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Clinical & Experimental Allergy

MALDI-TOF MS analysis of labile Lolium perenne major allergens in mixes


Vilma G. Duschak, Departamento de Investigación, Instituto Nacional de Parasitología “Dr. Mario Fatala Chaben”, ANLIS-Malbrán, Ministerio de Salud. Av. Paseo Colón 568, (1063) Ciudad de Buenos Aires, Buenos Aires, Argentina. E-mail:


Background 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.

Objective 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.

Methods 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 °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.

Results 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.

Conclusions 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.

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