Negm Ola H, Hamed Mohamed, Monaghan Tanya M
Breast Surgery Group, Division of Medical Sciences and Graduate Entry Medicine, School of Medicine, Queen's Medical Centre, University of Nottingham; Medical Microbiology and Immunology Department, Faculty of Medicine, Mansoura University;
Breast Surgery Group, Division of Medical Sciences and Graduate Entry Medicine, School of Medicine, Queen's Medical Centre, University of Nottingham; Medical Microbiology and Immunology Department, Faculty of Medicine, Mansoura University.
J Vis Exp. 2018 Jun 15(136):57399. doi: 10.3791/57399.
We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in human sera and in separate preparations of polyclonal intravenous immunoglobulin (IVIg). The protocol describes the methodological steps involved in sample preparation, printing of arrays, assay procedure, and data analysis. In addition, this protocol could be further developed to incorporate diverse clinical samples including plasma and cell culture supernatants. We show how protein microarray can be used to determine a combination of isotype (IgG, IgA, IgM), subclass (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), and strain-specific antibodies to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B-only expressing strain (CCUG 20309), a precursor form of a B fragment of binary toxin, pCDTb, ribotype-specific whole surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). During the experiment, microarrays are probed with sera from individuals with C. difficile infection (CDI), individuals with cystic fibrosis (CF) without diarrhea, healthy controls (HC), and from individuals pre- and post-IVIg therapy for the treatment of CDI, combined immunodeficiency disorder, and chronic inflammatory demyelinating polyradiculopathy. We encounter significant differences in toxin neutralization efficacies and multi-isotype specific antibody levels between patient groups, commercial preparations of IVIg, and sera before and following IVIg administration. Also, there is a significant correlation between microarray and enzyme-linked immunosorbent assay (ELISA) for antitoxin IgG levels in serum samples. These results suggest that microarray could become a promising tool for profiling antibody responses to C.difficile antigens in vaccinated or infected humans. With further refinement of antigen panels and a reduction in production costs, we anticipate that microarray technology may help optimize and select the most clinically useful immunotherapies for C. difficile infection in a patient-specific manner.
我们详细概述了一种新型高通量蛋白质微阵列分析方法，用于测定人血清和多克隆静脉注射免疫球蛋白（IVIg）单独制剂中的抗艰难梭菌抗体水平。该方案描述了样品制备、阵列打印、检测程序和数据分析所涉及的方法步骤。此外，该方案可进一步改进，以纳入包括血浆和细胞培养上清液在内的多种临床样本。我们展示了蛋白质微阵列如何用于确定同型（IgG、IgA、IgM）、亚类（IgG1、IgG2、IgG3、IgG4、IgA1、IgA2）以及针对高度纯化的全艰难梭菌毒素A和B（毒素型0，菌株VPI 10463，核糖体分型087）、仅表达艰难梭菌毒素B的菌株（CCUG 20309）的毒素B、二元毒素B片段的前体形式pCDTb、核糖体分型特异性全表面层蛋白（SLP；001、002、027）和对照蛋白（破伤风类毒素和白色念珠菌）的菌株特异性抗体的组合。在实验过程中，用来自艰难梭菌感染（CDI）患者、无腹泻的囊性纤维化（CF）患者、健康对照（HC）以及接受IVIg治疗前后的CDI、联合免疫缺陷病和慢性炎症性脱髓鞘性多发性神经根病患者的血清对微阵列进行检测。我们发现患者组、IVIg商业制剂以及IVIg给药前后的血清之间在毒素中和效力和多同型特异性抗体水平上存在显著差异。此外，血清样本中抗毒素IgG水平的微阵列检测与酶联免疫吸附测定（ELISA）之间存在显著相关性。这些结果表明，微阵列可能成为一种有前景的工具，用于分析接种疫苗或感染的人类对艰难梭菌抗原的抗体反应。随着抗原组的进一步优化和生产成本的降低，我们预计微阵列技术可能有助于以患者特异性方式优化和选择最具临床实用性的艰难梭菌感染免疫疗法。
