Bannantine John P, Stabel Judith R, Lippolis John D, Reinhardt Timothy A
Infectious Bacterial Diseases Research Unit, National Animal Disease Center, USDA-Agricultural Research Service, Ames, IA 50010, USA.
Ruminant Diseases Immunology Research Unit, National Animal Disease Center, USDA-Agricultural Research Service, Ames, IA 50010, USA.
Microorganisms. 2018 Dec 11;6(4):127. doi: 10.3390/microorganisms6040127.
Monoclonal antibodies against subspecies proteins are important tools in Johne's disease research and diagnostics. Johne's disease is a chronic inflammatory intestinal disease of cattle, sheep, and other ruminant animals. We have previously generated multiple sets of monoclonal antibodies (mAbs) in different studies; however, because many were generated and screened against a whole-cell extract of , the antigens that bind to these antibodies remained unknown. In this study, we used three different approaches to identify the corresponding antigens for 14 mAbs that could not be identified previously. In the first approach, a new -lambda phage expression library was screened to identify corresponding antigens for 11 mAbs. This approach revealed that mAbs 7C8, 9H3, 12E4, 3G5, and 11B8 all detect MAP_3404 encoding the biotin carboxylase subunit of acetyl-CoA carboxylase, while mAbs 7A6, 11F8, and 10C12 detect the GroEL2 chaperonin (MAP_3936), 6C9 detects electron transfer flavoprotein (MAP_3060c), and 14G11 detects MAP_3976, a lipoprotein anchoring transpeptidase. The epitopes to a selection of these mAbs were also defined. In a second approach, MAP_2698c bound monoclonal antibody (mAb) 14D4 as determined using protein arrays. When both of these approaches failed to identify the antigen for mAb 12C9, immunoprecipitation, mass spectrometry analysis, and codon optimization was used to identify the membrane protein, MAP_4145, as the reacting antigen. Characterized antibodies were used to quickly interrogate mycobacterial proteomic preps. We conclude by providing a complete catalog of available mAbs to proteins, along with their cognate antigens and epitopes, if known. These antibodies are now thoroughly characterized and more useful for research and diagnostic purposes.
针对亚种蛋白的单克隆抗体是约内氏病研究和诊断中的重要工具。约内氏病是牛、羊和其他反刍动物的一种慢性炎症性肠道疾病。我们之前在不同研究中产生了多组单克隆抗体（mAb）；然而，由于许多抗体是针对[未提及的某种细菌]的全细胞提取物产生和筛选的，与这些抗体结合的抗原仍然未知。在本研究中，我们使用了三种不同方法来鉴定之前无法鉴定的14种单克隆抗体的相应抗原。在第一种方法中，筛选了一个新的λ噬菌体表达文库以鉴定11种单克隆抗体的相应抗原。该方法表明，单克隆抗体7C8、9H3、12E4、3G5和11B8均检测到编码乙酰辅酶A羧化酶生物素羧化酶亚基的MAP_3404，而单克隆抗体7A6、11F8和10C12检测到GroEL2伴侣蛋白（MAP_3936），6C9检测到电子传递黄素蛋白（MAP_3060c），14G11检测到脂蛋白锚定转肽酶MAP_3976。还确定了这些单克隆抗体中一部分的表位。在第二种方法中，使用蛋白质阵列确定MAP_2698c与单克隆抗体（mAb）14D4结合。当这两种方法都未能鉴定出单克隆抗体12C9的抗原时，使用免疫沉淀、质谱分析和密码子优化来鉴定膜蛋白MAP_4145作为反应抗原。经过表征的抗体用于快速检测分枝杆菌蛋白质组学制剂。我们通过提供一份完整的针对[未提及的某种细菌]蛋白的可用单克隆抗体目录（如果已知，还包括它们的同源抗原和表位）来得出结论。这些抗体现在已得到充分表征，对研究和诊断目的更有用。
