Siman Isabella Lima, de Aquino Lais Martins, Ynoue Leandro Hideki, Miranda Juliana Silva, Pajuaba Ana Claudia Arantes Marquez, Cunha-Júnior Jair Pereira, Silva Deise Aparecida Oliveira, Taketomi Ernesto Akio
Laboratory of Allergy and Clinical Immunology, Institute of Biomedical Sciences, Federal University of Uberlândia, Avenida Pará 1720, Bloco 4 C, Campus Umuarama, 38400-902 Uberlândia, MG, Brazil.
Clin Dev Immunol. 2013;2013:657424. doi: 10.1155/2013/657424. Epub 2013 Aug 28.
One of the purposes of specific immunotherapy (SIT) is to modulate humoral immune response against allergens with significant increases in allergen-specific IgG levels, commonly associated with blocking activity. The present study investigated in vitro blocking activity of allergen-specific IgG antibodies on IgE reactivity to Dermatophagoides pteronyssinus (Dpt) in sera from atopic patients. Dpt-specific IgG antibodies were purified by ammonium sulfate precipitation followed by protein-G affinity chromatography. Purity was checked by SDS-PAGE and immunoreactivity by slot-blot and immunoblot assays. The blocking activity was evaluated by inhibition ELISA. The electrophoretic profile of the ammonium sulfate precipitated fraction showed strongly stained bands in ligand fraction after chromatography, compatible with molecular weight of human whole IgG molecule. The purity degree was confirmed by detecting strong immunoreactivity to IgG, negligible to IgA, and no reactivity to IgE and IgM. Dpt-specific IgG fraction was capable of significantly reducing levels of IgE anti-Dpt, resulting in 35%-51% inhibition of IgE reactivity to Dpt in atopic patients sera. This study showed that allergen-specific IgG antibodies purified from mite-allergic patients sera block the IgE recognition of Dermatophagoides pteronyssinus antigens. This approach reinforces that intermittent measurement of serum allergen-specific IgG antibodies will be an important objective laboratorial parameter that will help specialists to follow their patients under SIT.
特异性免疫疗法(SIT)的目的之一是调节针对变应原的体液免疫反应,使变应原特异性IgG水平显著升高,这通常与阻断活性相关。本研究调查了变应原特异性IgG抗体对特应性患者血清中IgE与尘螨(Dpt)反应性的体外阻断活性。通过硫酸铵沉淀,随后进行蛋白G亲和层析来纯化Dpt特异性IgG抗体。通过SDS-PAGE检查纯度,通过狭缝印迹和免疫印迹分析检查免疫反应性。通过抑制ELISA评估阻断活性。硫酸铵沉淀部分的电泳图谱显示,层析后配体部分有强染色条带,与人类全IgG分子的分子量相符。通过检测对IgG的强免疫反应性、对IgA可忽略不计以及对IgE和IgM无反应性来确认纯度。Dpt特异性IgG部分能够显著降低IgE抗Dpt水平,导致特应性患者血清中IgE与Dpt反应性受到35%-51%的抑制。本研究表明,从螨过敏患者血清中纯化的变应原特异性IgG抗体可阻断IgE对尘螨抗原的识别。这种方法进一步证明,间歇性检测血清变应原特异性IgG抗体将是一项重要的客观实验室参数,有助于专家在SIT治疗过程中跟踪患者情况。