Chapman M D, Aalberse R C, Brown M J, Platts-Mills T A
Department of Medicine, University of Virginia, Charlottesville 22908.
J Immunol. 1988 Feb 1;140(3):812-8.
Two mAb were used to develop new techniques for the purification and quantitation of the major feline salivary allergen, Felis domesticus allergen I (Fel d I). The allergen was purified from aqueous house dust extract with a high Fel d I content by affinity chromatography over a monoclonal immunosorbent and elution with 4 mM HCl, pH 2.5. This single step procedure gave 40 to 50% recovery of 90% pure allergen which, following final purification by size exclusion HPLC, showed a single line on immunodiffusion and crossed immunoelectrophoresis against monospecific anti-Fel d I and polyclonal anti-cat dander antibodies. The m.w. of native Fel d I was 39,000 on size exclusion HPLC, and 17,000 under nonreducing conditions on gel electrophoresis. The N-terminal amino acid sequence (33 residues) showed no homology with other known protein sequences. The combination of the SDS-PAGE and N-terminal sequence data suggests that Fel d I is a non-covalently linked homodimer. A two-site RIA was developed using mAb directed against different epitopes on Fel d I. This assay was species-specific, highly sensitive (0.0004 U/ml), and showed an excellent correlation with a polyclonal inhibition RIA (n = 27, r = 0.93, p less than 0.001). Cat allergen extracts used for immediate skin tests showed marked differences in Fel d I content (from 0.1 to 30 U/ml). Consistently high Fel d I levels were found at monthly intervals in six dust samples from four houses with cats (10 to 100 U/g of dust). Comparisons of Fel d I and mite and pollen allergen levels showed that house dust can contain greater than 100 micrograms/g of either of these allergens and is a potent source of foreign environmental antigens. Monoclonal affinity chromatography provides a major breakthrough in the purification of Fel d I, from a source material that would otherwise have been considered impossible (house dust). The mAb assay for Fel d I is both more sensitive and more easily standardized than existing techniques. These techniques will allow full structural and antigenic analysis of Fel d I and more detailed studies on the relationship between cat antigen exposure and the development of asthma.
使用两种单克隆抗体（mAb）开发了用于纯化和定量主要猫唾液过敏原——家猫过敏原I（Fel d I）的新技术。通过在单克隆免疫吸附剂上进行亲和层析，并用pH 2.5的4 mM盐酸洗脱，从含有高Fel d I含量的室内灰尘水提取物中纯化该过敏原。此单步程序可获得90%纯度过敏原40%至50%的回收率，经尺寸排阻高效液相色谱（HPLC）最终纯化后，在免疫扩散试验中显示为单一沉淀线，在用单特异性抗Fel d I和多克隆抗猫皮屑抗体进行的交叉免疫电泳中也显示为单一沉淀线。在尺寸排阻HPLC上，天然Fel d I的分子量为39,000，在非还原条件下凝胶电泳时为17,000。其N端氨基酸序列（33个残基）与其他已知蛋白质序列无同源性。SDS-PAGE和N端序列数据表明Fel d I是一种非共价连接的同型二聚体。利用针对Fel d I不同表位的单克隆抗体开发了一种双位点放射免疫分析（RIA）。该分析具有种属特异性、高度灵敏（0.0004 U/ml），并且与多克隆抑制RIA具有良好的相关性（n = 27，r = 0.93，p < 0.001）。用于即时皮肤试验的猫过敏原提取物在Fel d I含量上显示出显著差异（从0.1至30 U/ml）。在来自四户养猫家庭的六个灰尘样本中，每月定期检测到的Fel d I水平始终较高（10至100 U/g灰尘）。对Fel d I与螨虫和花粉过敏原水平的比较表明，室内灰尘中这些过敏原的含量可能超过100微克/克，是外来环境抗原的重要来源。单克隆亲和层析在从原本被认为不可能的来源材料（室内灰尘）中纯化Fel d I方面取得了重大突破。用于检测Fel d I的单克隆抗体分析比现有技术更灵敏且更易于标准化。这些技术将有助于对Fel d I进行全面的结构和抗原分析，并更详细地研究猫抗原暴露与哮喘发病之间的关系。
