Sukprasert Sophida, Nonkhwao Siriporn, Thanwiset Thitijchaya, Keller Walter, Daduang Sakda
Chulabhorn International College of Medicine, Thammasat University (Rangsit Campus), Pathum Thani 12120, Thailand.
Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI), Khon Kaen University, Khon Kaen 40002, Thailand.
Toxins (Basel). 2025 Jul 28;17(8):373. doi: 10.3390/toxins17080373.
Phospholipase A1 (Ves a 1), a major toxin from venom, poses significant risks to allergic individuals. Nevertheless, the epitope determinants of Ves a 1 have not been characterized. Thus, identifying its linear B-cell epitopes is crucial for understanding envenomation mechanisms. In this study, we predicted and identified B-cell epitopes EP5 and EP6 as potential candidates. EP5 formed an α-helix at the active site of Ves a 1, whereas EP6 adopted an extended loop conformation. Both synthetic peptides were synthesized and evaluated for their inhibitory effects using immune-inhibitory assays with polyclonal antibodies (pAbs) targeting both native (nVes a 1) and recombinant (rVes a 1) forms. The Ves a 1 polyclonal antibodies (pAb-nVes a 1 and pAb-Ves a 1) were produced, and their specificity binding to Ves a 1 was confirmed by Western blot. Next, ELISA inhibition assays showed that EP5 and EP6 significantly blocked pAb binding to both nVes a 1 and rVes a 1. Dot blot and Western blot assays supported these findings, particularly with stronger inhibition toward rVes a 1. Furthermore, enzymatic assays indicated that nVes a 1 and rVes a 1 retained phospholipase activity. Immunoinformatics docking showed that EP5 and EP6 specifically bind to a single-chain variable fragment antibody (scFv) targeting PLA2. Molecular analysis revealed similar amino acid interactions to the template, suggesting effective paratope-epitope binding. These results support the potential of EP5 and EP6 for future diagnosis and therapy of . venom allergy.
磷脂酶A1（Ves a 1）是毒液中的一种主要毒素，对过敏个体构成重大风险。然而，Ves a 1的表位决定簇尚未得到鉴定。因此，确定其线性B细胞表位对于理解中毒机制至关重要。在本研究中，我们预测并鉴定了B细胞表位EP5和EP6作为潜在候选表位。EP5在Ves a 1的活性位点形成α螺旋，而EP6呈现伸展环构象。两种合成肽均被合成，并使用针对天然（nVes a 1）和重组（rVes a 1）形式的多克隆抗体（pAbs）通过免疫抑制试验评估其抑制作用。制备了Ves a 1多克隆抗体（pAb - nVes a 1和pAb - Ves a 1），并通过蛋白质印迹法确认了它们与Ves a 1的特异性结合。接下来，酶联免疫吸附测定（ELISA）抑制试验表明，EP5和EP6显著阻断pAb与nVes a 1和rVes a 1的结合。斑点印迹和蛋白质印迹试验支持了这些发现，特别是对rVes a 1的抑制作用更强。此外，酶活性测定表明nVes a 1和rVes a 1保留了磷脂酶活性。免疫信息学对接显示，EP5和EP6特异性结合靶向磷脂酶A2的单链可变片段抗体（scFv）。分子分析揭示了与模板相似的氨基酸相互作用，表明抗原互补位与表位有效结合。这些结果支持了EP5和EP6在未来毒液过敏诊断和治疗中的潜力。
