采用质谱法测量过敏原提取物中的德国小蠊过敏原及其同型物。
Measurement of German cockroach allergens and their isoforms in allergen extracts with mass spectrometry.
机构信息
Laboratory of Immunobiochemistry, Division of Bacterial, Parasitic, and Allergenic Products, Office of Vaccines Research and Review, CBER/FDA, Silver Spring, MD, USA.
Department of Entomology and Plant Pathology, North Carolina State University, Raleigh, NC, USA.
出版信息
Clin Exp Allergy. 2020 Jun;50(6):741-751. doi: 10.1111/cea.13604. Epub 2020 May 20.
BACKGROUND
Allergen extracts are the primary tool for diagnosis and treatment of allergic diseases. In the United States, most allergen extracts are non-standardized. More sophisticated analytical approaches are needed to characterize these products and enable manufacturers and regulators to better determine potency.
OBJECTIVE
To expand the multiple reaction monitoring (MRM) assay for an in-depth characterization of German cockroach (GCr; Blattella germanica) allergen extracts.
METHODS
We applied advanced liquid chromatography (LC) and mass spectrometry (MS) techniques including MRM. The expanded LC/MRM-MS method was optimized to measure known GCr allergens and their isoforms/variants in commercial extracts and environmental samples. We performed isoform-specific allergen measurements in multiple extracts from four commercial sources and extracts prepared using environmental samples from urban homes. To investigate causes of heterogeneity, we examined over 30 extraction process variables.
RESULTS
Evaluation of the commercial extracts confirmed the variability of production lots and commercial sources. Commonly used defatting and extraction protocols yielded extracts with comparable allergen profiles and content. However, the identity and quality of source materials was a major contributor to variability. In comparing commercial GCr extracts to environmental samples, relative quantities of Bla g 1, Bla g 2, Bla g 3, Bla g 4 and Bla g 11 were similar, while Bla g 5, Bla g 6, Bla g 7 and Bla g 8 were present in the environmental samples but largely absent for the commercial extracts.
CONCLUSIONS AND CLINICAL RELEVANCE
LC/MRM-MS can be used to measure all known GCr allergens in commercial allergen extracts and environmental samples. Significant differences exist between allergen profiles of commercial extracts and the profiles of environmental samples from dwellings. This analytical platform can serve as a template to achieve better product characterization of similarly complex products.
背景
过敏原提取物是诊断和治疗过敏性疾病的主要工具。在美国，大多数过敏原提取物是非标准化的。需要更复杂的分析方法来描述这些产品，使制造商和监管机构能够更好地确定效力。
目的
扩展多重反应监测（MRM）测定法，以深入表征德国蟑螂（GCr；Blattella germanica）过敏原提取物。
方法
我们应用了先进的液相色谱（LC）和质谱（MS）技术，包括 MRM。优化了扩展的 LC/MRM-MS 方法，以测量商业提取物和环境样品中的已知 GCr 过敏原及其同工型/变体。我们在来自四个商业来源的多个提取物中进行了同工型特异性过敏原测量，并在使用来自城市家庭的环境样本制备的提取物中进行了测量。为了研究异质性的原因，我们检查了 30 多个提取过程变量。
结果
对商业提取物的评估证实了生产批次和商业来源的可变性。常用的脱脂和提取方案产生了具有可比过敏原谱和含量的提取物。然而，原材料的身份和质量是造成变异性的主要因素。在比较商业 GCr 提取物和环境样本时，Bla g 1、Bla g 2、Bla g 3、Bla g 4 和 Bla g 11 的相对数量相似，而 Bla g 5、Bla g 6、Bla g 7 和 Bla g 8 存在于环境样本中，但在商业提取物中基本不存在。
结论和临床相关性
LC/MRM-MS 可用于测量商业过敏原提取物和环境样本中的所有已知 GCr 过敏原。商业提取物的过敏原谱与住宅环境样本的过敏原谱之间存在显著差异。该分析平台可作为模板，实现类似复杂产品的更好产品特征描述。
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