■严重的马哮喘(SEA)是常见的,马的慢性呼吸道疾病,其特征是对干草粉尘的高反应性,与人类严重的嗜中性粒细胞性哮喘有许多相似之处。引发SEA的抗原尚未得到全面表征,但霉菌和螨虫被认为是相关来源。这里,我们使用免疫蛋白质组学和IgG同种型结合分析鉴定了相关候选抗原.
■通过二维凝胶电泳,然后进行免疫印迹(2D免疫印迹),分离出翼状螨(Derp)的蛋白质,得到440个斑点的特征性模式。血清孵育后,每个点对所有Ig(Pan-Ig)和IgG同种型(2型相关IgG3/5,1型相关IgG4/7)的抗体(Ig)结合进行定量,并比较哮喘和健康马血清(每组n=5).
■在30个斑点中检测到Ig结合差异。与健康马血清相比,哮喘患者的Pan-Ig结合在四个点上较高,和IgG3/5结合在18个斑点上更高。在10个斑点上检测到小的IgG4/7结合差异,在四个斑点上与哮喘患者血清结合较高,但在六个斑点上与健康马血清结合较高。通过液相色谱质谱法鉴定来自具有组差异的斑点的蛋白质,包括螨和酵母蛋白质。后者可能起源于Derp培养物的补料底物。鉴定的蛋白质中优先考虑的抗原候选物是Derp1,Derp11,第15组过敏原,肌球蛋白重链,和未表征的Derp蛋白。此外,酵母烯醇化酶,乙醇脱氢酶(ADH),磷酸甘油酸激酶(PGK),甘油醛-3-磷酸脱氢酶,和热休克蛋白被优先考虑。通过ELISA分别使用各自的蛋白质测试了11种抗原候选物的确认。哮喘患者与哮喘患者的差异健康马血清Ig与Derp1、Derp18和三种酵母酶(烯醇化酶,ADH,和PGK)证实这些是SEA中免疫应答的有希望的抗原。
■通过免疫蛋白质组学新鉴定了与SEA相关的抗原,酵母抗原首次被考虑用于SEA。血清IgG3/5与相关抗原的结合在SEA中增加,并且是新的特征,其指出在SEA中增加的2型应答,但需要确认相应的细胞应答。
Severe equine asthma (SEA) is a common, chronic respiratory disease of horses characterized by hyperreactivity to hay dust which has many similarities to severe neutrophilic asthma in humans. SEA-provoking antigens have not been comprehensively characterized, but molds and mites have been suggested as relevant sources. Here, we identified relevant antigen candidates using immunoproteomics with IgG isotype-binding analyses.
Proteins from Dermatophagoides pteronyssinus (Der p) were separated by two-dimensional gel electrophoresis followed by immunoblotting (2D immunoblots) resulting in a characteristic pattern of 440 spots. After serum incubation, antibody (Ig)-binding of all Ig (Pan-Ig) and IgG isotypes (type-2-associated IgG3/5, type-1-associated IgG4/7) was quantified per each spot and compared between asthmatic and healthy horses\' sera (n=5 per group).
Ig binding differences were detected in 30 spots. Pan-Ig binding was higher with asthmatics compared to healthy horses\' sera on four spots, and IgG3/5 binding was higher on 18 spots. Small IgG4/7 binding differences were detected on 10 spots with higher binding with asthmatics\' sera on four but higher binding with healthy horses\' sera on six spots. Proteins from the spots with group differences including mite and yeast proteins were identified by liquid chromatography mass spectrometry. The latter likely originated from the feeding substrate of the Der p culture. Prioritized antigen candidates amongst the proteins identified were Der p 1, Der p 11, group 15 allergens, myosin heavy chain, and uncharacterized Der p proteins. Additionally, yeast enolases, alcohol dehydrogenase (ADH), phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase, and heat shock proteins were prioritized. Eleven antigen candidates were tested for confirmation by ELISAs using the respective proteins separately. Differences in asthmatics vs. healthy horses\' serum Ig binding to Der p 1, Der p 18, and three yeast enzymes (enolase, ADH, and PGK) confirmed these as promising antigens of immune responses in SEA.
Antigens with relevance in SEA were newly identified by immunoproteomics, and yeast antigens were considered for SEA for the first time. Serum IgG3/5 binding to relevant antigens was increased in SEA and is a novel feature that points to increased type-2 responses in SEA but requires confirmation of the corresponding cellular responses.