烟曲霉变应原的鉴定——与哮喘肺损伤的潜在关联
2024/03/26
摘要
背景：与粗提物相比，组分解析诊断使IgE致敏性检测具有可重复性和标准化的优势。传统的一维或二维蛋白质印迹法和用患者的IgE筛选表达cDNA文库的过敏原鉴定方法的主要缺点是不能保持蛋白的天然结构。
方法：我们使用了一种新的免疫沉淀技术结合质谱鉴定烟曲霉新的过敏原。本文报道用磁珠结合抗人IgE抗体纯化人血清IgE及烟曲霉蛋白提取物过敏原。
结果：在随后的质量肽指纹图谱检测到的184个蛋白中，有13个重组表达并纯化。在52例烟曲霉致敏的哮喘患者、23例非真菌致敏的哮喘患者和18例健康者中，仅前者通过免疫印迹和(或)ELISA检测出IgE反应。我们发现了11种尚未被描述为烟曲霉变应原的蛋白质，其中最常见的是果糖二磷酸醛醇酶II (FBA2) (33%)， NAD依赖的苹果酸脱氢酶(31%)和铜锌超氧化物歧化酶(27%)。对于这三种变应原，天然蛋白和变性蛋白检测表明天然蛋白的识别能力更好。11种变应原中有7种符合WHO/IUIS标准并被接受为新的烟曲霉变应原。
结论：综上所述，我们介绍了一种通过免疫沉淀结合质谱鉴定烟曲霉等复杂变应原的方法，该方法比传统的通过保持蛋白结构来鉴定变应原的方法更有优势。
Identification of allergens from Aspergillus fumigatus—Potential association with lung damage in asthma
E. M. Rick, K. Woolnough, M. Richardson, W. Monteiro, M. Craner, M. Bourne, et al.
Abstract
BACKGROUND:Component- resolved diagnosis allows detection of IgE sensitization having the advantage of reproducibility and standardization compared to crude extracts. The main disadvantage of the traditional allergen identification methods, 1- or 2- dimensional western blotting and screening of expression cDNA libraries with patients' IgEs, is that the native structure of the protein is not necessarily maintained.
METHODS:We used a novel immunoprecipitation technique in combination with mass spectrometry to identify new allergens of Aspergillus fumigatus. Magnetic Dynabeads coupled with anti- human IgE antibodies were used to purify human serum IgE and subsequently allergens from A. fumigatus protein extract.
RESULTS:Of the 184 proteins detected by subsequent mass peptide fingerprinting, a subset of 13 were recombinantly expressed and purified. In a panel of 52 A. fumigatus- sensitized people with asthma, 23 non- fungal- sensitized asthmatics and 18 healthy individuals, only the former showed an IgE reaction by immunoblotting and/or ELISA. We discovered 11 proteins not yet described as A. fumigatus allergens, with fructose- bisphosphate aldolase class II (FBA2) (33%), NAD- dependent malate dehydrogenase (31%) and Cu/Zn superoxide dismutase (27%) being the most prevalent. With respect to these three allergens, native versus denatured protein assays indicated a better recognition of the native proteins. Seven of 11 allergens fulfilled the WHO/IUIS criteria and were accepted as new A. fumigatus allergens.
CONCLUSION:In conclusion, we introduce a straightforward method of allergen identification from complex allergenic sources such as A. fumigatus by immunoprecipitation combined with mass spectrometry, which has the advantage over traditional methods of identifying allergens by maintaining the structure of the proteins.
背景：与粗提物相比，组分解析诊断使IgE致敏性检测具有可重复性和标准化的优势。传统的一维或二维蛋白质印迹法和用患者的IgE筛选表达cDNA文库的过敏原鉴定方法的主要缺点是不能保持蛋白的天然结构。
方法：我们使用了一种新的免疫沉淀技术结合质谱鉴定烟曲霉新的过敏原。本文报道用磁珠结合抗人IgE抗体纯化人血清IgE及烟曲霉蛋白提取物过敏原。
结果：在随后的质量肽指纹图谱检测到的184个蛋白中，有13个重组表达并纯化。在52例烟曲霉致敏的哮喘患者、23例非真菌致敏的哮喘患者和18例健康者中，仅前者通过免疫印迹和(或)ELISA检测出IgE反应。我们发现了11种尚未被描述为烟曲霉变应原的蛋白质，其中最常见的是果糖二磷酸醛醇酶II (FBA2) (33%)， NAD依赖的苹果酸脱氢酶(31%)和铜锌超氧化物歧化酶(27%)。对于这三种变应原，天然蛋白和变性蛋白检测表明天然蛋白的识别能力更好。11种变应原中有7种符合WHO/IUIS标准并被接受为新的烟曲霉变应原。
结论：综上所述，我们介绍了一种通过免疫沉淀结合质谱鉴定烟曲霉等复杂变应原的方法，该方法比传统的通过保持蛋白结构来鉴定变应原的方法更有优势。
（中日友好医院呼吸与危重症医学科 李春晓 摘译 林江涛 审校）
（Allergy, 2024.DOI: 10.1111/all.16032）
（Allergy, 2024.DOI: 10.1111/all.16032）
Identification of allergens from Aspergillus fumigatus—Potential association with lung damage in asthma
E. M. Rick, K. Woolnough, M. Richardson, W. Monteiro, M. Craner, M. Bourne, et al.
Abstract
BACKGROUND:Component- resolved diagnosis allows detection of IgE sensitization having the advantage of reproducibility and standardization compared to crude extracts. The main disadvantage of the traditional allergen identification methods, 1- or 2- dimensional western blotting and screening of expression cDNA libraries with patients' IgEs, is that the native structure of the protein is not necessarily maintained.
METHODS:We used a novel immunoprecipitation technique in combination with mass spectrometry to identify new allergens of Aspergillus fumigatus. Magnetic Dynabeads coupled with anti- human IgE antibodies were used to purify human serum IgE and subsequently allergens from A. fumigatus protein extract.
RESULTS:Of the 184 proteins detected by subsequent mass peptide fingerprinting, a subset of 13 were recombinantly expressed and purified. In a panel of 52 A. fumigatus- sensitized people with asthma, 23 non- fungal- sensitized asthmatics and 18 healthy individuals, only the former showed an IgE reaction by immunoblotting and/or ELISA. We discovered 11 proteins not yet described as A. fumigatus allergens, with fructose- bisphosphate aldolase class II (FBA2) (33%), NAD- dependent malate dehydrogenase (31%) and Cu/Zn superoxide dismutase (27%) being the most prevalent. With respect to these three allergens, native versus denatured protein assays indicated a better recognition of the native proteins. Seven of 11 allergens fulfilled the WHO/IUIS criteria and were accepted as new A. fumigatus allergens.
CONCLUSION:In conclusion, we introduce a straightforward method of allergen identification from complex allergenic sources such as A. fumigatus by immunoprecipitation combined with mass spectrometry, which has the advantage over traditional methods of identifying allergens by maintaining the structure of the proteins.
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