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Notes: A female patient experienced a severe allergic reaction after consumption of vineyard snails. The patient proved to be sensitized to house-dust mite (HDM) and demonstrated a positive skin test and specific IgE to snail (Eobania vermiculata, Lofarma). The snail RAST was 〉 80% inhibited by HDM, whereas the mite RAST was 〈 10% inhibited by snail extract. This is possibly another example of food allergy related to primary sensitization by an aeroallergen.

Notes: For most foods, true standardization is not yet feasible because there is insufficient information on the relative importance of individual allergens and their variants. Standardization without sufficient information may easily be counterproductive because improvements are less likely to be implemented. In the analysis of natural test material, the following principles apply:〈list xml:id="l1" style="custom"〉1) RAST inhibition is usually inferior to other means of allergen quantitation.2) Immunoblot is inefficient for some “important” allergens and over-efficient for some “unimportant” allergens and may therefore be deceptive.3) Single-component assays are the only satisfactory way to describe complex mixtures.Improving the actual food-testing procedure is important, but will not alone result in a reliable diagnostic procedure. Tests for measuring “effect modifiers” will have to be developed in order to predict in vivo reactions to foods.

Notes: A group of 28 patients from Italy was studied who had asthma after consumption of snail. All patients also had asthma and/or rhinitis caused by house-dust mite. RAST analyses confirmed the combined sensitization to snail and mite. In a few sera, IgE antibodies reactive with other foods of invertebrate origin (mussel and shrimp) were detected. RAST inhibition showed that most IgE antibodies against snail were cross-reactive with house-dust mite. In contrast, the mite RAST was not significantly inhibited by snail. This indicates that house-dust mite was the sensitizing agent. Immunoblot analyses revealed multiple bands in snail extract recognized by IgE. In contrast to what has been described for cross-reactivity between shrimp and mite, tropomyosin played only a minor role as a cross-reactive allergen in these patients. The observations in this study indicate that snail consumption can cause severe asthmatic symptoms in house-dust-mite-allergic patients. It might, therefore, be advisable to screen mite-allergic asthma patients for allergy to snail and other invertebrate animal foods.

Notes: Background and Objective Chimeric mouse/human monoclonal IgGl and IgG4 antibodies were developed against the house dust mite allergen Der p 2. These chimeric IgG antibodies, hIgG1-Dp2 A and hIgG4-Dp2 A, have the same binding characteristics as the previously reported chimeric hIgE-Dp2 A and are composed of the heavy chain variable domains and light chains ot the original murine monoclonal antibody 2B12., whereas the heavy chain constant domains have been replaced by the human IgGl or IgG4 heavy chain. The expression level of hIgG1-Dp2 A and hIgG4-Dp2 A was 1 and 3.5 μg/mL, respectively.Methods and Results Since all IgG in these culture supernatants is allergen-specific. they are useful reference reagents and enable the calculation of the amount of allergen specific IgG l and IgG4 antibodies in absolute IgG amounts. The results obtained with two panels of sera from patients in immunotherapeutic treatment were evaluated and compared in Der p 2 IgE, IgGl and IgG4 RAST and with reversed lgG4 RAST using labelled purified Der p 2. Close agreement between the results for the two IgG4 assays was found.Conclusion With these chimeric reference reagents the quantities of isotype specific antiallergen antibodies can be calculated and compared.

Notes: IgE- and IgG4 antibodies were compared for reactivity with recombinant chain 1 and chain 2 of the cat allergen Felis domesticus (Fei d) I. Recombinanl chain 1 and chain 2 were coupled to sepharose and tested in IgE- and IgG4 radioallergosorbent test (RAST) experiments. Substantial IgE- and IgG4 binding was found. The fraction of Pel d I-specific antibody that bound to the recombinant chains was calculated. For chain 1, the mean value of this fraction was 0.30 for IgE and 0.23 for lgG4 (P= 0.05). For chain 2, the mean value of this fraction was 0.19 for IgE and 0.13 for IgG4 (P= 0.02). These results indicate that differences in fine specificity exist between IgE and IgG4 antibodies. Moreover, these findings support our results with chemically prepared peptides derived from these two chains and suggest that the B cells producing IgE antibodies are more likely to recognize a less ‘native’ form of Pel d I, compared with IgG4.

Notes: Background IgE titres tend to rise early after the start of immunotherapy, followed by a decline to pre-immunotherapy levels or lower.Objectives We were interested to ktiow whether the early increase in IgE antibodies includes new specificities of IgE, and whether these responses persist.Methods Sera of 64 patients undergoing grass pollen immunotherapy were tested for IgE against four purified grass pollen allergens: Lol p 1. 2, 3, and 5. At least two serum samples were taken, one before the start of therapy and one between 5 and 18 months after the first immunization (mean: 10 months).Results The mean IgE responses to Lol p 1, 2 and 3 showed a moderate but not significant increase. In contrast, the mean IgE response to Lol p 5 showed a significant decrease of 〉30%. IgE against total Lolium perenne pollen extract moderately increased (〉20%), showing that a RAST for total pollen is not always indicative for the development of IgE against its major allergens. For 〉40% of the patients it was found that IgE against one or more of the four allergens increased, while IgE against the remaining allergen(s) decreased. Eor 10 sera the ratio of IgE titres against at least two allergens changed by at least a factor of 5. The changes in specific IgE also included conversions from negative (〈 0.1 RU) to positive (0.6 to 5.0 RU) for five patients. For two patients, the induction of these ‘new’ IgE antibodies against major allergens was shown to result in a response that was persistent over several years.Conclusion Although active induction of new IgE specificities by immunotherapy was not really proven, the observations in this study indicate that monitoring of IgE against purified (major) allergens is necessary to evaluate changes in specific IgE in a reliable way.

Notes: Two assays have been developed to measure arthropod levels in house dust. The first assay measures silverfish antigens. The second assay measures invertebrate tropo-myosin and gives a global assessment of the level of atthropod-derived material. These assays and a Der p 1 and Der p 2 assay were used to analyse 53 dust samples. In most dust samples the ratio of tropomyosin/Der p 2 was higher than in mite body extract, indicating that the assay measures other arthropods besides mites. Silverfish antigen was detectable in most of the dust samples. In many homes in which the inhabitants were unaware of the presence of silverfish, silverfish antigen was detectable. Therefore for information on exposure an immunochemical analysis is superior to a questionnaire.

Notes: Recombinant Der p 2, expressed in yeast, lacked reactivity with 5 monoclonal antibodies against natural Der p 2.〈section xml:id="abs1-2"〉〈title type="main"〉ObjectiveThe aim of this study was to investigate whether the lack of reactivity with recombinant Der p 2 can be explained by the existence of isoforms.〈section xml:id="abs1-3"〉〈title type="main"〉MethodsBy site-directed mutagenesis three recombinant isoforms of Der p 2 were produced. Reactivity with monoclonal antibodies and human IgE was analysed by means of RAST and RAST-inhibition.〈section xml:id="abs1-4"〉〈title type="main"〉ResultsAll five monoclonals that lacked reactivity with the originally selected isoform, showed reactivity upon replacement of aspartic acid by asparagine at position 114. The other two substitutions (at position 26 and 47) had no effect. Binding of human IgE (n = 10) was not significantly influenced by the isogenetic variation at position 114.〈section xml:id="abs1-5"〉〈title type="main"〉ConclusionsMonoclonal antibodies raised against natural Der p 2 can sometimes discriminate between different isoforms, allowing the study of the natural occurrence of isoforms. For application in allergen-measurement assays, non-discriminating monoclonal antibodies should be selected.

Notes: Current diagnostic tests for Fagales tree pollen allergy are often composed of mixtures of pollen of birch, alder and hazel. Their complex composition hampers accurate standardization.〈section xml:id="abs1-2"〉〈title type="main"〉ObjectiveThe aim of this study was to investigate whether mixtures of tree pollen extracts can be replaced by a single pollen species, and whether a single pollen species can be replaced by a limited number of purified natural or recombinant major allergens.〈section xml:id="abs1-3"〉〈title type="main"〉MethodsSera (n = 1725) were selected on ground of a general suspicion for inhalant allergy, and tested in a RAST for birch, alder and hazel pollen. Sera with 〉 0.5 RU/mL for any of the three species were tested in a RAST for natural Bet v 1 and Bet v 2 as well as for recombinant versions of both allergens.〈section xml:id="abs1-4"〉〈title type="main"〉ResultsSpecific IgE antibodies (〉 0.3 RU/mL) against birch, alder and hazel were found in 242, 298 and 292 sera, respectively. All sera with a positive RAST for alder and/or hazel and a negative RAST for birch were low-responder sera on alder and hazel, only five sera having a RAST value 〉 1.0 (all 〈 2.0). For all sera with a RAST 〉 0.5 RU/mL (n = 250), the mean of individual ratio's alder/birch and hazel/birch was 1.02 and 0.54, respectively. Of 223 of these sera, 63.2% had specific IgE against natural Bet v 1 and 63.7% against natural Bet v 2. When responses to both allergens were combined 93.7% were positive. The mean ratios Bet v 1 + 2/extract were 1.00, 1.04 and 2.11 in case of birch, alder and hazel, respectively. For 211 sera the same analysis was performed with recombinant Bet v 1 and Bet v 2. Only six sera with Bet v 1-specific IgE (all 〈 0.5 RU/mL) were negative (〈 0.3 RU/mL) on recombinant Bet v 1. For Bet v 2, 77/132 sera with specific IgE to the natural allergen did not react to the recombinant version. Twelve false-negatives had RAST values 〉 1.0 RU/mL. The mean of the individual recombinant/natural ratios was 0.98 for Bet v 1 and 0.38 for Bet v 2 (P 〈 0.001). The mean ratio rBet v 1 + 2/birch was 0.75 with 17.5% false-negatives on the combination of recombinant allergens.〈section xml:id="abs1-5"〉〈title type="main"〉ConclusionReliable in vitro diagnosis is possible with a single tree pollen extract (birch or alder). The same is true for purified natural Bet v 1 and Bet v 2. A combination of recombinant molecules is slightly less efficient.

Notes: Six monoclonal antibodies against Bet v I, the major cross-reactive allergen of birch pollen (Betula verrucosa), were obtained. Four did not react with fruits, but two monoclonal antibodies (mAbs) (5H8 and 9C11) were reactive with apple and other fruits. These two cross-reactive antibodies reacted with identical or overlapping sites, but differed in their relative degree of cross-reactivity toward various fruits and hazelnut. Cross-reactive human IgE antibodies reacted with a nonoverlapping epitope, as indicated by results of a two-site radioimmunoassay (RIA) with the fruit-reactive mAb 9C11. By isoelectric focusing (IEF) in conjunction with immunoblotting, a maximum of seven isoforms could be distinguished. Depletion of birch-pollen extract for Bet v I with the most reactive mAb (7F7) removed approximately 95% of the IgE cross-reactivity between birch pollen and apple extract. The remaining 5% cross-reactive material was still capable of inhibiting the binding of IgE to apple allergen completely, and was reactive with mAbs 5H8 and 3C4. By means of IEF/immunoblot, it was shown that these mAbs recognize an isoform of Bet v I that is poorly, if at all, recognized by mAb 7F7. These results illustrate the heterogeneity of Bet v I, both with respect to the cross-reactive sites as well as to the backbone structure. This type of heterogeneity has possible implications for the use of monoclonal antibodies in allergen standardization.