Reactivity to IgE‐dependent histamine‐releasing factor is due to monomeric IgE

Background: Lipid transfer proteins (LTP) are stable, potentially life-threatening allergens in fruits and many other vegetable foods. The aim of this study was to clone and express recombinant apple LTP (Mal d 3), as has previously been done for peach LTP (Pru p 3) and set up quantitative tests for measuring fruit LTPs. Methods: cDNA for Mal d 3 and Pru p 3 was cloned, expressed in the yeast Pichia pastoris and the resulting proteins were purified via cation exchange chromatography. The immune reactivity of rMal d 3 was compared to nMal d 3 by RAST (inhibition), immunoblotting and basophil histamine release testing. To obtain monoclonal and monospecific polyclonal antibodies, mice and rabbits were immunized with purified nMal d 3. Results: The deduced amino acid sequence of Mal d 3 was identical to the published sequence, Pru p 3 differed at two positions (S9A and S76H). The rMal d 3 had an IgE-binding potency and biological activity close to its natural counterpart. One sandwich ELISA selectively detecting apple LTP and another cross-reactive with cherry, nectarine and hazelnut LTP were developed. In addition, a competitive RIA was developed with polyclonal rabbit antiserum and labeled nMal d 3. Conclusion: rMal d 3 (as shown before for rPru p 3) may be a useful tool for application in component-resolved diagnosis of food allergy. Assays for the measurement of LTP will increase the traceability of this potentially dangerous allergen.

Section: Methodsmentioning

confidence: 99%

Lipid Transfer Proteins from Fruit: Cloning, Expression and Quantification

Zuidmeer

Leeuwen

Budde

et al. 2005

“…White blood cells were isolated from blood of a nonallergic donor by Percoll centrifugation and stripped from IgE by lactic acid treatment as described elsewhere [39, 40]. Subsequently, cells were resensitized with affinity-purified group-5-specific IgE.…”

Section: Methodsmentioning

confidence: 99%

The Major Grass Pollen Group 5 Allergen from Dactylis glomerata and Its C-Terminal Split Product Both Behave as Dimers: Implications for Allergen Standardization

Oort

Dieker

Heer

et al. 2005

Background: On SDS-PAGE grass pollen group-5 allergens migrate as a doublet with an apparent molecular mass (M_r) of 25 kDa. Immunoblot analysis revealed additional group 5 reactivity at double and half this M_r. The aim of this study was to investigate these group 5 molecular entities and to compare their allergenicity and behavior in quantitative immunoassays. Methods: Group-5-specific monoclonal antibodies were produced and used for the development of a group-5-specific sandwich ELISA. Affinity-purified Dac g 5 was separated by SDS-PAGE/Western blotting; individual bands were analyzed by N-terminal sequencing. Size exclusion chromatography (SEC) in conjunction with group-5-specific ELISA, competitive RIA and RAST inhibition were used to analyze the size distribution of Dac g 5. Basophil histamine release assays were used to assess biological activity. Results: The lower band of the typical group 5 doublet was identified as a truncated form lacking the typical group 5 N-terminus AD^L/_AGY, observed in the upper band. The 12-kDa peptide was shown to be the C-terminal half of Dac g 5 (amino acid 127 onwards). SEC in conjunction with competitive RIA revealed that around 45% of Dac g 5 is represented by the 12-kDa peptide. Both the C-terminal half and the whole allergen dimerize under nondenaturing conditions. In competitive RIA and RAST inhibition both forms are equally well detected. In contrast, the half molecule is poorly recognized in sandwich ELISA and displays negligible biological activity in basophil histamine release tests with purified IgE. Conclusions: These observations stress the need to evaluate the performance of allergen standardization protocols in detail, with special attention to allergen size distribution.

“…White blood cells were isolated from blood of a nonallergic donor by Percoll centrifugation and stripped from IgE by lactic acid treatment as described elsewhere [35, 36]. Subsequently, cells were resensitized with patients’ sera (n = 6) testing positive (RAST) for group 5 allergen.…”

Section: Methodsmentioning

confidence: 99%

Immunochemical Characterization of Two Pichia pastoris-Derived Recombinant Group 5 Dactylis glomerata Isoallergens

Oort

Heer

Lerouge

et al. 2001

Background: Grass pollen of the Poaceae grasses are known to be highly allergenic. Major allergens from the species Lolium, Phleum, Poa and Holcus have been cloned and expressed as recombinant proteins, but of the important species Dactylis glomerata no recombinants are available. Methods: Dac g 5 was cloned by PCR on the basis of homology with Lol p 5 and expressed in Pichia pastoris. Recombinant Dac g 5 (rDac g 5) was affinity purified and compared to natural Dac g 5 (nDac g 5) by immunoblot, radioallergosorbent test (RAST), RAST inhibition, basophil histamine release assay (HRA), competitive radioimmunoassay (RIA) and sandwich enzyme-linked immunosorbent assay (ELISA). In addition, N-terminal sequencing, concanavalin A (Con A) binding, circular dichroism spectrum measurements and matrix-assisted laser desorption ionization-time of flight mass-spectrometric analysis were performed. Results: Clones were obtained that coded for pro-Dac g 5 and two mature isoforms of Dac g 5; the deduced amino acid sequences of both isoforms differed by 4 amino acids. Both mature isoforms were expressed in Pichia at a concentration of ∼15 mg/l. SDS-PAGE analysis showed that rDac g 5 had an apparent M_r approximately 10 kD above nDac g 5. By mass spectrometry this difference was shown to be around 2.5 kD. Positive Con A staining suggested (O-linked) glycosylation as an explanation for this increase in M_r. Whereas both purified recombinants showed a tendency to dimerize, purified nDac g 5 contained a 12-kD peptide not observed for rDac g 5. RAST, RAST inhibition and HRA showed that the IgE reactivity of rDac g 5 was similar to that of nDac g 5. A small subgroup, however, clearly demonstrated decreased IgE reactivity to rDac g 5.02. Differences in immune reactivity of both isoforms were confirmed by monoclonal antibody (mAb)-based sandwich ELISA. Conclusions: Dac g 5 was successfully cloned and expressed in P. pastoris. Minor differences in primary structure between isoforms influence their immune reactivity.