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Background Allergoids were first used in the decades of the 60s and 70s of the last century as an effective treatment of allergic respiratory diseases. Allergoids can be modified with formaldehyde or glutaraldehyde. Modified allergens, or allergoids, decrease the risk of adverse reactions while administering higher allergen doses. The objective of this study was to analyse specific IgE and IgG binding to glutaraldehyde modified and non-modified allergen extracts of Phleum pratense. Methods The sera of 69 patients sensitized to P. pratense were tested. All these patients had signs and symptoms of rhinoconjunctivitis with, or without, asthma in May and June of 2011. All these patients had positive skin prick tests to a standardized extract of P. pratense, and other grass species. Most patients were also sensitized to olive pollen. Specific IgE and IgG binding were analysed by direct ELISA against P. pratense native (non-modified) and allergoid extracts. Relative potencies were evaluated through ELISA inhibition assays, and the protein composition of non-modified and allergoid samples was determined by Mass Spectrometry (MS/MS). Results Mean Specific IgE levels against the native extract was 16.68 ± 11.65 Units (U) and against the allergoid: 7.26 ± 8.24 U (P < 0.0001; Mann-Whitney). On the other hand, mean specific IgG binding against the non-modified extract was 90.34 ± 75.57 U versus 76.19 ± 70.31 U against the allergoid (P = 0.16; Mann-Whitney). Linear regression coefficients obtained between immunoglobulin reactivity against both extracts were: r2 = 0.51 for specific IgE and r2 = 0.83 for specific IgG. An important decrease in the allergenic activity, measured by inhibition ELISA, was clearly observed. The MS/MS assay revealed the presence of the mayor allergen, and some isoforms, in non-modified and allergoid extracts. Conclusions Results obtained demonstrate that the glutaraldehyde polymerization process induces an important decrease in specific IgE binding to allergoids of P. pratense while there are no significant differences in specific IgG binding. The allergenic composition of the P. pratense allergoid was equivalent to the non-modified pollen extract.

Background D. pteronyssinus and B. tropicalis are the most common house dust mite species worldwide. D. pteronyssinus and B. tropicalis are the most prevalent species in the tropics. Twelve allergens have been described in B. tropicalis and fifteen in D. pteronyssinus. The objectives of this study were: 1) to analyze the prevalence of sensitization to B. tropicalis and D. pteronyssinus on the tropical Island of Martinique; 2) to evaluate the allergenic crossreactivity between D. pteronyssinus and B. tropicalis and 3) to identify common, and species specific allergens, using mass spectrometry (MS). The serum of patients sensitized to a single, or to both species, was further analyzed by western blots. Methods The sera of 1,243 consecutive patients evaluated for allergic rhinitis and/or asthma were evaluated for specific IgE using direct ELISA. All patients resided on the tropical island of Martinique. Crossreactivity between both species was analyzed by ELISA Inhibitions. Proteins of both species were digested with trypsin and analyzed by MS/MS (MALDI TOF/TOF). Different databases were used for the identification of the proteins. Results Sensitization to B. tropicalis was detected in 868 sera and to D. pteronyssinus in 919 sera; 135 were sensitized exclusively to B. tropicalis and 177 to D. pteronyssinus. Specific IgE levels to D. pteronyssinus were significantly higher than to B. tropicalis (Mean 0.67 OD ± 0.89 vs. 0.86 ± 1.04; P < 0.001). The correlation coefficient between specific IgE levels to both species was 0.135. ELISA Inhibition studies demonstrated minimal crossreactivity between both species. Western blots done with the serum of poly or monosensitized patients revealed specific IgE binding to similar proteins. Proteomic analysis revealed the presence of the following allergens: B. tropicalis: groups 2, 3, 4, 6, 9, 14 and groups 1, 2, 3, 4, 8, 11, 14, 15, 16 and 18 in D. pteronyssinus. Species specific, as well as common proteins were detected. Conclusions There is limited crossreactivity between B. tropicalis and D. pteronyssinus. Sensitization to their allergens seems to be a parallel phenomenon. Allergen extracts of both species are needed for the correct diagnosis and treatment of mite allergic individuals in the Caribbean.

Background Allergoids have been successfully used in the treatment of respiratory allergic diseases. They are modified allergen extracts that allow the administration of high allergen doses, due to their reduced IgE binding capacity.They maintain allergen-specific T-cell recognition. Since they are native allergen extracts that have been polymerized with glutaraldehyde, identification of the allergenic molecules requires more complicated methods. The aim of the study was to determine the qualitative composition of different polymerized extracts and investigate the presence of defined allergenic molecules using Mass spectrometry. Methods Proteomic analysis was carried out at the Proteomics Facility of the Hospital Nacional de Parapléjicos (Toledo, Spain). After reduction and alkylation, proteins were digested with trypsin and the resulting peptides were cleaned using C18 SpinTips Sample Prep Kit; peptides were separated on an Ultimate nano-LC system using a Monolithic C18 column in combination with a precolumn for salt removal. Fractionation of the peptides was performed with a Probot microfraction collector and MS and MS/MS analysis of offline spotted peptide samples were performed using the Applied Biosystems 4800 plus MALDI TOF/TOF Analyzer mass spectrometer. ProteinPilot Software V 2.0.1 and the Paragon algorithm were used for the identification of the proteins. Each MS/MS spectrum was searched against the SwissProt 2010_10 database, Uniprot-Viridiplantae database and Uniprot_Betula database. Results Analysis of the peptides revealed the presence of native allergens in the polymerized extracts: Der p 1, Der p 2, Der p 3, Der p 8 and Der p 11 in D. pteronyssinus; Bet v 2, Bet v 6, Bet v 7 and several Bet v 1 isoforms in B. verrucosa and Phl p 1, Phl p 3, Phl p 5, Phl p 11 and Phl p 12 in P. pratense allergoids. In all cases, potential allergenic proteins were also identified, including ubiquitin, actin, Eenolase, fructose-bisphosphate aldolase, luminal-binding protein (Heat shock protein 70), calmodulin, among others. Conclusions The characterization of the allergenic composition of allergoids is possible using MS/MS analysis. The analysis confirms the presence of native allergens in the allergoids. Mayor allergens are preserved during polymerization.