Andalusian Centre for Nanomedicine and Biotechnology-BIONAND, Málaga, Spain.
Allergy Research Group, Instituto de Investigación Biomédica de Málaga-IBIMA, Málaga, Spain.
Allergy. 2021 Oct;76(10):3183-3193. doi: 10.1111/all.14834. Epub 2021 May 5.
Amoxicillin (AX) is nowadays the β-lactam that more frequently induces immediate allergic reactions. Nevertheless, diagnosis of AX allergy is occasionally challenging due to risky in vivo tests and non-optimal sensitivity of in vitro tests. AX requires protein haptenation to form multivalent conjugates with increased size to be immunogenic. Knowing adduct structural features for promoting effector cell activation would help to improve in vitro tests. We aimed to identify the optimal structural requirement in specific cellular degranulation to AX using well-precised nanoarchitectures of different lengths.
We constructed eight Bidendron Antigens (BiAns) based on polyethylene glycol (PEG) linkers of different lengths (600-12,000 Da), end-coupled with polyamidoamine dendrons that were terminally multi-functionalized with amoxicilloyl (AXO). In vitro IgE recognition was studied by competitive radioallergosorbent test (RAST) and antibody-nanoarchitecture complexes by transmission electron microscopy (TEM). Their allergenic activity was evaluated using bone marrow-derived mast cells (MCs) passively sensitized with mouse monoclonal IgE against AX and humanized RBL-2H3 cells sensitized with polyclonal antibodies from sera of AX-allergic patients.
All BiAns were recognized by AX-sIgE. Dose-dependent activation responses were observed in both cellular assays, only with longer structures, containing spacers in the range of PEG 6000-12,000 Da. Consistently, greater proportion of immunocomplexes and number of antibodies per complex for longer BiAns were visualized by TEM.
BiAns are valuable platforms to study the mechanism of effector cell activation. These nanomolecular tools have demonstrated the importance of the adduct size to promote effector cell activation in AX allergy, which will impact for improving in vitro diagnostics.
阿莫西林（AX）是目前最常引起即刻过敏反应的β-内酰胺类抗生素。然而，由于体内试验风险大、体外试验灵敏度不理想，AX 过敏的诊断偶尔具有挑战性。AX 需要与蛋白质发生半抗原化反应，形成具有增加尺寸的多价缀合物，从而具有免疫原性。了解促进效应细胞活化的加合物结构特征将有助于改进体外试验。我们旨在使用不同长度的精确纳米结构，确定 AX 特异性细胞脱颗粒的最佳结构要求。
我们构建了 8 种基于不同长度（600-12000 Da）聚乙二醇（PEG）接头的双端抗原（BiAns），末端偶联有多胺树枝状大分子，末端多功能化有阿莫西林酰基（AXO）。通过竞争放射过敏原吸附试验（RAST）研究体外 IgE 识别，通过透射电子显微镜（TEM）研究抗体-纳米结构复合物。使用骨髓来源的肥大细胞（MCs）和人源化 RBL-2H3 细胞，被动致敏抗 AX 单克隆 IgE 和 AX 过敏患者血清多克隆抗体，评估其变应原活性。
所有 BiAns 均被 AX-sIgE 识别。在两种细胞试验中均观察到剂量依赖性的激活反应，仅在较长结构中观察到，其包含 PEG 6000-12000 Da 范围内的间隔物。通过 TEM 也可以观察到较长的 BiAns 具有更大比例的免疫复合物和每个复合物的抗体数量。
BiAns 是研究效应细胞活化机制的有价值的平台。这些纳米分子工具证明了加合物大小对促进 AX 过敏效应细胞活化的重要性，这将对改进体外诊断产生影响。
