METHODS: Raw and cooked extracts of the giant freshwater prawn were prepared. The IgE reactivity pattern was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting technique with the sera of 20 skin prick test (SPT) positive patients. The major allergen identified was then characterized using the proteomics approach involving a combination of two-dimensional (2-DE) electrophoresis, mass spectrometry and bioinformatics tools.
RESULTS: SDS-PAGE of the raw extract showed 23 protein bands (15-250 kDa) but those ranging from 40 to 100 kDa were not found in the cooked extract. From immunoblotting experiments, raw and cooked extracts demonstrated 11 and 5 IgE-binding proteins, respectively, with a molecular mass ranging from 15 to 155 kDa. A heat-resistant 36 kDa protein was identified as the major allergen of both extracts. In addition, a 42 kDa heat-sensitive protein was shown to be a major allergen of the raw extract. The 2-DE gel fractionated the prawn proteins to more than 50 different protein spots. Of these, 10 spots showed specific IgE reactivity with patients' sera. Matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis led to identification of 2 important allergens, tropomyosin and arginine kinase.
CONCLUSIONS: It can be concluded that the availability of such allergens would help in component-based diagnosis and therapy of prawn allergies.
Methods: The antibody binding pattern of the epitope was analyzed using bioinformatics tools. The IgG production in mice were examined by FACS Calibur™ Flow cytometer.
Results: The epitope bound the 72A1 monoclonal antibody at the same site as GP350/220 protein, indicating that the epitope should stimulate B cells to produce antibody. Moreover, in vivo administration of EBVepitope successfully induced IgG expression from B cells, compared with controls. Further investigation indicated that the relative number of B cells expressing IgE in EBVepitope-treated mice was lower than controls.
Conclusions: Our data suggest that this EBV GP350 epitope is able to induce IgG expression in vivo without causing allergic reactions, and represents a potential EBV vaccine candidate.
OBJECTIVE: The aim of this study was to clone and express Hev b 3 and to obtain the immunologic active and soluble recombinant allergen for diagnosis of SB-associated latex allergy.
METHODS: A complementary DNA (cDNA) coding for Hev b 3 was amplified from RNA of fresh latex collected from Malaysian rubber trees (Hevea brasiliensis). PCR primers were designed according to sequences of internal peptide fragments of natural (n) Hev b 3. The 5'-end sequence was obtained by specific amplification of cDNA ends. The recombinant (r) Hev b 3 was produced in Escherichia coli as a 6xHis tagged protein. Immunoblotting and inhibition assays were performed to characterize the recombinant allergen.
RESULTS: An Hev b 3 cDNA clone of 922 bp encoding a protein of 204 amino acid residues corresponding to a molecular weight of 22.3 kd was obtained. In immunoblots 29/35, latex-allergic patients with SB revealed IgE binding to rHev b 3, as did 4 of 15 of the latex-sensitized group. The presence of all IgE epitopes on rHev b 3 was shown by its ability to abolish all IgE binding to nHev b 3. Hev b 3 is related to Hev b 1 by a sequence identity of 47%. Cross-reactivity between these 2 latex allergens was illustrated by the large extent of inhibition of IgE binding to nHev b 1 by rHev b 3.
CONCLUSION: rHev b 3 constitutes a suitable in vitro reagent for the diagnosis of latex allergy in patients with SB. The determination of the full sequence of Hev b 3 and the production of the recombinant allergen will allow the epitope mapping and improve diagnostic reagents for latex allergy.
METHODS: A prospective multicentre assessment of stable COPD (n=614) was undertaken in five hospitals across three countries: Singapore, Malaysia and Hong Kong. Clinical and serological assessment was performed against a panel of 35 fungal allergens including crude and recombinant Aspergillus and non-Aspergillus allergens. Unsupervised clustering and topological data analysis (TDA) approaches were employed using the measured sensitisation responses to elucidate if sensitisation subgroups exist and their related clinical outcomes.
RESULTS: Aspergillus fumigatus sensitisation was associated with increased exacerbations in COPD. Unsupervised cluster analyses revealed two "fungal sensitisation" groups. The first was characterised by Aspergillus sensitisation and increased exacerbations, poorer lung function and worse prognosis. Polysensitisation in this group conferred even poorer outcome. The second group, characterised by Cladosporium sensitisation, was more symptomatic. Significant numbers of individuals demonstrated sensitisation responses to only recombinant (as opposed to crude) A. fumigatus allergens f 1, 3, 5 and 6, and exhibited increased exacerbations, poorer lung function and an overall worse prognosis. TDA validated these findings and additionally identified a subgroup within Aspergillus-sensitised COPD of patients with frequent exacerbations.
CONCLUSION: Aspergillus sensitisation is a treatable trait in COPD. Measuring sensitisation responses to recombinant Aspergillus allergens identifies an important patient subgroup with poor COPD outcomes that remains overlooked by assessment of only crude Aspergillus allergens.