Ronald L. Rabin, Derek Croote, Aaron Chen, E. Dobrovolskaia, Joyce Jia Wen Wong, Jessica Grossman, Robert G. Hamilton
{"title":"A human monoclonal antibody based immunoenzymetric assay to measure Fel d 1 concentrations in cat hair and pelt allergenic extracts","authors":"Ronald L. Rabin, Derek Croote, Aaron Chen, E. Dobrovolskaia, Joyce Jia Wen Wong, Jessica Grossman, Robert G. Hamilton","doi":"10.3389/falgy.2024.1417879","DOIUrl":null,"url":null,"abstract":"In the United States, 19 allergen extracts of different specificities are standardized, which means that their potencies are determined in comparison to a US reference standard. For cat allergen extracts, potency is determined by measuring Fel d 1 content expressed in in Fel d 1 units, and with a unitage that correlates with skin test reactions (bioequivalent allergy units or BAU). Currently, Fel d 1 content is measured with a radial immunodiffusion (RID) assay that uses polyclonal sheep antisera to detect the allergenic protein by producing a white precipitin line in agar gel. However, the RID is considered cumbersome, and the polyclonal sera may qualitatively vary among animals and may recognize epitopes irrelevant to human allergic disease. In this report, we describe a quantitative two-site immunoenzymetric assay (IEMA) for Fel d 1 that uses immobilized capture and soluble biotin-labeled detection Fel d 1-specific human IgE monoclonal antibodies (mAb) that have been class-switched to IgG4. Together, they sandwich Fel d 1 molecules from extracts. Using purified natural Fel d 1 as a calibrator, the historically reported ∼4 micrograms Fel d 1/Fel d 1 unit assignment was directly measured in this mAb-based IEMA at 3.12 ± 0.24 micrograms of Fel d 1 per Fel d 1 unit. This IEMA appears to be equivalent to RID in the measurement of biological potencies of commercial cat hair and cat pelt extracts marketed in the United States.","PeriodicalId":73062,"journal":{"name":"Frontiers in allergy","volume":null,"pages":null},"PeriodicalIF":3.3000,"publicationDate":"2024-07-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Frontiers in allergy","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.3389/falgy.2024.1417879","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"ALLERGY","Score":null,"Total":0}
引用次数: 0
Abstract
In the United States, 19 allergen extracts of different specificities are standardized, which means that their potencies are determined in comparison to a US reference standard. For cat allergen extracts, potency is determined by measuring Fel d 1 content expressed in in Fel d 1 units, and with a unitage that correlates with skin test reactions (bioequivalent allergy units or BAU). Currently, Fel d 1 content is measured with a radial immunodiffusion (RID) assay that uses polyclonal sheep antisera to detect the allergenic protein by producing a white precipitin line in agar gel. However, the RID is considered cumbersome, and the polyclonal sera may qualitatively vary among animals and may recognize epitopes irrelevant to human allergic disease. In this report, we describe a quantitative two-site immunoenzymetric assay (IEMA) for Fel d 1 that uses immobilized capture and soluble biotin-labeled detection Fel d 1-specific human IgE monoclonal antibodies (mAb) that have been class-switched to IgG4. Together, they sandwich Fel d 1 molecules from extracts. Using purified natural Fel d 1 as a calibrator, the historically reported ∼4 micrograms Fel d 1/Fel d 1 unit assignment was directly measured in this mAb-based IEMA at 3.12 ± 0.24 micrograms of Fel d 1 per Fel d 1 unit. This IEMA appears to be equivalent to RID in the measurement of biological potencies of commercial cat hair and cat pelt extracts marketed in the United States.
在美国,有 19 种不同特异性的过敏原提取物被标准化,这意味着它们的效价是根据与美国参考标准的比较来确定的。对于猫过敏原提取物,其效价是通过测量 Fel d 1 含量(以 Fel d 1 单位表示)和与皮试反应相关的单位(生物等效过敏单位或 BAU)来确定的。目前,Fel d 1 含量是通过径向免疫扩散(RID)测定法来测量的,该方法使用多克隆羊抗血清,通过在琼脂凝胶中产生白色沉淀线来检测过敏原蛋白。然而,放射免疫扩散法操作繁琐,而且不同动物的多克隆血清可能存在定性差异,并可能识别与人类过敏性疾病无关的表位。在本报告中,我们介绍了一种针对 Fel d 1 的定量双位点免疫酶测定法(IEMA),它使用固定捕获和可溶性生物素标记的 Fel d 1 特异性人类 IgE 单克隆抗体(mAb)进行检测,这些抗体已被分级为 IgG4。它们共同夹住了提取物中的 Fel d 1 分子。使用纯化的天然 Fel d 1 作为校准物,在这种基于 mAb 的 IEMA 中直接测得每 Fel d 1 单位的 Fel d 1 含量为 3.12 ± 0.24 微克,即历史上报道的 Fel d 1 含量为 4 微克/Fel d 1 单位。在测量美国市场上销售的商品猫毛和猫皮提取物的生物效力时,这种 IEMA 似乎等同于 RID。