Irañeta S G, Acosta D M, Duran R, Apicella C, Orlando U D, Seoane M A, Alonso A, Duschak V G
División Alergia, Hospital de Clínicas Gral. San Martín, Buenos Aires, Argentina.
Clin Exp Allergy. 2008 Aug;38(8):1391-9. doi: 10.1111/j.1365-2222.2008.03004.x. Epub 2008 May 22.
It is well known that allergen extracts used for specific therapy of allergic disorders are commonly stored as mixtures, causing an alteration of its stability.
The aim of this report is to identify pollen allergens susceptible to degradation during storage of mixtures containing different sources of proteases in the absence of glycerol as a preserving agent.
Mixes containing Lolium perenne (Lol p) pollen extract with either Aspergillus fumigatus or Periplaneta americana extracts were prepared and co-incubated for 90 days at 4 degrees C. Samples were taken off at fixed times and comparatively tested by in vitro and in vivo assays with atopic patients. Selected pollinic allergens were subjected to MALDI-TOF MS analysis.
ELISA inhibition evidenced the loss of potency from ryegrass extract, and immunoblotting assays showed the degradation of specific pollinic allergens during storage of mixtures containing protease-rich sources. An in vivo intradermal skin assay confirmed the gradual loss of the biological activity of L. perenne pollen extract co-incubated with non-related protease-rich extracts in comparison with that of the control pollen extract. MALDI-TOF MS analysis allowed us to determine that Lol p 1 and Lol p 5 are susceptible to proteolysis whereas Lol p 4 was found to be resistant to degradation during storage.
Lol p 1 and Lol p 5 degradation is responsible for the loss of the biological activity of L. perenne pollen extract when co-incubated with protease-rich fungal and cockroach extracts in the same vial for months in the absence of glycerol as a preserving agent. The integrity of these major allergens must be preserved to increase the vaccine stability and to assure efficacy when mixes are used for immunotherapy.
众所周知，用于过敏性疾病特异性治疗的变应原提取物通常作为混合物储存，这会导致其稳定性改变。
本报告旨在确定在不含甘油作为防腐剂的情况下，与不同来源蛋白酶混合储存期间易降解的花粉变应原。
制备含有黑麦草（Lol p）花粉提取物与烟曲霉或美洲大蠊提取物的混合物，并在4℃下共同孵育90天。在固定时间取样，并通过体外和体内试验对特应性患者进行比较测试。对选定的花粉变应原进行基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）分析。
酶联免疫吸附测定（ELISA）抑制表明黑麦草提取物效力丧失，免疫印迹分析显示在含有丰富蛋白酶来源的混合物储存期间，特定花粉变应原发生降解。体内皮内皮肤试验证实，与对照花粉提取物相比，与非相关富含蛋白酶提取物共同孵育的黑麦草花粉提取物的生物活性逐渐丧失。MALDI-TOF MS分析使我们能够确定Lol p 1和Lol p 5易受蛋白水解作用影响，而Lol p 4在储存期间对降解具有抗性。
当在同一小瓶中与富含蛋白酶的真菌和蟑螂提取物共同孵育数月且不含甘油作为防腐剂时，Lol p 1和Lol p 5的降解导致黑麦草花粉提取物生物活性丧失。必须保持这些主要变应原的完整性，以提高疫苗稳定性，并确保在将混合物用于免疫治疗时的疗效。
