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Background Evidence indicates that infection with Ascaris lumbricoides may promote development of allergy and asthma. Objective To study the role of tropomyosin, a pan-allergen in invertebrates, in IgE responses to A lumbricoides. Methods Recombinant A lumbricoides and Periplaneta americana tropomyosins were expressed in Pichia pastoris . Levels of IgE to tropomyosins from A lumbricoides and P americana were determined by chimeric ELISA in sera from 119 children living in a parasite-endemic area and 112 patients with cockroach allergy from the allergy clinics. Presence of tropomyosin in A lumbricoides larvae at L3 stage was evaluated by immunofluorescence using mAb 1A6, directed against mite tropomyosin. Molecular modeling of P americana and A lumbricoides tropomyosins was performed by using the MODELLER program. Results A lumbricoides tropomyosin showed 69% to 98% sequence identity to tropomyosins from other invertebrates. The predicted structure of A lumbricoides tropomyosin was similar to that of P americana tropomyosin and showed the characteristic coiled-coil structure. Strong correlation was found for IgE antibodies to tropomyosins from A lumbricoides and P americana in sera from children living in a parasite-endemic area and from patients with cockroach allergy. Larvae of A lumbricoides reacted strongly with mAb 1A6. Conclusion Tropomyosin induces IgE responses in A lumbricoides –infected children and in patients allergic to cockroach.

Background Production of soluble correctly folded recombinant group 1 allergens has proven to be difficult. Purified natural group 1 allergens could be an alternative for application in immunotherapy. Objective Cloning and expression of recombinant Dac g 1; purification of natural Dac g 1 variants and immunochemical characterization of these molecules. Methods Dac g 1 was cloned and expressed in the yeast Pichia pastoris . Hydrophobic interaction (HIC), size exclusion, and/or affinity chromatography were used to purify Dac g 1 from Dactylis glomerata pollen extract. Dac g 1 variants were analyzed by N-terminal sequencing. Immune reactivity was assessed by sandwich ELISA, competitive RIA, RAST (inhibition), and in vitro basophil histamine release tests. Results Dac g 1 was cloned, revealing up to 98% amino acid sequence homology to other group 1 allergens. Purification of natural Dac g 1 revealed at least 3 variants, with an apparent molecular mass (M r ) on SDS-PAGE of 33 kd (HM r ), 30 kd (IM r ) and 28 kd (LM r ). Extraction of IM r Dac g 1 required 0.9% saline, whereas the other 2 variants were also extractable in water. The N-terminus of HM r and IM r Dac g 1 differs at 2 positions, and LM r Dac g 1 was shown to be N-terminally truncated, lacking the first 30 amino acids. The nonretarded fraction of HIC commonly used in group 1 purification protocols does not contain this LM r molecule. IM r Dac g 1 was poorly recognized in 2 of 3 sandwich ELISAs and competitive RIA but demonstrated similar biological activity compared with HM r Dac g 1. Conclusions Natural Dac g 1 variants can be separated by extraction of pollen in the presence or absence of saline followed by HIC and size exclusion chromatography. Thus, purified Dac g 1 is an alternative to recombinant group 1 allergens.

The synthesis of IgE by B cells occurs at a low rate compared with that of other antibodies, even in allergic subjects. One rate-limiting step is the class switch, by which B lymphocytes switch to produce immunoglobulin epsilon heavy chains rather than micro or gamma heavy chains. We propose an additional rate-limiting step: survival of the B lymphocyte after the switch to IgE. The hypothesis we present here is that the survival of the IgE-switched B cell is compromised, particularly in an active germinal center. Antigenic stimulation in the absence of a danger signal fails to induce a mature germinal center, which allows IgE-switched B cells to escape and mature into plasma cells. Antigenic stimulation in the presence of a danger signal (or under nonhygienic conditions) induces germinal centers, which eliminate IgE-switched B cells. Thus the essence of an allergen is antigenic stimulation in the absence of conditions that generate mature germinal centers. Because germinal centers are important for the generation of B-cell memory, the IgE immune response is characteristically poor in memory (but might be long lasting because of the generation of long-lived plasma cells). In addition to this direct route to IgE, typical for atopic sensitization, another type of T(H)2 response exists. On chronic allergen exposure with the concomitant induction of germinal centers, IgG4-switched B memory cells are induced that are slow to differentiate into plasma cells. These IgG4-switched B memory cells might occasionally undergo a secondary switch to IgE.

A chimeric human IgE monoclonal antibody was developed against the house dust mite allergen Der p 2. This chimeric antibody (hIgE-Dp2A) was composed of the heavy-chain variable domains and light chains of the original murine monoclonal antibody retaining its binding characteristics, whereas the heavy-chain constant domains were exchanged with the human IgE heavy chain. The chimeric IgE expression level was IgE 600 IU/ml (1 IU = 2.4 ng/ml). The binding of the chimeric hIgE-Dp2A to mite extract was indistinguishable from that of the original mouse monoclonal antibody. Parallel dose-response curves were found when the binding of hIgE-Dp2A to mite extract and anti-IgE coupled to sepharose were compared. Binding levels were not identical; however, hIgE-Dp2A bound significantly better to the mite-extract sepharose. This result indicates that the commonly used anti-IgE on solid phase calibration systems may lead to an overestimation of the amount of allergen-specific IgE present in the serum sample. The less efficient binding of the detector anti-IgE in case of the anti-IgE sepharose is likely to be because of the occupation of epitopes of the IgE by the sepharose-bound anti-IgE. Dose-response curves of serial dilutions of patient samples were parallel with the hIgE-Dp2A dose-response curve, which indicates that hIgE-Dp2A behaves like natural IgE antibodies in binding to allergen coupled to solid phase. This antibody is well suited for use as a reference reagent in the RAST and enables the expression of the amount of allergen-specific IgE present in a patient sample in absolute amounts.