IgG-binding Epitopes Research Articles

Prediction, identification, and analysis of major B-cell linear epitopes of bomb m 6 from silkworm pupa

Bomb m 6 is one of the most representative allergens in silkworm pupae. The aim of the present study was to investigate the B-cell linear epitopes of Bomb m 6. Sequence propensity– and machine learning–based strategies were first used to predict 12 and 8 candidate epitopes, respectively. Then, 46 overlapping peptides [P1–P46; length, 15 amino acids (AAs); offset, 5 AAs] spanning the Bomb m 6 sequence were synthesized and tested for IgE/IgG reactivity by dot blotting. Seven IgE-binding epitopes and six IgG-binding epitopes were identified. AA61–80, AA136–150, and AA146–150 were identified as the shared dominant IgE/IgG-binding epitopes. The epitopes contain mainly α-helix and β-sheet structures and high proportions of hydrophobic AAs, and are distributed in charged regions. The major peptides of the epitopes were poorly resistant to simulated gastrointestinal fluid digestion. These findings provide a reference for the prevention of and immunotherapy for silkworm pupa allergy.

Investigating the Mechanism of Microwave-Assisted Enzymolysis Synergized with Magnetic Bead Adsorption for Reducing Ovalbumin Allergenicity through Biomass Spectrometry Analysis.

This study found that, after microwave treatment at 560 W for 30 s, alkaline protease enzymolysis significantly reduced the allergenicity of ovalbumin (OVA). Furthermore, specific adsorption of allergenic anti-enzyme hydrolyzed peptides in the enzymatic products by immunoglobulin G (IgG) bound to magnetic bead further decreased the allergenicity of OVA. The results indicated that microwave treatment disrupts the structure of OVA, increasing the accessibility of OVA to the alkaline protease. A comparison between 17 IgG-binding epitopes identified through high-performance liquid chromatography-higher energy collisional dissociation-tandem mass spectrometry and previously reported immunoglobulin E (IgE)-binding epitopes revealed a complete overlap in binding epitopes at amino acids (AA)125-135, AA151-158, AA357-366, and AA373-381. Additionally, partial overlap was observed at positions AA41-59, AA243-252, and AA320-340. Consequently, these binding epitopes were likely pivotal in eliciting the allergic reaction to OVA, warranting specific attention in future studies. In conclusion, microwave-assisted enzymolysis synergized with magnetic bead adsorption provides an effective method to reduce the allergenicity of OVA.

Investigation of the mechanism underlying the influence of mild glycation on the digestibility and IgG/IgE-binding abilities of β-lactoglobulin and its digests through LC orbitrap MS/MS

This study aimed to investigate the mechanism underlying the influence of mild glycation on the digestibility and anaphylactic effect of β-lactoglobulin (βLG) and its digests through LC Orbitrap MS/MS. Three glycated sites were discovered after glycation with fructose, whereas only lysine47 (K47) was glycated with maltose. After digestion, the intrinsic fluorescence intensity decreased and red shifted, and the degree of hydrolysis (DH) values showed no significant difference among digests. βLG glycated with Fru showed the lowest IgG and IgE-binding abilities. Meanwhile, the IgG and IgE-binding abilities further decreased and showed no apparent difference among digests. These results were attributed to the proximity to the IgG-binding epitope, the high glycation degree of K47, and mild glycation. Therefore, mild glycation could reduce the anaphylaxis of proteins before digestion, but did not influence the anaphylactic effect of the digests.

Immunoglobulin E and immunoglobulin G cross-reactive allergens and epitopes between cow milk αS1-casein and soybean proteins

Some infants allergic to cow milk-based formula are also sensitive to soybean-based formula. This paper aimed to explore the association of IgE and IgG cross-reactivity between αS1-casein in cow milk (CM) and soybean proteins. The IgE and IgG cross-reactive allergens and epitopes were identified using sera from infants allergic to CM or mice monoclonal antibodies. The AA sequence alignment was performed using bioinformatics software. Finally, the digestion and heating stability of the cross-reactive allergen were explored by sodium dodecyl sulfate (SDS)-PAGE and Western blotting. The results showed that the IgE and IgG cross-reactive allergen was α subunit of β-conglycinin named Gly m Bd 60K. The IgE and IgG epitopes were the sequences at AA 319-341 and AA 164-182. No intact Gly m Bd 60K allergen could be observed after 2 min in simulated gastric fluid by SDS-PAGE. Heating did not change IgE and IgG cross-reactivity by Western blotting. Therefore, the existence of cross-reactivity between CM αS1-casein and soybean proteins possibly contributes to the frequently observed cosensitization for these allergens in cow milk-allergic patients. The same IgE- and IgG-binding epitopes of cross-reactive allergens may provide important information for elucidation of the association between IgG and IgE antibody generation.

Boiling down the cysteine-stabilized LTP fold - loss of structural and immunological integrity of allergenic Art v 3 and Pru p 3 as a consequence of irreversible lanthionine formation

BackgroundNon-specific lipid transfer proteins (LTPs) are important allergens in fruits, pollen, vegetables, nuts and latex. Due to their compact structure, LTPs are highly resistant to heat treatment. Here, Art v 3 from mugwort pollen and Pru p 3 from peach were used as model allergens to in-depth investigate structural and immunological properties upon thermal treatment at different buffer conditions. MethodsRecombinant Art v 3 and Pru p 3 were purified from E. coli and incubated at 95 °C up to 120 min using sodium phosphate buffer pH 3.4 or 7.3. Physicochemical properties of allergens were analyzed in circular dichroism spectroscopy, Fourier transform infrared spectroscopy, dynamic light scattering, size exclusion chromatography, and mass spectrometry. The crystal structure of Art v 3.0201 was determined to 1.9 Å resolution. IgG and IgE binding was investigated in ELISA using murine and LTP allergic patients’ sera. ResultsHighly pure and homogenous recombinant allergens were obtained from bacterial production. The crystal structure of Art v 3.0201 revealed an antiparallel four helix bundle with a C-terminal extension mediating an asymmetric, transient dimer interface and differently sized cavities. Both allergens showed high thermal stability at acidic conditions. In contrast, extensive heat treatment in neutral buffer induced irreversible structural changes due to lanthionine-based cysteine rearrangement. This fostered loss of the typical α-helical structure, increased molecular size and abrogation of IgG and IgE binding epitopes. Pru p 3 lost its structural integrity at shorter heat stress duration than Art v 3, which did however only partially affect the molecule’s IgE binding epitopes. ConclusionDuring thermal treatment, susceptibility to structural changes of the LTP-fold is highly dependent on the surrounding environment but also on intrinsic features of individual LTPs. This is a crucial fact to consider when processing LTP-containing food or food products as this will directly influence their allergenic potential.

Der p 5 allergen from house dust mite: first epitope mapping of rabbit IgG blocking antibodies.

Der p 5 is one of the important house dust mite allergens in Algeria; this allergen is frequently recognized by patients with allergic asthma. However, there is no information on its IgG–binding epitopes. In the present study, rabbits were immunized with recombinant Der p 5 allergen, and serum samples were obtained. Recognition of linear IgG epitopes of Der p 5 was determined using synthesized peptides derived from the allergen sequence. The results showed that serum from immunized rabbits recognized three linear epitopes from Der p 5 (28EDKKHDYQNEFDFLLMERIHEQIK43), (37IHEQIKKGELALFYLQEQ55) and (92LMQRKDLDIFEQYNLEMAKKS112). More interestingly, we observed that the 92L-S112 amino acid sequence is well recognized by both IgE and IgG antibodies. Der p 5 stimulates the synthesis of specific IgG antibodies which recognize common but also novel epitopes compared to IgE antibody binding. Indeed, the potential to induce IgG antibodies can be used to inhibit human IgE binding to allergens which may be part of the mechanism of action of specific immunotherapy.

Molecular fingerprinting of complex grass allergoids: size assessments reveal new insights in epitope repertoires and functional capacities

Background Subcutaneous allergen immunotherapy (SCIT) is a well-documented treatment for allergic disease which involves injections of native allergen or modified (allergoid) extracts. The use of allergoid vaccines is a growing sector of the allergy immunotherapy market, associated with shorter-course therapy. The aim of this study was the structural and immunological characterisation of group 1 (Lol p 1) IgG-binding epitopes within a complex mix grass allergoid formulation containing rye grass.Methods HP-SEC was used to resolve a mix grass allergoid preparation of high molecular weight into several distinct fractions with defined molecular weight and elution profiles. Allergen verification of the HP-SEC allergoid fractions was confirmed by mass spectrometry analysis. IgE and IgG immunoreactivity of the allergoid preparations was explored and Lol p 1 specific IgG-binding epitopes mapped by SPOT synthesis technology (PepSpot™) with structural analysis based on a Lol p 1 homology model.Results Grass specific IgE reactivity of the mix grass modified extract (allergoid) was diminished in comparison with the mix grass native extract. A difference in IgG profiles was observed between an intact mix grass allergoid preparation and HP-SEC allergoid fractions, which indicated enhancement of accessible reactive IgG epitopes across size distribution profiles of the mix grass allergoid formulation. Detailed analysis of the epitope specificity showed retention of six Lol p 1 IgG-binding epitopes in the mix grass modified extract.Conclusion The structural and immunological changes which take place following the grass allergen modification process was further unravelled revealing distinct IgG immunological profiles. All epitopes were mapped on the solvent exposed area of Lol p 1 homology model accessible for IgG binding. One of the epitopes was identified as an ‘immunodominant’ Lol p 1 IgG-binding epitope (62-IFKDGRGCGSCFEIK-76) and classified as a novel epitope. The results from this study support the concept that modification allows shorter-course therapy options as a result of providing an IgG epitope repertoire important for efficacy. Additionally, the work paves the way to help further develop methods for standardising allergoid platforms.

Epitope mapping and identification of amino acids critical for mouse IgG-binding to linear epitopes on Gly m Bd 28K.

Gly m Bd 28K is one of the major allergens in soybeans, but there is limited information on its IgG-binding epitopes. Thirty-four overlapping peptides that covered the entire sequence of Gly m Bd 28K were synthesized, and 3 monoclonal antibodies against Gly m Bd 28K were utilized to identify the IgG-binding regions of Gly m Bd 28K. Three dominant peptides corresponding to (28)GDKKSPKSLFLMSNS(42)(G28-S42), (56)LKSHGGRIFYRHMHI(70)(L56-I70), and (154)ETFQSFYIGGGANSH(168)(E154-H168) were recognized. L56-I70 is the most important epitope, and a competitive ELISA indicated that it could inhibit the binding of monoclonal antibody to Gly m Bd 28K protein. Alanine scanning of L56-I70 documented that F64, Y65, and R66 were the critical amino acids of this epitope. Two bioinformatics tools, ABCpred and BepiPred, were used to predict the epitopes of Gly m Bd 28K, and the predictions were compared with the epitopes that we had located by monoclonal antibodies.

Identification of the Critical Amino Acid Residues of Immunoglobulin E and Immunoglobulin G Epitopes in α-Lactalbumin by Alanine Scanning Analysis.

α-Lactalbumin represents one of the major allergens causing cow milk allergy. Few studies have clearly evaluated immunological relationships between immunoglobulin E (IgE) and immunoglobulin G (IgG)-binding epitopes of α-lactalbumin. IgE- and IgG-binding epitopes were immunolabeled with individual sera from cow milk-allergic patients. Alanine scanning of immunodominant epitopes was used to identify the critical amino acid (aa). Our initial data revealed Val8 , Phe9 , Arg10 , Tyr103 , Leu105 , and His107 were the critical aa for IgE-binding epitope. The critical aa of IgG-binding epitopes were Phe9 , Leu15 , Pro24 , Trp26 , and His32 . This study will provide necessary information to alter the cDNA to encode a protein capable of activating milk-specific T cells, but with reduced IgE- or IgG-binding capacity.

IgE and IgG epitope mapping by microarray peptide-immunoassay reveals the importance and diversity of the immune response to the IgG3 equine immunoglobulin

The presence of whole horse IgG in therapeutic snake antivenom preparations of high purity is a contamination that can cause IgE-mediated allergic reactions in patients. In this study, the immunodominant IgE and IgG-binding epitopes in horse heavy chain IgG3 were mapped using arrays of overlapping peptides synthesized directly onto activated cellulose membranes. Pooled human sera from patients with and without horse antivenom allergies were used to probe the membrane. We have demonstrated that, for both cases, individuals produce antibodies to epitopes of sequential amino acids of horse heavy chain IgG3, although the signal strength and specificity appear to be distinct between the two groups of patients. A single region was found to contain the dominant allergic IgE epitope. The critical residues involved in the binding of human IgE to the epitope were determined to include four hydrophobic amino acids followed by polar and charged residues that formed a coil structure. This is the first study to describe the specific amino acid sequences involved with the immune recognition of human IgG and IgE to horse antivenom.

Identification of the critical amino acid residues of immunoglobulin E and immunoglobulin G epitopes on αs1-casein by alanine scanning analysis

αs1-Casein represents one of the major allergens causing cow milk allergy. Few studies have clearly evaluated immunological relationships between IgE- and IgG-binding epitopes of αs1-casein. This study aimed to map IgE- and IgG-binding epitopes of αs1-casein by the serology method, and identify the critical amino acids of αs1-casein by alanine scanning analysis. Our initial data revealed IgE-binding epitopes located in the sequences of AA 126 to 140, AA 6 to 20, AA 171 to 185, and AA 11 to 25. The sequences at AA 21 to 35, AA 56 to 70, and AA 161 to 175 were recognized by IgG antibodies. The alanine scanning analysis showed that IgE- and IgG-binding epitopes share the same critical AA: arginine at position 22 and phenylalanine at position 23. Results obtained from this study will provide necessary information to alter the cDNA to encode a protein with reduced IgE- or IgG-binding capacity.

Linear B-cell epitopes in BthTX-1, BthTX-II and BthA-1, phospholipase A2's from Bothrops jararacussu snake venom, recognized by therapeutically neutralizing commercial horse antivenom

The benefits from treatment with antivenom sera are indubitable. However, the mechanism for toxin neutralization has not been completely elucidated. A mixture of anti-bothropic and anti-crotalic horse antivenom has been reported to be more effective in neutralizing the effects of Bothrops jararacussu snake venom than anti-bothropic antivenom alone. This study determined which regions in the three PLA2s from B. jararacussu snake venom are bound by antibodies in tetravalent anti-bothropic and monovalent anti-crotalic commercial horse antivenom. Mapping experiments of BthTX-I, BthTX-II and BthA-I using two small libraries of 69 peptides each revealed six major IgG-binding epitopes that were recognized by both anti-bothropic and anti-crotalic horse antivenom. Two epitopes in BthTX-I were only recognized by the anti-bothropic horse antivenom, while anti-crotalic horse antivenom recognized four unique epitopes across the three PLA2s. Our studies suggest that the harmful activities of the PLA2s present in the venom of B. jararacussu are neutralized by the combinatorial treatment with both antivenom sera through their complementary binding sites, which provides a wide coverage on the PLA2s. This is the first peptide microarray of PLA2s from B. jararacussu snake venom to survey the performance of commercial horse antiophidic antivenom. Regions recognized by the protective antivenom sera are prime candidates for improved venom cocktails or a chimeric protein encoding the multiple epitopes to immunize animals as well as for designing future synthetic vaccines.

Identification of the critical amino acid residues of immunoglobulin E and immunoglobulin G epitopes in β-lactoglobulin by alanine scanning analysis

β-Lactoglobulin represents one of the major allergens causing cow milk allergy. Few studies have clearly evaluated immunological relationships between IgE- and IgG-binding epitopes of β-lactoglobulin. For characterization of immunological epitopes, peptides of 15 amino acids (AA) in length were synthesized. Immunoglobulin E- and IgG-binding epitopes were immunolabeled with individual sera from cow milk-allergic patients. Alanine scanning of immunodominant epitopes was used to identify the critical AA of IgE- and IgG-binding epitopes. The results showed that 4 IgE-binding epitopes were identified. Our initial data revealed IgE-binding epitopes at AA 17 to 31, AA 72 to 86, AA 92 to 106, and AA 152 to 166. Threonine 20, Met23, and Asp27 are the critical AA of IgE-binding epitopes. Two IgG epitopes were identified, which were located at AA 22 to 36 and AA 127 to 141. The critical AA of IgG-binding epitopes were Leu26 and Val31. Results obtained from this study will provide necessary information to alter the cDNA to encode a protein capable of activating milk-specific T cells, but with reduced IgE- or IgG-binding capacity.

Characterization of the 41 kDa allergen Asp v 13, a subtilisin-like serine protease from Aspergillus versicolor

Aspergillus versicolor is common on moldy building materials. Asp v 13, the principal allergen is produced by strains collected from across Canada. In this paper, we report a 1833bp Asp v 13 open reading frame predicted to encode a protein of 403 amino acids in length with three introns. A BLAST search of Asp v 13, a phylogenic tree calculation and alignment with its homologous proteins from other species indicated that Asp v 13 is a secretory, subtilisin-like serine protease widely distributed in Aspergillus species. His-tagged Asp v 13 was over-expressed in Escherichia coli and purified using Ni-NTA columns with a yield of 1mg/L. Based on immuno binding assay of recombinant protein both antibodies developed against the natural protein, and human sera IgE, the recombinant protein was similar to the natural form. Six IgE- and seven IgG-binding epitopes were also identified with selected human sera along the entire amino acid sequence of Asp v 13. Most residues binding these epitopes are exposed on the surface and correspond to charged regions of the molecule.

Dietary antigens, epitope recognition, and immune complex formation in recent onset psychosis and long-term schizophrenia

Peptides derived from dietary antigens such as bovine milk caseins are opioid receptor ligands and contribute to schizophrenia-associated hyperpeptidemia and hyperpeptiduria. The IgG antibody response to bovine caseins is increased in schizophrenia and recent onset psychosis. To identify specific casein peptide sequences that are antigenic in patients vs controls, we measured serum IgG binding to 10–26 amino acid long linear epitopes of casein with immunoassays for the entire group (n = 95 recent onset psychosis; n = 103 long-term schizophrenia; n = 65 control), and with peptide microarray libraries in a casein-sensitive subset (n = 14 recent onset; n = 10 control). In the entire group, we compared anti-casein peptide IgG vs anti-whole casein IgG and evaluated whether peptide immune complexes contributed to IgG binding results. Anti-whole casein IgG levels correlated with anti-casein peptide IgG in controls only (R 2 = 0.17–0.25, p ≤ 0.002–0.03). In recent onset psychosis, IgG binding to linear peptide sequences was significantly decreased 3.8–5.7-fold compared to controls in immunoassays (OR 0.18–0.26, p ≤ 0.0001–0.001). In peptide microarrays, recent onset patients again showed significantly reduced IgG binding and fewer epitopes than controls (p ≤ 0.00001–0.05). Anti-peptide IgG levels did not differ between patients with long-term schizophrenia and controls. Finally, significantly more recent onset individuals had casein peptide-IgG immune complexes than controls (OR 4.96, p ≤ 0.001). These findings suggest an immunological specificity that differs in early vs later stages of neuropsychiatric diseases and an IgG saturation by casein-derived peptides that may in part explain the reduced IgG binding to small linear epitopes observed in these patients.

Studies on the immunoreactivity of the major allergen tropomyosin in mantis shrimp (<I>Squilla oratoria</I>)

Crustacean is one of the eight kinds of allergen sources in coastal areas,which causes the IgE-mediated hypersensitive reactions with clinical manifestations including urticaria,angioedema,asthma,and even fatal anaphylaxis. Tropomyosin (TM) is the major allergen of decapod crustaceans with highly conserved amino acid sequences. The purpose of this study is to confirm whether TM is a major cross-reactive allergen in mantis shrimp (Squilla oratoria) which is taxonomically distinct from decapods and largely consumed as a delicacy in China. Quantification of TM in different species of crustaceans was also conducted. Muscle sample from mantis shrimp was homogenized with phosphate buffer to prepare heated extracts and separated by 12 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). A protein band with molecular mass of about 36 ku was detected by IgE-immunoblotting by all of the sera with crustacean allergy,suggesting it is the major allergen of mantis shrimp. This protein was further purified to homogeneity by acetone powder preparation,isoelectric point precipitation,ammonium sulfate fractionation (40%-60% saturation),and heat treatment. Purified protein was demonstrated to be TM by Western blot using polyclonal antibody against TM from Chinese mitten crab (Eriocheir sinensis). Heated extracts from other crustaceans including mud crab (Scylla serrata),Pacific white shrimp (Penaeus vannamei) and short necked clam (Venerupis variegata) were also prepared,and the cross-reactivity of TM from mantis shrimp with TMs from these crustaceans was confirmed by inhibition immunoblotting using polyclonal antibody against TM and inhibition ELISA using sera with crustacean allergy. Quantification by ELISA using polyclonal antibody against TM revealed that TM content in mantis shrimp muscle is much lower than that in Pacific white shrimp and mud crab muscle,which was about 45 times lower in mantis shrimp muscle than that in Pacific white shrimp muscle. However,its allergenicity seems equivalent to other decapod crustaceans as showed by inhibition ELISA using sera with crustacean allergy. This may be due to the degradation of TM in mantis shrimp by its endogenous serine proteinases and cathepsins which destroyed the IgG-binding epitope but not the IgE-binding epitopes. In conclusion,this study demonstrated that allergenicity of mantis shrimp is almost equivalent to decapods and TM is the major allergen in mantis shrimp with high cross-reactivity to decapod crustaceans.

The design and characterization of two proteins with 88% sequence identity but different structure and function

To identify a simplified code for conformational switching, we have redesigned two natural proteins to have 88% sequence identity but different tertiary structures: a 3-alpha helix fold and an alpha/beta fold. We describe the design of these homologous heteromorphic proteins, their structural properties as determined by NMR, their conformational stabilities, and their affinities for their respective ligands: IgG and serum albumin. Each of these proteins is completely folded at 25 degrees C, is monomeric, and retains the native binding activity. The complete binding epitope for both ligands is encoded within each of the proteins. The IgG-binding epitope is functional only in the alpha/beta fold, and the albumin-binding epitope is functional only in the 3-alpha fold. These results demonstrate that two monomeric folds and two different functions can be encoded with only 12% of the amino acids in a protein (7 of 56). The fact that 49 aa in these proteins are compatible with both folds shows that the essential information determining a fold can be highly concentrated in a few amino acids and that a very limited subset of interactions in the protein can tip the balance from one monomer fold to another. This delicate balance helps explain why protein structure prediction is so challenging. Furthermore, because a few mutations can result in both new conformation and new function, the evolution of new folds driven by natural selection for alternative functions may be much more probable than previously recognized.

Specificity of IgE antibodies to sequential epitopes of hen's egg ovomucoid as a marker for persistence of egg allergy

Approximately two-thirds of egg-allergic infants become tolerant within the first 5 years of life. We sought (1) to compare the recognition of sequential (linear) and conformational binding sites of ovomucoid, ovalbumin and ovotransferrin, by IgE antibodies of children with persistent and transient egg allergy, (2) to identify immunodominant IgE-and IgG-binding epitopes of ovomucoid, and (3) to compare epitope-specificity of IgE antibodies between patients with differing natural histories of egg allergy. Using immunodot-blots or ImmunoCAPs, IgE-antibodies against conformational (native) and sequential (reduced and alkylated) egg proteins were determined at the time of clinical reactivity in patients who retained their allergy and in those who developed clinical tolerance. IgE- and IgG-binding epitopes were mapped for ovomucoid using overlapping decapeptides on SPOTs membranes. Recognition of the major IgE-binding epitopes were compared between patients with differing natural histories of egg allergy. The patients with long-lasting egg allergy had a higher concentrations of IgE antibodies against sequential and native ovomucoid and ovalbumin than the children who subsequently gained tolerance (P < 0.01). Four major IgE-binding epitopes were identified in ovomucoid at amino acid 1-10, 9-20, 47-56, and 113-124. IgE antibodies of all seven patients with persistent egg allergy recognized these epitopes whereas none of the 11 children who outgrew their egg allergy did so. Patients with persistent egg allergy develop IgE antibodies against more sequential and conformational epitopes of ovomucoid and ovalbumin. The presence of serum IgE antibodies to specific sequential epitopes of ovomucoid may be used as a screening instrument for persistent egg allergy.

Molecular cloning and characterization of full‐length cDNAs encoding a novel high‐molecular‐weight Dermatophagoides pteronyssinus mite allergen, Der p 11

Dermatophagoides pteronyssinus (Dp) and D. farinae (Df) mites are the most important source of indoor aeroallergens. Most Dp mite allergens identified to date have relatively low molecular weights (MWs). Identification of high-MW mite allergens is a crucial step in characterizing the complete spectrum of mite allergens and to provide appropriate tools for diagnostic and therapeutic application. The full-length Der p 11 cDNA clone was isolated using cDNA library immunoscreening, the 5'-3' rapid amplification of cDNA ends (RACE) system and polymerase chain reactions (PCR). The whole cDNA insert and its PCR-derived DNA fragments (p1 to p4) were generated and expressed in the Escherichia coli expression system. The allergenicity of the recombinant protein and its peptide fragments was examined by IgE immunodot assays. The IgE-binding reactivity of rDer p 11 was analyzed in the serum of 50 asthmatic children with positive reactivity to Dp mite extract. Its recombinant peptide fragments were also examined by immunodot assays in 30 mite-allergic children. Der p 11 cDNA consists of a 2625-bp open reading frame encoding a 103-kDa protein with 875 amino acids. It exhibits significant homology with the paramyosin of other invertebrates. The protein sequence alignment of this newly identified Dp mite allergen (denominated as Der p 11) revealed over 89% identity with Der f 11 and Blo t 1. Among 50 Dp-sensitive asthmatic children, rDer p 11 showed positive IgE-binding reactivity to 39 patients (78%). Using immunodot assays, multiple human IgE-binding activities were demonstrated in all four fragments of Der p 11. Using immunoblot assays, the dominant IgG-binding epitope for monoclonal antibody (mAb642) was located in fragment p3 only. In immunoblot assays, cross-inhibition between rDer p 11 and rDer f 11 was up to 73-80% at concentrations of 100 microg/ml. This study confirms that the newly identified recombinant Der p 11 is a novel and important high-MW Dp mite allergen for asthmatic children. Our data also indicates that human IgE-binding major epitopes are scattered over the entire molecule of Der p 11.

IgG binding of mugwort pollen allergens and allergoids exposed to simulated gastrointestinal conditions measured by a self-developed ELISAtest

This study considers the influence of exposure to simulated gastrointestinal conditions (saliva, gut, intestine and acidic conditions of the gut) on IgG binding of unmodified allergens and three types of LMW allergoids of Artemisia vulgaris pollen extract obtained by means of potassium cyanate succinic and maleic anhydride. It also concerns the optimization of a self-developed ELISA assay for comparison of the specific IgG binding of mugwort pollen extract and modified mugwort pollen derivatives. The ELISA was conducted with a mugwort pollen extract coupled to the plate, using the sera from 12 mugwort- pollen allergic patients. The exposure to saliva fluid for 2 min did not influence the IgG binding properties of allergens and allergoids. Exposure of mugwort pollen allergens and LMW allergoids to the acidic conditions of the gut did not dramatically change their IgG binding properties. By exposing mugwort pollen extract and LMW derivatives to the SGF conditions for 1 h, the percent of IgG binding epitopes was reduced to a half of its starting value in the extract and to about 30%in all the allergoid samples. After prolonged exposure only the carbamyl derivative showed reduced IgG binding. Changes of the IgG binding potential of all four samples after exposure in SIF followed a similar pattern.