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Biochemical and immunological analysis of mould skin prick test solution: Current status of standardization

机译：霉菌皮刺试验溶液的生化和免疫学分析：标准化现状

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Background: Sensitization prevalence to moulds reached from less than 10% in the general population to more than 25% in atopic and/or asthmatic subjects. To diagnose IgE-mediated mould sensitization, skin prick test (SPT) and specific IgE (sIgE) measurement are recommended. However, concordance of SPT and sIgE results is often less than 50% and standardization of the extracts is required to achieve reliable test results. Objective: The aim of our study was to analyse mould SPT solutions (SPTs) with respect to quantity and quality of protein, antigen and human IgE-binding content as a prerequisite for further in vivo studies. Methods: Commercial SPTs of Alternaria alternata, Aspergillus fumigatus, Cladosporium herbarum and Penicillium chrysogenum from six manufacturers as well as two in-house extracts from Aspergillus versicolor were investigated. Protein-, antigen- and IgE-binding contents were quantified by Bradford assay, sandwich ELISA and IgE-ImmunoCAP-inhibition tests. Protein composition and IgE and IgG binding were analysed by SDS-PAGE and immunoblotting, respectively. Results: Median protein concentrations were similar in all mould SPT extracts (90-110 μg/mL). In contrast, antigen contents and IgE-binding capacity showed a high variability with median antigen values from 4 to 118 μg/mL and IgE inhibition results between 30 to 95%. Whereas almost all SPTs of A. alternata and A. versicolor showed complete sIgE inhibition with mean values > 80%, only three extracts for A. fumigatus, two extracts for C. herbarum and none of the tested extracts for P. chrysogenum exceeded 50% sIgE reduction. Quantitative amounts of protein, antigenic and IgE-binding structures were not comparable with the quality of the corresponding protein or immunoblot pattern, with the exception of A. alternata SPTs. Conclusions and Clinical Relevance: Commercially available mould SPT extracts showed high variability raising the question of comparability and reliability of SPT results. Possible consequences for diagnostic test outcome will be investigated in the next step.
机译：背景：霉菌的致病率从普通人群的不到10％到特应性和/或哮喘患者的25％以上。为了诊断IgE介导的霉菌敏化，建议进行皮肤点刺试验（SPT）和特定的IgE（sIgE）测量。但是，SPT和sIgE结果的一致性通常小于50％，并且需要对提取物进行标准化才能获得可靠的测试结果。目的：我们的研究目的是分析霉菌SPT溶液（SPT）的蛋白质，抗原和人类IgE结合含量的数量和质量，作为进行进一步体内研究的前提。方法：调查了六株制造商的链格孢菌，烟曲霉，标本草和青霉青霉的商业SPT，以及两种室内提取的杂色曲霉。通过Bradford分析，夹心ELISA和IgE-ImmunoCAP抑制试验对蛋白质，抗原和IgE的结合含量进行定量。分别通过SDS-PAGE和免疫印迹分析蛋白质组成以及IgE和IgG结合。结果：所有霉菌SPT提取物中的蛋白质中位数浓度相似（90-110μg/ mL）。相比之下，抗原含量和IgE结合能力表现出较高的变异性，中位抗原值为4至118μg/ mL，IgE抑制结果在30％至95％之间。几乎所有的链球菌和杂色曲霉的SPT均显示出完全的sIgE抑制，平均值> 80％，烟曲霉的只有3种提取物，草炭疽菌的有2种提取物，而产黄青霉的测试提取物均没有超过50％减少信号。蛋白质，抗原和IgE结合结构的定量数量与相应蛋白质或免疫印迹模式的质量不相上下，除了链格孢菌SPTs。结论与临床意义：市售的霉菌SPT提取物显示出高变异性，提出了SPT结果的可比性和可靠性问题。下一步将对诊断测试结果可能产生的后果进行调查。

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