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- Elfman, LHM, et al.
(författare)
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IgE binding capacity of synthetic and recombinant peptides of the major storage mite (Lepidoglyphus destructor) allergen, Lep d 2
- 1998
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Ingår i: International archives of allergy and immunology. - : S. Karger AG. - 1018-2438 .- 1423-0097. ; 117:3, s. 167-173
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Tidskriftsartikel (refereegranskat)abstract
- <b>Background:</b> Lepidoglyphus destructor is an important non–pyroglyphid mite species in Europe and a dominant allergen in farming environments. The major allergen of L. destructor, Lep d 2, is a protein of 13.2 kD that is recognised by about 90% of sera RAST positive to this mite species. <b>Methods:</b> The cDNA of two isoallergens of the Lep d 2 has previously been sequenced and the protein expressed in different protein expression systems. In order to map the B–cell epitopes, the full length protein and the truncated forms of the protein have been expressed in Escherichia coli as glutathione–S–transferase (GST) fusion proteins. Recombinant Lep d 2 fragments and synthetic overlapping 15 mer peptides spanning Lep d 2 were probed with sera from patients allergic to storage mite. <b>Results:</b> The full–length (125 amino acids) GST fusion protein reacted strongly with patient IgE in Western blots and dot blots. Synthetic peptides failed to react with IgE antibodies from mite–allergic patients and the truncated fusion proteins displayed weak IgE–binding capacity. <b>Conclusion:</b> We conclude that there are no dominant linear IgE–binding epitopes in Lep d 2. Recombinant or synthetic Lep d 2 fragments may, however, be further evaluated as hypoallergenic candidate molecules for specific immunotherapy.
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| 4. |
- Johansson, E, et al.
(författare)
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Evaluation of specific IgE to the recombinant group 2 mite allergens Lep d 2 and Tyr p 2 in the Pharmacia CAP system
- 1999
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Ingår i: International archives of allergy and immunology. - : S. Karger AG. - 1018-2438 .- 1423-0097. ; 120:1, s. 43-49
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Tidskriftsartikel (refereegranskat)abstract
- <b>Background:</b> Several recombinant allergens have been shown to be potentially useful for diagnosis of IgE–mediated allergy, but only a few recombinant allergens are at present commercially available in serological assays for detection of specific IgE. The aim of this study was to evaluate the IgE binding to the recombinant major dust mite allergens rLep d 2 and rTyr p 2 and compare it with the IgE binding to the commercial mite extracts <i>Lepidoglyphus destructor</i> and <i>Tyrophagus putrescentiae</i> in the Pharmacia RAST CAP System. <b>Methods:</b> The recombinant allergens rLep d 2 and rTyr p 2 were immobilised on ImmunoCAPs, and sera from 461 Swedish farmers who are frequently exposed to mites were analysed for specific IgE antibodies. Immunoblotting was performed to evaluate discrepancies between the results obtained with the recombinant and the commercial CAP assays. <b>Results:</b> The IgE values of each recombinant assay significantly correlated with the IgE values of the corresponding commercial CAP assay. The sensitivity of the rLep d 2 assay was 73.3% and that of the rTyr p 2 assay, 60.5% of that provided by the commercial <i>L. destructor</i> and <i>T. putrescentiae</i> assays. Two subjects out of 416, who tested negative in the commercial <i>L. destructor assay</i>, were positive to rLep d 2. The corresponding figures for rTyr p 2 and the <i>T. putrescentiae</i> extract were 5/418. The possibility that these subjects were sensitised to <i>L. destructor</i> and <i>T. putrescentiae</i> could not be excluded. <b>Conclusions:</b> The data suggest that it may be possible to use rLep d 2 and rTyr p 2 on ImmunoCAPs to detect and quantify IgE antibodies to these, the major allergens of <i>L. destructor</i> and <i>T. putrescentiae</i>. It appears likely that the addition of just a few more recombinant <i>L. destructor</i> and <i>T. putrescentiae</i> allergens in the CAP assay will be sufficient for in vitro diagnosis of IgE mediated allergy to <i>L. destructor</i> and <i>T. putrescentiae</i>.
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| 8. |
- Eriksson, TLJ, et al.
(författare)
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Identification and characterisation of two allergens from the dust mite Acarus siro, homologous with fatty acid-binding proteins
- 1999
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Ingår i: International archives of allergy and immunology. - : S. Karger AG. - 1018-2438 .- 1423-0097. ; 119:4, s. 275-281
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Tidskriftsartikel (refereegranskat)abstract
- <b>Background:</b> Dust mites are a major cause of allergic disease worldwide. The dust mite <i>Acarus siro</i> is an inducer of occupational allergy among farmers, but sensitisation has also been found in non–farming populations. <b>Methods:</b> A degenerate primer was designed to the N–terminal amino acid sequence of a 15–kD IgE–binding protein in <i>A. siro</i> extract. The cDNA sequence was obtained by using reverse transcriptase polymerase chain reaction, standard cloning and sequencing techniques. The protein was expressed in <i>Escherichia coli</i> with a 6–histidine tag at its C–terminus. Immunoblotting of the recombinant protein and whole extract was performed using patient sera. <b>Results and conclusion:</b> 15 and 17–kD allergens were identified in a fraction of <i>A. siro</i> extract. The cDNA of the 15–kD allergen was isolated, cloned and sequenced and the allergen was expressed as a recombinant protein. The calculated molecular weight of the cDNA–encoded protein is 14.2 kD. The predicted amino acid sequence has one potential N–glycosylation site at position 4–6 and a cytosolic fatty acid–binding protein signature at position 5–22. The protein has 64% sequence identity with Blo t 13, an allergen from the dust mite <i>Blomia tropicalis</i>, as well as homology with several other fatty acid–binding proteins (FABPs) from different organisms. The allergen was named Aca s 13 and was recognised strongly by 3 of 13 (23%) of the subjects investigated. The amino acid sequence of the 17–kD protein was partly determined and it also showed high sequence homology with Blo t 13 and FABPs.
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