Said Elsayed

Immunochemical analysis of cod fish allergen M: locations of the immunoglobulin binding sites as demonstrated by the native and synthetic peptides.

The major allergen of codfish (Allergen M) is a muscle protein belonging to the family of calcium binding parvalbumins. The primary structure of the molecule was established and the molecular weight was estimated from the sequence data to be 12,328. Allergen M consists of 113 amino acid residues and one residue of glucose. A molecular arrangement of three domains (AB, CD and EF—the latter two bind one Ca2+ ion each) was described for Allergen M, analogous to carp parvalbumin pI 4.25. The suggested strucutre was based on the extensive intramolecular amino acid homologies and the immunochemical cross‐reactivities of the intact molecule and the two major isolated fragments.

Cloning of Two Distinct cDNAs Encoding Parvalbumin, the Major Allergen of Atlantic Salmon (Salmo salar)

Allergy to fish is common in Northern Europe. Variable reactions to different fish species are usually experienced among fish allergic patients. The allergens of cod fish and particularly the major allergen parvalbumin β (Gadus callarias) have been extensively studied in Norway. In the present communication, the white muscle parvalbumin was similarly found to be a major allergen in Atlantic salmon (Salmo salar, Sal s1 ). A purified salmon parvalbumin was obtained by anion exchange chromatography, gel filtration chromatography (GFC) and high‐performance liquid chromatography (HPLC) of the muscle extracts. The antigenicity and allergenicity of salmon parvalbumin were confirmed using various immunologic and electrophoretic techniques. The protein is representative for several isoallergens judged by the amino acid (AA) sequence variance at certain sites in the AA sequence of CNBr cleavage peptides. Using sera from patients with cod and salmon allergy Sal s1 was demonstrated to be the major allergen of Atlantic salmon, as judged by RAST‐ and ELISA‐inhibitions and crossed radioimmunoelectrophoresis (CRIE) techniques. The protein was also demonstrated to be antigenic by the use of polyclonal cod and salmon antibodies in IgG ELISA and immunoelectrophoretic methods. Cloning of parvalbumin cDNA from Atlantic salmon was performed based on an alignment of parvalbumin AA sequences from other species. A probe was generated by PCR and used for screening a salmon muscle cDNA‐library. Subcloning and sequencing of two hybridizing clones revealed transcripts from two different parvalbumin genes. The translated sequences of both clones belong to the β‐lineage of parvalbumins and include the entire coding region.

Antigenic and allergenic determinants of ovalbumin. I. Peptide mapping, cleavage at the methionyl peptide bonds and enzymic hydrolysis of native and carboxymethyl OA.

The effects of enzymic cleavage and perturbing the conformation of the allergenic and antigenic determinants of hens egg white albumin (OA) were examined. Hens egg white extract of a total protein concentration 8.43 g/l was prepared. Isoelectric focusing in sodium dodecyl sulfate and polyacrylamide gel peptide maps for the crude egg white extract showed 26 spots visualized by staining with Coomassie blue. The OA was purified using a TSK-2000 gel filtration chromatography column. The specific allergenic reactivity of the purified OA as measured by RAST inhibition and direct RAST was relatively high: 3 micrograms gave an inhibition of approximately 10%. The cleavage of OA with cyanogen bromide resulted in 4 fractions, all capable of binding specific IgE with the first peak showing the highest inhibition. Thermal denaturation of OA had no direct effect on the antigenic reactivity. RAST inhibition values for the denatured protein were similar to those of the native protein. Carboxymethylation of OA gave a product with only 20% of the inhibition reactivity. Further treatment with trypsin did not abolish the allergenic and antigenic reactivities as shown by RAST inhibition and by deflection of OA line in rocket line immunoelectrophoresis. On the other hand, limited pepsin hydrolysis destroyed the antigenic structure of the molecule. The reactivity of OA is thus relatively stable and could easily be retained making it possible to identify the allergenic determinants of enzymic hydrolysates used for elucidating the antigenic structure of the molecule.

The major allergen (parvalbumin) of codfish is encoded by at least two isotypic genes: cDNA cloning, expression and antibody binding of the recombinant allergens.

The major allergen (parvalbumin) from cod, designated Allergen M Gad c 1, has been intensively studied both from the structural and immunological sides. In the present study, transcripts of two isotypic parvalbumin genes in Atlantic cod were identified and characterized. Subsequently, subfragments were inserted into the expression vector pET-19b, generating plasmids with coding capacity for complete parvalbumin polypeptides fused to an N-terminal his(10) tag. Most of the recombinant products were found in the soluble fraction of the expression host Escherichia coli. The target proteins showed to react with polyclonal antibodies raised against Allergen M and demonstrated binding to specific IgE from 12 sera of patients allergic to cod in ELISA inhibition experiments. Sera with classes 4 and 5 CAP FEIA exhibited also strong binding to recombinant parvalbumins in immunoblots.

Evaluation of the Allergenicity and Antigenicity of Bovine‐Milk αs1‐Casein Using Extensively Purified Synthetic Peptides

αs1‐Casein (CAS1_BOVIN), the major allergen of cows milk (CM), is widely used as hydrolysates in infant diet formulae and additive to other processed food items. To date, most of the reported B‐cell epitope mapping were performed on polyethylene pins or cellulose‐derivative membrane. We sought to locate the motifs critical for human‐specific IgE and rabbit polyclonal IgG binding using extensively purified CAS1_BOVIN, synthetic peptides and derivatives. Thirteen overlapping peptides covering the whole CAS1_BOVIN encompassing 17 : 20 amino acid (AA) were synthesized by f‐moc AA solid‐phase polyamide peptide synthesis. In addition, six cyanogen bromide (CNBr) cleavage fragments were prepared. Limited hydrolysis, oxidized and reduced/alkylated derivatives were also produced. The preparations were purified by ion exchange, gel filtration chromatography, reversed phase and high‐performance liquid chromatography. The homogeneity was visualized by sodium dodecyl sulfate (SDS) and poly acryl amide gel electrophoresis (PAGE) followed by IgE and IgG immunoblotting. IgE binding was measured by Biotin Streptavidin (Bio/strep) fluoro enzyme immuno assay (FEIA) or ELISA‐inhibition. Eighteen CM allergy (CMA) sera from 45 clinically examined children (Melbourne) and five adults (Bergen) were selected. Individual sera and pools were used for mapping IgE‐binding epitopes. Rabbit IgG sera and pools were used for locating the antigenic sites of the molecule. Results indicated that all the individual CMA sera and pools recognized the intact molecule and three of the CNBr fragments as major antibody‐binding allergens. The N‐ and C‐terminal peptides (CAS 16‐35; CAS 136‐155) showed high IgE‐binding affinity. CAS 1‐18 and CAS 181‐199 showed high IgG bindings. Considering the diversity of the antibody specificities, a reasonable agreement between IgE and IgG epitopes were found at the N‐ and C‐terminals of CAS1_BOVIN. Mapping IgE B‐cell epitopes by direct Bio/strep FEIA allowed the development of a sensitive modified technique for detecting unlabelled, casein immune dominant peptides in food products.

Food allergy and intolerance: response to intestinal provocation monitored by endosonography

OBJECTIVE Objective assessment of adverse reactions to food is a long-felt want. We report our preliminary experience with a new endosonographic allergen provocation test. METHODS Twenty patients were examined, seven patients having food allergy and 13 having food intolerance. The duodenal mucosa was challenged with allergen extracts via a nasoduodenal tube. The responses were recorded using a miniprobe for endosonography through the tube. Thereafter, intestinal lavage was performed by giving 2 l PEG solution containing micro Ci (51)CrEDTA. The gut lavage fluid and urine for 5 h were collected. RESULTS Increased mucosal thickness in response to provocation was recorded in 11 patients, but not more often or pronounced in the allergic than in the intolerance group. Interestingly, increased mucosal thickness associated with a new echogenic layer was seen in two patients and a sustained duodenal contraction, lasting 15-20 min associated with pain, in another two. Intestinal permeability and inflammatory mediators were not significantly different in the two groups. CONCLUSION In patients with self-reported adverse reactions to food abnormal responses to duodenal provocation may be recognised by endosonography. However, neither endosonography nor intestinal permeability or faecal calprotectin responses were able to distinguish between food allergy and intolerance. Sustained duodenal contractions in response to food might be a cause of abdominal pain.

Airway Inflammation and Bronchial Remodelling in Toluene Diisocyanate‐exposed BALB/c Mouse Model

Toluene diisocyanate (TDI), a highly reactive industrial chemical, is one of the leading causes of occupation‐related asthma in industrialized countries. The pathogenesis of TDI‐induced asthma, however, remains not fully understood, in part due to lack of appropriate animal models. Twenty five female BALB/c mice (age: 8 weeks) were randomly divided into 5 groups: Ovabumin (OVA); OVA peptide amino acid residues No. 323–339 (Pep); TDI; alum and physiological saline. Mice were intraperitoneally injected with 25 μg OVA or pep absorbed on 300 μg alum, 300 μg alum or saline on days 0, 7 and 14. For the TDI group, mice were sensitized subcutaneously with 20 μl neat TDI on day 0; 20 μl of TDI in olive oil (1:10) on days 7 and 14; on days 21–23. Then each group was challenged intranasally with 20 μl of 1% OVA, 1% Pep, 1% TDI, 10% alum and saline respectively. On day 28, mice were killed under pentothal anesthesia. The results demonstrated that neutrophil‐dominant inflammation with a few eosinophil infiltration occurred in the peri‐bronchial and peri‐vascular regions of the lungs. This was accompanied by hyperplasia/hypertrophy of cells lining the airways and mucus production as shown by HE staining. Positive immunohistochemical MBP staining in parenchyma was also shown. Th2 cytokine IL‐4 and IgE production were significant increased 5 days after last challenge while IFN‐γ level was below the detection limit. Conclusion: the clear elevation of IL‐4 and IgE could allow to conclude a possible Th2‐like dominated allergic response in TDI‐exposed BALB/c mouse model.

Characterization of the immunological cross reactivity of fragments TM 1 and TM 2 of allergen M from cod.

Abstract A strong immunological cross reactivity was demonstrated for the polypeptide fragments TM 1 and TM 2 from cod Allergen M for both human reaginic and rabbit precipitating antibodies. The findings were interpretative in terms of the molecular domain hypothesis postulated for parvalbumins. since the cross reactivity was consistent with the high interdomain periodicity found in the amino acid sequence of parvalbumins. In other words, the immunologically active sequences must be repetitive along the polypeptide chains. In the rabbit precipitationg antibody system, fragment TM 1 (analogous to two molecular domains) could absorb most or all antibodies specific for fragment TM 2. In addition, it could neutralize a major portion of antibodies against the intact molecule. Furthermore, fragment TM 2 (analogous to. one domain) could partially neutralize anti-TM 1 or anti-Allergen M. The system was similarly valid tor human IgE-mediated reactions applying radio-allergosorbent cross inhibition tests. PK inhibition and cross neutralization tests. Both Allergen M and the monomer form of fragment TM l were immunogenic in the rabbit, in contrast to fragment TM 2 which was immunogenically active only after conjugation to human serum albumin through a carbodiimide ligand.

The effect of amino acid modification and polymerization on the immunochemical reactivity of cod Allergen M

Abstract The modification of the lysyl, tyrosyl, tryptophyl or arginyl side chains and the release of the two calcium ions, respectively, degraded the immunochemical reactivity of Allergen M. The IgE and IgG mediated activities were similarly diminished as assessed by RAST/RAST-inhibition for the former, and Farr/precipitin tests for the latter. The findings suggested, therefore, that the allergenic and antigenic sites on the Allergen M molecule, if distinct, are structurally homologous. Qualitative degradation of the native tertiary structure of Allergen M following specific chemical modification or removal of Ca 2+ only somewhat reduced the immunochemical reactivity, emphasizing the sequential character of the determinants. Polymerization of Allergen M strongly reduced its immunochemical reactivity as well as its immunogenic potential in the rabbit. Carbodiimide cross linking of fragment TM 1 reduced its primary interaction with antibody while enhancing the formation of immune-complexes. In contrast, in all test systems used, cross linking of fragment TM 2 to a trimer polypeptide enhanced both allergenic and antigenic reactivity.

Common antigenic and allergenic determinants on codfish proteins detected with mouse monoclonal IgG and human IgE antibodies

The antigenic and allergenic profiles of codfish extract have been examined and a comparison made of the specificities of the determinants defined by mouse monoclonal antibodies and human IgE antibodies. By gel electrophoresis, codfish extract was found to comprise a heterogeneous mixture of proteins, in which the principal component and allergen was Gad cI (allergen M). Using monoclonal antibodies and sera from human cod-allergic subjects as immunological probes, common antigenic and allergenic determinants were demonstrated on some codfish proteins. It was also established that, although two monoclonal antibodies recognized the same determinant on Gad cI, there was no cross-reactivity between this determinant and those specified by IgE antibodies in the sera of cod-allergic patients. The specificity of IgE populations directed against Gad cI was found to vary from patient to patient, and was indicative of the existence of two types of allergenic determinants: those unique to a particular allergen and those shared by other proteins in the extract. These studies promote speculation regarding the relative immunogenicity of antigenic and allergenic determinants and the size and diversity of the IgE repertoire, given the potential immunogenicity of the entire protein surface.