, taking advantage of the cell biology principle, imitates the activation of sensitized cells in vivo and thus can well reflect the activity of sIgE antibody. However, many limitations have restricted it from wide application clinically. There are studies that have shown the close correlation of the pathogenic activity of sIgE antibody with the epitope it recognizes. The "linear epitopes" of food allergens were found to play an important role in the induction of sIgE antibody production and the activation of sensitized cells. Our previous studies have also demonstrated the good reactivity of recombinant linear epitope fusion protein with sIgE antibodies in serum. Therefore, we propose a hypothesis that the bioinformatic epitopes of allergens can distinguish the pathogenic activity of the sIgE antibody, and by using recombinant bioinformatic epitopes fusion protein as known antigen, we can realize the accurate determination of the pathogenic activity of sIgE antibody. For this, we will use the major allergens from milk and hen’s egg as the research object and screen for bioinformatic epitopes. By using recombinant bioinformatic epitopes fusion protein as the coating antigen, we will establish a light initiated chemiluminescent assay for accurate measurement of the pathogenic activity of sIgE antibody.

血清sIgE抗体的致病活性测定对食物过敏诊疗具有重要价值。目前仅嗜碱性粒细胞激活试验能够实现sIgE抗体致病活性的精准检测，它是基于细胞生物学原理，模拟体内致敏细胞的激活过程，结果反映sIgE抗体的活性，但因诸多限制尚不能广泛应用于临床。有研究显示，体内sIgE抗体的致病活性与其所识别的抗原表位密切相关。食物过敏原的“线性表位”在诱导sIgE抗体产生、致敏细胞激活过程中发挥了重要作用。我们前期实验证明：人工重组的线性表位“串联体”与血清sIgE抗体显示出较好反应性。为此，我们提出假设：生物信息表位能够区分过敏原sIgE抗体的致病活性，用重组的生物信息表位“嵌合体”作为已知抗原，可实现对sIgE抗体致病活性的精准检测。本课题以牛奶和禽蛋的主要过敏原为研究对象，筛选获得与sIgE抗体致病活性相关的生物信息表位，制备生物信息表位嵌合体，建立光激化学发光分析的sIgE抗体致病活性检测方法。

血清过敏原特异性IgE（specific IgE，sIgE）是诊断食物过敏（food allergy，FA）重要实验指标，但现有实验手段不能精准区分sIgE抗体的致病活性，以致出现检测结果与临床表现相矛盾的现象。本研究总体目标是证实“血清sIgE抗体的致病活性与其所识别的线性表位相关”的科学假设，筛选与血清sIgE抗体致病活性相关的生物信息表位。采用由生物信息表位串联组成的“嵌合体”重组蛋白作为已知抗原，基于光激化学发光（LICA）平台，建立血清sIgE抗体致病活性的精准检测方法。本项目首先从分子水平上完成了禽蛋5种过敏组分（Gal d 1、Gal d 2、Gal d 3、Gal d 4、Gal d 5）、牛奶5种过敏组分（Bos d 4、Bos d 5、Bos d 9、Bos d 11、Bos 12）组分诊断（CRD）的临床价值研究。从亚分子水平分别了完成Bos d 5 、Gal d 1、Pen a 1的sIgE和sIgG4的表位谱分析，证实存在个体差异性和生物信息表位，提出sIgE抗体独特型异质性程度和临床表现具有一定相关性。然后，构建了高表达IgE Fc受体和荧光蛋白分子的细胞系，建立了血清样本sIgE-BAT方法，用于评估sIgE的生物活性。此方法操作复杂，受体表达欠稳定，不易推广。此外，基于光激化学发光分析，建立了tIgE、sIgE、CRD-sIgE、sIgG4检测方法，分析性能符合临床实验室要求。本项目从分子水平上，证实食物过敏患者体内过敏原sIgE抗体存在异质性，各组分sIgE抗体出现频率和诊断价值存在差异，CRD可以提高检测准确性并为精准脱敏治疗提供依据。从亚分子水平上，食物过敏患者体内同一过敏原sIgE抗体的独特型存在异质性，sIgE独特型异质性程度和临床表现相关联并有望成为食物过敏诊断的新型指标。光激化学发光分析作为均相免疫分析技术，具有“免分离”、“高敏感”、“高通量”等特点，完全适合食物过敏tIgE、sIgE、CRD-sIgE、sIgG4等指标的检测，具有较好转化价值。