Morón Belén, Bethune Michael T, Comino Isabel, Manyani Hamid, Ferragud Marina, López Manuel Carlos, Cebolla Angel, Khosla Chaitan, Sousa Carolina
Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad de Sevilla, Sevilla, Spain.
PLoS One. 2008 May 28;3(5):e2294. doi: 10.1371/journal.pone.0002294.
Celiac disease is a permanent intolerance to gluten prolamins from wheat, barley, rye and, in some patients, oats. Partially digested gluten peptides produced in the digestive tract cause inflammation of the small intestine. High throughput, immune-based assays using monoclonal antibodies specific for these immunotoxic peptides would facilitate their detection in food and enable monitoring of their enzymatic detoxification. Two monoclonal antibodies, G12 and A1, were developed against a highly immunotoxic 33-mer peptide. The potential of each antibody for quantifying food toxicity for celiac patients was studied.
Epitope preferences of G12 and A1 antibodies were determined by ELISA with gluten-derived peptide variants of recombinant, synthetic or enzymatic origin.
The recognition sequences of G12 and A1 antibodies were hexameric and heptameric epitopes, respectively. Although G12 affinity for the 33-mer was superior to A1, the sensitivity for gluten detection was higher for A1. This observation correlated to the higher number of A1 epitopes found in prolamins than G12 epitopes. Activation of T cell from gluten digested by glutenases decreased equivalently to the detection of intact peptides by A1 antibody. Peptide recognition of A1 included gliadin peptides involved in the both the adaptive and innate immunological response in celiac disease.
The sensitivity and epitope preferences of the A1 antibody resulted to be useful to detect gluten relevant peptides to infer the potential toxicity of food for celiac patients as well as to monitor peptide modifications by transglutaminase 2 or glutenases.
乳糜泻是对来自小麦、大麦、黑麦以及部分患者对燕麦中的麦醇溶蛋白产生永久性不耐受。在消化道中产生的部分消化的麸质肽会导致小肠炎症。使用针对这些免疫毒性肽的特异性单克隆抗体进行高通量免疫分析，将有助于在食物中检测它们，并能够监测其酶解毒过程。针对一种高度免疫毒性的33肽开发了两种单克隆抗体G12和A1。研究了每种抗体对乳糜泻患者食物毒性定量的潜力。
通过酶联免疫吸附测定（ELISA），使用重组、合成或酶解来源的麸质衍生肽变体，确定G12和A1抗体的表位偏好。
G12和A1抗体的识别序列分别是六聚体表位和七聚体表位。尽管G12对33肽的亲和力优于A1，但A1对麸质检测的灵敏度更高。这一观察结果与醇溶蛋白中发现的A1表位数量多于G12表位有关。由谷氨酰胺转氨酶消化麸质激活的T细胞与A1抗体对完整肽的检测同样降低。A1的肽识别包括参与乳糜泻适应性和先天性免疫反应的麦醇溶蛋白肽。
A1抗体的灵敏度和表位偏好可用于检测与麸质相关的肽，以推断食物对乳糜泻患者的潜在毒性，以及监测转谷氨酰胺酶2或谷氨酰胺转氨酶对肽的修饰。
