Movérare Robert, Blume Karin, Lind Peter, Crevel René, Marknell DeWitt Åsa, Cochrane Stella
Thermo Fisher Scientific, ImmunoDiagnostics, Uppsala University, Uppsala, Sweden.
Int Arch Allergy Immunol. 2017;172(1):1-10. doi: 10.1159/000455098. Epub 2017 Feb 21.
Knowledge of human IgG subclass antibody responses to various allergens has been hampered by a lack of reliable standardized assays. The aim here was to develop quantitative immunoassays for human IgG1, IgG2, and IgG3 antibodies using ImmunoCAP® technology and to evaluate their application.
Enzyme conjugates with isotype-specific monoclonal antibodies and calibrators composed of purified myeloma paraproteins were developed for each assay and used together with other standardized assay reagents for the Phadia® 100 instrument. The calibrators were adjusted to the international reference preparation IRP 67/86. The assays were characterized and used together with other standard ImmunoCAP assays to measure antibodies to various allergens in preliminary studies.
The new assays had limits of quantitation of 1.0 (IgG1), 4.6 (IgG2), and 0.04 mgA/L (IgG3), and coefficients of variation of <20%. Only some minor cross-reactivity with IgG2 was observed for the specific IgG1 assay. The specific IgG2 assay showed a bias for the allotype G2m(23) and compensation factors were used to adjust the measured concentrations accordingly. Preliminary studies indicated a strong and stable IgG4 antibody response to β-lactoglobulin in healthy individuals, a high IgG1 and even higher IgG2 antibody response to house dust mite in sensitized and nonsensitized subjects, and a mixed IgG subclass response to venom allergens in allergic patients with increasing IgG4 antibody levels during venom immunotherapy.
The new research assays are valuable tools for immunological studies, enabling the characterization of antibody profiles using a standardized approach, and facilitating data interpretation and the comparison of results across studies.
由于缺乏可靠的标准化检测方法，对人类IgG亚类抗体对各种过敏原反应的了解受到了阻碍。本研究的目的是利用免疫捕获（ImmunoCAP®）技术开发针对人类IgG1、IgG2和IgG3抗体的定量免疫检测方法，并评估其应用。
针对每种检测方法开发了与同种型特异性单克隆抗体结合的酶结合物以及由纯化的骨髓瘤副蛋白组成的校准品，并与其他标准化检测试剂一起用于Phadia® 100仪器。校准品已根据国际参考制剂IRP 67/86进行了调整。在初步研究中，对这些检测方法进行了特性鉴定，并与其他标准免疫捕获检测方法一起用于测量针对各种过敏原的抗体。
新检测方法的定量限分别为1.0（IgG1）、4.6（IgG2）和0.04 mgA/L（IgG3），变异系数<20%。特异性IgG1检测仅观察到与IgG2有一些轻微的交叉反应。特异性IgG2检测对同种异型G2m(23)有偏差，因此使用补偿因子相应地调整测量浓度。初步研究表明，健康个体对β-乳球蛋白有强烈且稳定的IgG4抗体反应，致敏和未致敏受试者对屋尘螨有高IgG1和更高的IgG2抗体反应，在接受毒液免疫治疗期间，过敏患者对毒液过敏原的IgG亚类反应混合，且IgG4抗体水平升高。
新的研究检测方法是免疫学研究的宝贵工具，能够使用标准化方法对抗体谱进行表征，并有助于数据解释和跨研究结果的比较。
