Hoff Michael, Krail Marion, Kästner Marion, Haustein Dieter, Vieths Stefan
Paul-Ehrlich-Institut, Department of Allergology, Langen.
J Allergy Clin Immunol. 2002 Jan;109(1):96-101. doi: 10.1067/mai.2002.120560.
Therapeutic allergen extracts are frequently stored as mixtures to provide preparations used for specific immunotherapy. Substantial protease activity, found in certain mold extracts, has been suspected to cause a loss of allergenic activity as a result of self-degradation, as well as by means of degradation of allergens from pollen and other allergenic sources.
This study was performed to address possible deterioration of individual pollen allergens during storage of extract mixtures, with a mold extract as the source of proteolytic activity.
Aqueous birch and timothy pollen extracts were stored for 60 days at 6 degrees C with and without addition of an extract derived from the mold Fusarium culmorum. The stability of the pollen allergens Bet v 1, Bet v 6, Phl p 1, and Phl p 5, as well as 2 to-date-undefined F culmorum allergens was examined by using immunoblotting analysis with sera from allergic patients and allergen-specific mAbs. Furthermore, the residual allergenic activity of the pollen extracts was monitored by using the rat basophilic leukemia cell-mediator release assay. Proteolytic activity of extracts was determined by using a commercial protease assay and gelatinase zymography.
Pollen extracts were very stable, corresponding to the low proteolytic activity of these extracts. In contrast, high proteolytic activity was found for the F culmorum extract, resulting in self-degradation of mold proteins and deactivation of allergens. Similarly, the mixtures showed a strong decrease of allergenic potency in the mediator release assay. Bet v 1 and Phl p 1 were relatively stable, whereas Bet v 6 and Phl p 5 were almost entirely degraded within 1 day.
Proteases of the mold F culmorum clearly affected the overall allergenic activity of pollen extracts within a short time period. Apart from general objections against the use of mixtures of non-cross-reacting allergens, mixing of pollen extracts with extracts derived from molds for immunotherapy is not recommended unless they are applied directly after preparation of the mixture.
治疗性变应原提取物常作为混合物储存，以提供用于特异性免疫治疗的制剂。在某些霉菌提取物中发现的大量蛋白酶活性，被怀疑会因自身降解以及花粉和其他变应原来源的变应原降解而导致变应原活性丧失。
本研究旨在探讨以霉菌提取物作为蛋白水解活性来源时，提取物混合物储存期间单个花粉变应原可能的降解情况。
将桦树和梯牧草花粉水提取物在6℃下储存60天，分别添加和不添加来自禾谷镰刀菌的提取物。通过使用过敏患者血清和变应原特异性单克隆抗体进行免疫印迹分析，检测花粉变应原Bet v 1、Bet v 6、Phl p 1和Phl p 5以及2种迄今未明确的禾谷镰刀菌变应原的稳定性。此外，通过大鼠嗜碱性白血病细胞介质释放试验监测花粉提取物的残留变应原活性。使用商业蛋白酶测定法和明胶酶谱法测定提取物的蛋白水解活性。
花粉提取物非常稳定，这与这些提取物的低蛋白水解活性相对应。相比之下，禾谷镰刀菌提取物具有高蛋白水解活性，导致霉菌蛋白自身降解和变应原失活。同样，在介质释放试验中，混合物的变应原效力显著降低。Bet v 1和Phl p 1相对稳定，而Bet v 6和Phl p 5在1天内几乎完全降解。
禾谷镰刀菌的蛋白酶在短时间内明显影响了花粉提取物的整体变应原活性。除了对使用非交叉反应变应原混合物的一般反对意见外，除非在混合物制备后直接应用，否则不建议将花粉提取物与霉菌提取物混合用于免疫治疗。
