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首页> 外文学位 >Production, characterization and possible applications of monoclonal antibodies generated against toluene diisocyanate-conjugated proteins.
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Production, characterization and possible applications of monoclonal antibodies generated against toluene diisocyanate-conjugated proteins.

机译：抗甲苯二异氰酸酯偶联蛋白的单克隆抗体的生产，表征和可能的应用。

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摘要

Diisocyanates are very reactive low molecular weight chemicals that are widely used in the manufacture of polyurethane products. Diisocyanate exposure is one of the most commonly reported causes of occupational asthma. Although diisocyanates have been identified as causative agents of respiratory diseases, the specific mechanisms by which these diseases occur remain largely unknown.;Tandem mass spectrometry was used to unambiguously identify the binding site of isocyanates within four model peptides (Leu-enkephalin (Leu-enk, YGGFL), Angiotensin I (DRVYIHPFHL), Substance P-amide (RPKPQQFFGLM-NH2), and Fibronectin-adhesion promoting peptide (FAPP, WQPPRARI)). In each case, isocyanates were observed to react to the N-terminus of the peptide. No evidence of side chain/isocyanate adduct formation exclusive of the N-terminus was observed. However, significant intra-molecular diisocyanate crosslinking between the N-terminal amine and a side chain amine group was observed for arginine, when located within two residues of the N-terminus. Addition of multiple isocyanates to the peptide occurs via polymerization at the N-terminus, rather than addition of multiple isocyanate molecules to varied residues within the peptide.;Toluene diisocyanate (TDI)-specific monoclonal antibodies (mAbs) with potential use in immunoassays for exposure and biomarker assessments were produced. A total of 59 unique mAbs were produced (29 IgG1, 14 IgG2a, 4 IgG2b, 2 IgG3 and 10 IgM) against 2,4 and 2,6 TDI bound protein. The reactivities of these mAbs were characterized by a solid phase indirect enzyme-linked immunosorbent assay (ELISA), Dot ELISA and Western immunoblot against various monoisocyanate, diisocyanate and dithioisocyanate protein conjugates. A subset of the mAbs were specific for 2,4 or 2,6 TDI-conjugated proteins only while others reacted to multiple dNCO conjugates including methylene diphenyl diisocyanate- and hexamethelene diisocyanate-human serum albumin . Western blot analyses demonstrated that some TDI conjugates form inter- and intra-molecular links resulting in multimers and a change in the electrophoretic mobility of the conjugate.;In general, 2,4/2,6 TDI reactive mAbs displayed (1) stronger recognition of monoisocyanate haptenated proteins when the isocyanate was in the ortho position relative to the tolyl group, and were able to discriminate between (2) isocyanate and isothiocyanate conjugates (i.e. between the urea and thiourea linkage); and (3) between aromatic and aliphatic diisocyanates. The mAbs produced were not carrier protein specific with estimated affinity constants toward toluene diisocyanate conjugated human serum albumin ranging from 2.21 x 107 to 1.07 x 1010 M-1 for IgG mAbs. Studies using TDI vapor exposed lung and epithelial cell lines suggest potential utility of these mAbs for both research and biomonitoring of isocyanate exposure.
机译：二异氰酸酯是非常活泼的低分子量化学品，已广泛用于制造聚氨酯产品。二异氰酸酯暴露是最常见的职业哮喘病原因之一。尽管二异氰酸酯已被确定为呼吸系统疾病的病原体，但这些疾病发生的具体机制仍不清楚。;串联质谱法用于明确鉴定四种模型肽（Leu-enkephalin（Leu-enk ，YGGFL），血管紧张素I（DRVYIHPFHL），物质P-酰胺（RPKPQQFFGLM-NH2）和纤连蛋白粘附促进肽（FAPP，WQPPRARI））。在每种情况下，观察到异氰酸酯与肽的N-末端反应。没有观察到除N末端外形成侧链/异氰酸酯加合物的证据。然而，当精氨酸位于N-末端的两个残基内时，在N-末端胺和侧链胺基团之间观察到明显的分子内二异氰酸酯交联。通过在N端进行聚合，可将多种异氰酸酯添加到肽中，而不是将多种异氰酸酯分子添加到肽中的各种残基上。甲苯二异氰酸酯（TDI）特异性单克隆抗体（mAb），可能在免疫测定中用于暴露并进行了生物标志物评估。针对2,4和2,6 TDI结合蛋白，总共产生了59种独特的mAb（29 IgG1、14 IgG2a，4 IgG2b，2 IgG3和10 IgM）。通过针对各种单异氰酸酯，二异氰酸酯和二硫代异氰酸酯蛋白缀合物的固相间接酶联免疫吸附测定（ELISA），Dot ELISA和Western免疫印迹来表征这些mAb的反应性。一部分mAb仅对2,4或2,6 TDI偶联蛋白具有特异性，而其他mAb与多种dNCO偶联物反应，包括亚甲基二苯基二异氰酸酯和六亚甲基二异氰酸酯-人血清白蛋白。 Western印迹分析表明，某些TDI共轭物形成分子间和分子内连接，从而导致多聚体以及共轭物的电泳迁移率发生变化;一般而言，显示2,4 / 2,6 TDI反应性单克隆抗体（1）较强的识别能力当异氰酸酯相对于甲苯基位于邻位时，单异氰酸酯半抗原化的蛋白质，并且能够区分（2）异氰酸酯和异硫氰酸酯结合物（即尿素和硫脲键之间）； （3）芳族和脂族二异氰酸酯之间。产生的单克隆抗体不是特异性的载体蛋白，其对与甲苯二异氰酸酯偶联的人血清白蛋白的亲和常数估计为IgG mAb，范围为2.21 x 107至1.07 x 1010 M-1。使用TDI蒸气暴露的肺和上皮细胞系进行的研究表明，这些mAb具有潜在的实用性，可用于异氰酸酯暴露的研究和生物监测。

著录项

  • 作者

    Ruwona, Tinashe Blessing.;

  • 作者单位

    Portland State University.;

  • 授予单位 Portland State University.;
  • 学科 Health Sciences Toxicology.;Chemistry Analytical.;Health Sciences Immunology.
  • 学位 Ph.D.
  • 年度 2010
  • 页码 169 p.
  • 总页数 169
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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