Fine structural recognition specificities of IgE antibodies distinguishing amoxicilloyl and amoxicillanyl determinants in allergic subjects.

IgE antibodies in the sera of subjects allergic to beta-lactam antibiotics detect a spectrum of specificities ranging from side-chain groups to an entire penicillin or cephalosporin molecule. In addition to such structural heterogeneity of allergenic determinants, IgE antibodies in the sera of different allergic subjects show heterogeneous recognition responses. Detailed immunochemical studies were carried out on the sera of penicillin-allergic subjects that showed selective and unexpected reactions with the frequently prescribed penicillin, amoxicillin. Antibodies from one subject reacted only with the amoxicilloyl determinant while IgE from another subject showed multiple reactivity with penicilloyl and penicillanyl determinants of different penicillins but not with the amoxicilloyl determinant. Quantitative hapten inhibition studies revealed that the combining sites of the former antibodies were complementary to amoxicillin in a form that permits binding to the hydroxyaminobenzyl side-chain and the thiazolidine ring carboxyl. These conditions are satisfied with the drug in the '-oyl' but not in the '-anyl' form which involves linkage through the 2-carboxyl of the thiazolidine ring. With the second serum, adsorption studies showed that the wide-ranging reactivity of IgE was due to a single population of antibodies that detected a common specificity on the different penicillins. Combining site studies revealed clear recognition of the benzyl portion of the side-chain of benzylpenicilloyl, benzylpenicillanyl, ampicilloyl, ampicillanyl and amoxicillanyl determinants when free antibody access to the side-chain was possible but little or no recognition of the ring hydroxyl of amoxicillin. Such uninhibited access may not occur, however, when amoxicillin is conjugated in the '-oyl' form since opening the beta-lactam ring allows increased flexibility and rotation of the molecule and the possibility of close association of the hydroxyaminobenzyl side-chain of amoxicillin with the linked peptide carrier. In such close steric association, H-bonding involving the ring hydroxyl and amino acids of the carrier may prevent antibody access to the side-chain region of the amoxicilloyl determinant.     

Detection and side-chain specificity of IgE antibodies to flucloxicillin in allergic subjects

Unlike studies on the antigenicity of penicillins in laboratory animals, limited information is available on the allergenicity of penicillins in man, especially with regard to fine structural allergenic differences between the many different penicillins. Inconsistent with the earlier conclusions of others, our studies suggest that side-chain structures to flucloxacillin-reactive IgE antibodies. Quantitative hapten inhibition studies revealed potent inhibition by flucloxacillin and three structurally related penicillins: oxacillin, cloxacillin and dicloxacillin. Analysis of the results showed that the side-chain group of flucloxacillin, 3-2(-chloro-6-fluorophenyl)-5-methyl-4-isoxazolyl, is recognized by some antibodies and that the 5-methyl-3-phenyl-4-isoxazolyl group, with or without halogen substituents, accounts for the reactivity of other antibodies and for the cross-reactions seen with some other penicillins. Since it is the side-chain group that distinguishes the many different types of penicillin, and since IgE antibodies in many of the allergic reactions recognize the side-chain groups, it is becoming clear that specific assays are required for the detection of IgE antibodies to each of the different penicillins.     

Synthesis,characterization and immunochemical evaluation of cephalosporin antigenic determinants

Lack of knowledge of the exact chemical structure of cephalosporin antigenic determinants has hindered clinical interpretation of adverse reactions to these drugs and delayed understanding of the mechanisms involved in the specific recognition and binding of IgE molecules to these antigenic determinants. We further resolve the relationship between structure and activity of proposed antigenic chemicals, including the rational design and synthesis of these haptenic structures. Comparative RAST inhibition studies of the synthesized molecules revealed that they were recognized by IgE antibodies induced by cephalosporin antibiotics. Thus, these data indicate that recognition is mainly directed to the acyl side chain and to the beta-lactam fragment that remains linked to the carrier protein in the cephalosporin conjugation course.     

Purification of allergenic proteins from peanut for preparation of the reactive solid phase of a specific IgE radioimmunoassay

Peanut is one of the most allergenic foods. Detection of specific IgE in the serum of allergic patients requires the purification of allergenic proteins. In the present work, proteins were recovered from peanut kernel after successive treatment in acetone and diethy ether. The proteins were dissolved in 0.05% TFA and analysed by RP-HPLC with a 0–100% gradient of methanol containing 0.05% TFA. The protein peaks were recovered and tested in SDS-PAGE. Eleven proteins were identified with a M_r ranging from 13 to 81. Western blotting was performed with sera from allergic patients. Allergenic proteins had a M_r of 15, 18, 19, 33, 41 and 67. By comparison, a protein fraction from peanut shell contained seven proteins with M_r ranging from 15 to 81. Only two proteins with M_r of 18 and 41 were detected in a Western blot. The protein fractions were coupled to epoxy-Sepharose and the gels were used as a solid reactive phase for detection by IgE-RIA of specific IgE from the serum of allergic patients.     

Allergenicity of Acroptilon repens and Juglans regia pollen in rats

Pollen proteins that are located in the cytoplasm or on the surface of the exine can function as allergens and evoke immune system responses in sensitive patients, leading to allergic rhinitis and asthma. In this research, the pollen allergenicity and ability to induce IgE response of the pollen of two plant species were studied in rats. Acroptilon repens is an herbaceous, invasive plant with entomophilous pollen, while Juglans regia which is a tree crop produces anemophilous pollen. Immunoblot analysis using sera of sensitised rats revealed IgE reactivity to three protein bands including the 70, 41 and 25.12 kDa bands present in the A. repens pollen extract, while only one single immunogenic band of 11 kDa was detected in J. regia pollen extract. Both pollen extracts increased the eosinophil content and caused some clinical signs of allergy in treated rats. The results showed that both entomophilous and anemophilous pollen can be allergenic.     

Measurement of IgG blocking antibodies by interference in the radioallergosorbent test

We previously found that sera of patients immunized with ragweed pollen extract contained a factor that interfered with the binding of IgE antibodies to solid-phase allergens in the radioallergosorbent test (RAST). We now describe an assay, RAST interference, to measure this factor, and we present evidence that the factor is IgG blocking antibody. Sera from immunized allergic patients were heated at 56 degrees C for 4 hr to destroy heat-labile Fc determinants on IgE and were tested for their ability to prevent binding of additional IgE antibody to solid-phase allergens in the RAST. Eight of 10 sera from allergic immunized patients gave RAST interference dose-response curves that did not differ from the arbitrary standard. The factor causing interference showed specificity for the immunizing antigen, was heat-stable, eluted from Sephadex G-200 in the 7S peak, was present only in sera of immunized patients, and rose after initiation of immunization. These results indicated that RAST interference can be used to measure IgG blocking antibodies with the same reagents employed for the measurement of IgE antibodies, provided the antiserum to IgE is specific for the heat-labile FC determinants on IgE.     

Detection of IgG and IgE antibodies to Aspergillus fumigatus in human sera by immunogold assay

An immunogold assay (IGA) was developed to detect IgG and IgE antibodies to Aspergillus fumigatus. Sixteen sera from patients with allergic bronchopulmonary aspergillosis (ABPA), aspergilloma, and normal controls were studied. All sera were also evaluated for antibodies against A. fumigatus by biotin-avidin linked enzyme immunosorbent assay (BALISA) and by agar gel double diffusion method. A. fumigatus specific IgG and IgE antibodies could be detected by IGA in all the patients' sera but not in the sera of normal controls. Both IgG and IgE antibodies to A. fumigatus could be demonstrated in all the sera by BALISA and normal controls showed only low levels of these antibodies. There was a positive correlation between the degree of reactivity detected by IGA, the BALISA titer and the precipitins by agar gel diffusion. It can be concluded that IGA is a reliable, sensitive and simple method capable of detecting both IgG and IgE antibodies against A. fumigatus in patient serum.     

Diagnosis of <Emphasis Type="Italic">Chenopodium album</Emphasis> allergy with a cocktail of recombinant allergens as a tool for component-resolved diagnosis

Chenopodium album pollen is one of the main sources of pollen allergy in desert and semi-desert areas and contains three identified allergens, so the aim of this study is comparison of the diagnostic potential of C. album recombinant allergens in an allergenic cocktail and C. album pollen extract. Diagnostic potential of the allergenic cocktail was investigated in 32 individuals using skin prick test and obtained results were compared with the acquired results from C. album pollen extract. Specific IgE reactivity against the pollen extract and allergenic cocktail was determined by ELISA and western blotting tests. Inhibition assays were performed for the allergenic cocktail characterization. The exact sensitization profile of all patients was identified which showed that 72, 81 and 46% of allergic patients had IgE reactivity to rChe a 1, rChe a 2 and rChe a 3, respectively. Almost all of C. album allergic patients (30/32) had specific IgE against the allergenic cocktail. In addition, there was a high correlation between IgE levels against the allergenic cocktail and IgE levels against the pollen extract. The allergenic cocktail was able to completely inhibit IgE binding to natural Che a 1, Che a 2 and Che a 3 in C. album extract. In addition, positive skin test reactions were seen in allergic patients that tested by the allergenic cocktail. The reliable results obtained from this study confirmed that the allergenic cocktail with high diagnostic potential could be replaced with natural C. album allergen extracts in skin prick test and serologic tests.     

Identification of Two Metallothioneins as Novel Inhalative Coffee Allergens Cof a 2 and Cof a 3

BackgroundDust of green coffee beans is known to be a relevant cause for occupational allergic disorders in coffee industry workers. Recently, we described the first coffee allergen (Cof a 1) establishing an allergenic potential of green coffee dust.ObjectiveOur aim was to identify allergenic components of green coffee in order to enhance inhalative coffee allergy diagnosis.MethodsA Coffea arabica pJuFo cDNA phage display library was created and screened for IgE binding with sera from allergic coffee workers. Two further coffee allergens were identified by sequence analysis, expressed in E. coli, and evaluated by Western blots. The prevalence of sensitization to recombinant Cof a 1, Cof a 2, and Cof a 3 and to commercially available extract was investigated by ELISA (enzyme-linked immunosorbent assay) respectively CAP (capacity test) screening in 18 sera of symptomatic coffee workers.ResultsIn addition to the previously described chitinase Cof a 1, two Coffea arabica cysteine-rich metallothioneins of 9 and 7 kDa were identified and included in the IUIS Allergen Nomenclature as Cof a 2 and Cof a 3. Serum IgE antibodies to at least one of the recombinant allergens were found in 8 out of 18 symptomatic coffee workers (44%). Only 2 of the analysed sera (11%) had reacted previously to the commercial allergy test.ConclusionsIn addition to the previously described Cof a 1 we have identified two further coffee proteins to be type I coffee allergens (Cof a 2 and Cof a 3) which may have a relevant potential for the specific diagnosis and/or therapy of coffee allergy.     

Specificities of haemagglutinating antibodies evoked by members of the cephalosporin C family and benzylpenicillin

1. Antisera have been produced in rabbits to benzylpenicillin and four members of the cephalosporin C family and to conjugates of these substances with bovine gamma-globulin. 2. Deacetoxycephalosporin C reacted less readily and deacetylcephalosporin C lactone more readily with bovine gamma-globulin than did benzylpenicillin, cephalosporin C or deacetylcephalosporin C. 3. Antisera to free or conjugated benzylpenicillin agglutinated red cells sensitized with a variety of penicillins, but only reacted to a significant extent with cells sensitized with the cephalosporins tested when the latter contained an N-phenylacetyl or chemically related side chain. 4. Antisera to members of the cephalosporin C family agglutinated cells sensitized with these cephalosporins or with penicillin N, but did not react with cephalosporins whose side chains were chemically unrelated to alpha-aminoadipic acid. 5. Members of the cephalosporin C family and products of hydrolysis of cephalosporin C behaved as hapten inhibitors of antisera to cephalosporin C, but 7-aminocephalosporanic acid was relatively ineffective. 6. These findings are discussed in relation to differences in the chemical properties of penicillins and cephalosporins.     

Improved Synthesis of 3-14C-3-Hydroxy-3-methylglutaryl Coenzyme A

A new polysaccharide with a molecular weight of 5.0×10⁴ was isolated as a possible wheat allergen from a water-soluble fraction of flour by affinity chromatography and gel filtration. The isolated polysaccharide was found to be a possible wheat allergen, as it bound specifically to IgE antibodies in the sera of patients allergic to the water-soluble fraction of flour. Chemically, the sugar moiety of the polysaccharide consisted of D-glucose and D-mannose with β-1,4-linkages in a molar ratio of 4.4:1. Since this mannoglucan is thought to be stable in our body, it would act as a remaining allergen to cause a long-lasting allergic reaction to wheat flour.     

Mapping of IgE-binding regions on recombinant Cyn d 1, a major allergen from Bermuda Grass Pollen (BGP)

Background

Bermuda grass (Cynodon dactylon; subfamily Chloridoideae) is an important source of seasonal aeroallergens in warm tropical and sub-tropical areas worldwide. Improved approaches to diagnosis and therapy of allergic diseases require a thorough understanding of the structure and epitopes on the allergen molecule that are crucial for the antigen-antibody interaction. This study describes the localization of the human IgE-binding regions of the major group 1 pollen allergen Cyn d 1 from Bermuda grass.

Methods

A cDNA library was constructed from Bermuda grass pollen (BGP) using a Lambda gt11 expression vector. The gene encoding the Cyn d 1 allergen was isolated by screening the library with a mouse monoclonal antibody raised against grass group 1 allergen. In order to characterize the IgE epitopes on Cyn d 1, seven overlapping fragments and three deletion mutants were cloned and over-expressed in E. coli. The recombinant fragments and deletion mutants were evaluated for their comparative IgE reactivity with sera of non atopic individuals and grass pollen allergic patients by ELISA and a dot-blot assay.

Results

Analysis of IgE binding regions by overlapping fragments and deletion mutants identified two major allergenic regions corresponding to amino acids 120–170 and 224–244. Deletion of either or both regions led to a significant reduction in IgE binding, emphasizing the importance of the C-terminal region on Cyn d 1 in epitope-IgE interaction.

Conclusion

Anti-Cyn d 1 IgE antibodies from allergic human sera recognize two epitopes located at the C-terminal end of the molecule. These data will enable the design of improved diagnostic and therapeutic approaches for BGP hypersensitivity.     

Allergenic lipid transfer proteins from plant-derived foods do not immunologically and clinically behave homogeneously: the kiwifruit LTP as a model

Background

Food allergy is increasingly common worldwide. Tools for allergy diagnosis measuring IgE improved much since allergenic molecules and microarrays started to be used. IgE response toward allergens belonging to the same group of molecules has not been comprehensively explored using such approach yet.

Objective

Using the model of lipid transfer proteins (LTPs) from plants as allergens, including two new structures, we sought to define how heterogeneous is the behavior of homologous proteins.

Methods

Two new allergenic LTPs, Act d 10 and Act c 10, have been identified in green (Actinidia deliciosa) and gold (Actinidia chinensis) kiwifruit (KF), respectively, using clinically characterized allergic patients, and their biochemical features comparatively evaluated by means of amino acid sequence alignments. Along with other five LTPs from peach, mulberry, hazelnut, peanut, mugwort, KF LTPs, preliminary tested positive for IgE, have been immobilized on a microarray, used for IgE testing 1,003 allergic subjects. Comparative analysis has been carried out.

Results

Alignment of Act d 10 primary structure with the other allergenic LTPs shows amino acid identities to be in a narrow range between 40 and 55%, with a number of substitutions making the sequences quite different from each other. Although peach LTP dominates the IgE immune response in terms of prevalence, epitope recognition driven by sequence heterogeneity has been recorded to be distributed in a wide range of behaviors. KF LTPs IgE positive results were obtained in a patient subset IgE positive for the peach LTP. Anyhow, the negative results on homologous molecules allowed us to reintroduce KF in patients'' diet.

Conclusion

The biochemical nature of allergenic molecule belonging to a group of homologous ones should not be taken as proof of immunological recognition as well. The availability of panels of homologous molecules to be tested using microarrays is valuable to address the therapeutic intervention.     

Polymorphism in allergens

Allergens are antigens that elicit an IgE-mediated immune response; they originate from diverse sources such as pollens, mites, molds, mammal exudates, insects and food. Allergenic molecules can contain several antigenic determinants, termed epitopes. Allergenic proteins have been discovered with polymorphisms, i.e., a mixture of similar molecules with minor variations in their amino acid sequences. These are called isoallergens or allergenic variants depending on the degree of similarity. Polymorphism may be defined by the presence of several alleles of the same gene or as families of related genes. Polymorphisms can have an important effect on the epitopes recognized by T lymphocytes, monoclonal antibodies and IgE of allergic patients. Individual polymorphisms can affect the basal level of allergenicity as well as the cross-reactivity with other allergens. The use of isoforms with low or total absence of IgE binding capacity but with high capacity to stimulate T cell response has been suggested as an alternative to the conventional immunotherapy for allergic diseases. Standardization of allergenic compounds can be affected by the differing proportions of isoforms in allergenic sources from different regions.     

Allergen Microarray Indicates Pooideae Sensitization in Brazilian Grass Pollen Allergic Patients

Background

Grass pollen, in particular from Lolium multiflorum is a major allergen source in temperate climate zones of Southern Brazil. The IgE sensitization profile of Brazilian grass pollen allergic patients to individual allergen molecules has not been analyzed yet.

Objective

To analyze the IgE sensitization profile of a Brazilian grass pollen allergic population using individual allergen molecules.

Methods

We analyzed sera from 78 grass pollen allergic patients for the presence of IgE antibodies specific for 103 purified micro-arrayed natural and recombinant allergens by chip technology. IgE-ELISA inhibition experiments with Lolium multiflorum, Phleum pratense extracts and a recombinant fusion protein consisting of Phl p 1, Phl p 2, Phl p 5 and Phl p 6 were performed to investigate cross-reactivities.

Results

Within the Brazilian grass pollen allergic patients, the most frequently recognized allergens were Phl p 1 (95%), Phl p 5 (82%), Phl p 2 (76%) followed by Phl p 4 (64%), Phl p 6 (45%), Phl p 11 (18%) and Phl p 12 (18%). Most patients were sensitized only to grass pollen allergens but not to allergens from other sources. A high degree of IgE cross-reactivity between Phleum pratense, Lolium multiflorum and the recombinant timothy grass fusion protein was found.

Conclusions

Component-resolved analysis of sera from Brazilian grass pollen allergic patients reveals an IgE recognition profile compatible with a typical Pooideae sensitization. The high degree of cross-reactivity between Phleum pratense and Lolium multiflorum allergens suggests that diagnosis and immunotherapy can be achieved with timothy grass pollen allergens in the studied population.     

Chironomid midge sensitization in sewage workers: case study

Non‐biting chironomid midges (Diptera: Chironomidae) may cause sensitization and allergic reactions in humans and have recently been identified as a potential health problem in Swedish municipal sewage treatment plants. To investigate, on a pilot scale, the allergenic potential of chironomids in sewage workers, all workers (n = 8) at a sewage treatment plant and local controls (n = 16) completed a symptom questionnaire, underwent measurement of the fraction of nitric oxide in exhaled air, spirometry, and provided serum samples for the determination of atopy status and the prevalence of specific immunoglobulin E (IgE) antibodies against Chironomus thummi (Chi t) using a commercial fluorescence enzyme immunoassay (FEIA). Three sewage workers (38%) but no controls (0%) were FEIA positive for C. thummi‐specific IgE antibodies (P < 0.05). No other health‐related findings were significantly different between the groups. The study suggested that occupational exposure to Chironomids may cause sensitization with circulating IgE‐antibodies in sewage workers.     

Study on major mite allergens in Italian homes

Summary There has always been a great interest for allergologists in the relation between the exsposure to allergic pollens a/o perennials and allergic symptomatology. Indeed the allergologist is constantly looking for clear evidence to what is shown or suggested by clinical history and routine allergologic tests.At present the availability of reliable quantitative analytical methods has given us the possibility of making a study on the presence of house mite and cat allergens in Italian homes.The study involved 5 Italian towns and was carried out in autumn months and the collection of samples has been made with a simple retail electric vacuum cleaner. The dosage of the allergenic content of the dust has been carried out by an immunoassay using specific monoclonal antibodies and a mixture of allergic patients' sera and a monoclonal anti IgE antibody coupled with an enzyme. The results suggest a wide variability of the allergenic content of the samples coming from the same geographical area but a constant ratio between the major mite allergens (Der I/Der II) ranging around a value 21.The prevalence of one mite species over another is variable, and depends on the geographical locality.     

Search for Allergens from the Pollen Proteome of Sunflower (Helianthus annuus L.): A Major Sensitizer for Respiratory Allergy Patients

Background

Respiratory allergy triggered by pollen allergens is increasing at an alarming rate worldwide. Sunflower pollen is thought to be an important source of inhalant allergens. Present study aims to identify the prevalence of sunflower pollinosis among the Indian allergic population and characterizes the pollen allergens using immuno-proteomic tools.

Methodology

Clinico-immunological tests were performed to understand the prevalence of sensitivity towards sunflower pollen among the atopic population. Sera from selected sunflower positive patients were used as probe to detect the IgE-reactive proteins from the one and two dimensional electrophoretic separated proteome of sunflower pollen. The antigenic nature of the sugar moiety of the glycoallergens was studied by meta-periodate modification of IgE-immunoblot. Finally, these allergens were identified by mass-spectrometry.

Results

Prevalence of sunflower pollen sensitization was observed among 21% of the pollen allergic population and associated with elevated level of specific IgE and histamine in the sera of these patients. Immunoscreening of sunflower pollen proteome with patient sera detected seven IgE-reactive proteins with varying molecular weight and pI. Hierarchical clustering of 2D-immunoblot data highlighted three allergens characterized by a more frequent immuno-reactivity and increased levels of IgE antibodies in the sera of susceptible patients. These allergens were considered as the major allergens of sunflower pollen and were found to have their glycan moiety critical for inducing IgE response. Homology driven search of MS/MS data of these IgE-reactive proteins identified seven previously unreported allergens from sunflower pollen. Three major allergenic proteins were identified as two pectate lyases and a cysteine protease.

Conclusion

Novelty of the present report is the identification of a panel of seven sunflower pollen allergens for the first time at immuno-biochemical and proteomic level, which substantiated the clinical evidence of sunflower allergy. Further purification and recombinant expression of these allergens will improve component-resolved diagnosis and therapy of pollen allergy.     

The expression of a mountain cedar allergen comparing plant-viral apoplastic and yeast expression systems

Jun a 3, a major allergenic protein in mountain cedar pollen, causes seasonal allergic rhinitis in hypersensitive individuals. Recombinant Jun a 3 was expressed in Nicotiana benthamiana interstitial fluid (300 microg/g leaf material) and Pichia pastoris (100 microg/ml media). Polyclonal anti-Jun a 3 and IgE antibodies from the sera of allergic patients both reacted with the recombinant protein. Of the two systems, recombinant protein from the plant apoplast contained fewer contaminating proteins. This method allows for a more convenient and inexpensive expression of the recombinant allergen, which will allow for further structural studies and may prove useful in diagnostic and/or immunotherapeutic strategies for cedar allergy.     

IgE binding to proteins from sesame and assessment of allergenicity: implications for biotechnology?

Successful prediction of the potential allergenicity of a protein may be a key factor in the development of novel, genetically modified foods. The use of the decision tree approach for the prediction of allergenicity is discussed. The methods currently used for identifying allergenic proteins (including use of IgE from patient sera for recognition of proteins) are reviewed. Finally, a specific review of the literature concerning identification of allergens from sesame leads to the conclusion that in the absence of validated animal models, identification of allergenicity (and, consequently, prediction of allergenicity) may be problematic.