Liu T, Lin Y
Laboratory of Immunobiochemistry, Division of Allergenic Products and Parasitology, Food and Drug Administration, Rockville, Maryland, USA.
Ann Allergy Asthma Immunol. 1998 Feb;80(2):177-83. doi: 10.1016/S1081-1206(10)62952-3.
Commercial mite allergenic extracts sold in the US are prepared with whole body mites in buffer solutions containing 50% glycerol. Mite extracts were reported to contain large number of proteolytic enzymes and their structural integrity in aqueous solutions have not been evaluated.
To evaluate the epitope stabilities of Group 1 and 2 allergens in two commercial mite extracts used by Center for Biologics Evaluation and Research (CBER) as reference extracts, E5-Dp (Dermatophagoides pteronyssinus) and E5-Df (D. farinae).
Epitope stability was determined by using monoclonal antibodies in a Sandwich ELISA. Samples were stored at four different temperatures and the amounts of Der p 1, Der p 2, Der f 1, and Der f 2 were determined at different time intervals. The overall stability of mite extracts was evaluated by immunoblot and competition ELISA.
The epitope stability of these allergens varies: Der f 1 was stable for at least 3 years and Der f 2 for 1 year when stored at 4 degrees C; Der p 1 and 2 were less stable. None of the Group 1 and 2 allergens remained intact when stored at 50 degrees C. Immunoblot and competition ELISA data also showed similar trend of degradation as compared with extracts stored at 4 degrees C for same length of time.
With the exception of Der f 1, the amount of detectable epitopes in Group 1 and 2 allergens reduce rapidly after 1 year, especially at elevated temperatures. The changes in allergen composition were also observed by immunoblotting and in relative potency by ELISA competition assay. These findings are highly relavent to the users of CBER's mite extracts as standards.
在美国销售的商业性螨变应原提取物是用保存在含50%甘油的缓冲溶液中的全螨制备的。据报道，螨提取物含有大量蛋白水解酶，且其在水溶液中的结构完整性尚未得到评估。
评估生物制品评估和研究中心（CBER）用作参考提取物的两种商业螨提取物E5-Dp（粉尘螨）和E5-Df（屋尘螨）中1组和2组变应原的表位稳定性。
通过夹心ELISA法使用单克隆抗体测定表位稳定性。样品在四个不同温度下储存，并在不同时间间隔测定Der p 1、Der p 2、Der f 1和Der f 2的含量。通过免疫印迹和竞争ELISA评估螨提取物的总体稳定性。
这些变应原的表位稳定性各不相同：在4℃储存时，Der f 1至少稳定3年，Der f 2稳定1年；Der p 1和Der p 2较不稳定。在50℃储存时，1组和2组变应原均无完整保留。免疫印迹和竞争ELISA数据也显示出与在4℃储存相同时间的提取物相比类似的降解趋势。
除Der f 1外，1组和2组变应原中可检测表位的量在1年后迅速减少，尤其是在高温下。通过免疫印迹观察到变应原组成的变化，并通过ELISA竞争测定法观察到相对效价的变化。这些发现与使用CBER螨提取物作为标准的用户高度相关。
