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S Elsayed

Publications and source records attributed to S Elsayed.

At least 73 records · Page 4Linked to original sources
PubMed1989

Synthetic allergenic epitopes from the amino-terminal regions of the major allergens of hazel and birch pollen.

Five immunogenic peptides from the major allergens of tree pollen extracts (residues 23-38) were synthesized by Merrifield solid-phase peptide synthesis. The primary structures of these peptides were deduced from the known N-terminal amino acid sequence of the major allergen of hazel, HIa, and birch, Bet v I. In a segment of 16 amino acids (23-38), 2 residues were the difference between birch and hazel. The selection of the peptides was on the basis of optimal hydropathicity. The segment 29-34 of these peptides was predicted to prefer a helical rather than beta-sheet conformational state. The insertion of amino acid residues during the synthesis was confirmed by the analysis of amino acid composition prior to coupling the preceding residue. The linearities of the synthetic chains were assigned by the primary structure analysis of the entire chain by automatic Edman degradation. The synthetic peptides were purified by gel filtration chromatography and reversed-phase high-performance liquid chromatography, resulting in satisfactory homogeneity of the preparations. The immunogenicity of the peptides was tested in rabbits using conjugated peptide-bovine serum albumin through a carbodiimide spacer arm. The results indicated that all constituents of the conjugate could elicit an immune response. The peptides gave precipitation lines in crossed immunoelectrophoresis when using rabbit antibodies against the conjugated preparation. All the synthetic peptides and analogues from region 23-38 could inhibit, in dose-response patterns, the binding of specific IgE to the intact molecules. Similar results were shown for the shorter segment 25-34, suggesting that the C-terminal tetrapeptide did not contribute to the activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1989

Reestimations of the protein concentrations of birch pollen allergen extracts selected as candidates for the international standard (IS) preparation.

The total protein concentrations of six selected candidates for an international standard (IS) of birch pollen were previously estimated (Ann Allergy 1987;58:71-77). The results of analyses showed large variations depending both on the method and the reference protein used. More than 2-fold variations in the protein content were obtained using Bradford's protein binding method. In the present communication the protein contents of these birch IS-candidates were reestimated by Lowry, a modified Lowry technique, Bradford's protein-dye binding, bicinchoninic acid reagent and amino acid compositions. Great variations were obtained for the protein contents, depending on the technique employed, emphasizing the difficulties of choosing a proper method of protein analyses of pollen allergens. The Lowry and the bicinchoninic acid reagent methods gave the highest obtained protein estimates, while the modified Lowry and Bradford's protein-dye binding methods showed the lowest values. The amino acid analysis gave concentrations similar to those obtained by Lowry and bicinchoninic acid reagent methods. The Lowry method seemed to be adequate for the analysis of plant proteins. The amino acid analysis is currently the most precise method for estimation of the protein concentration irrespective of additional prosthetic groups. A serious shortcoming of the method is that it requires an amino acid analyser or other liquid chromatrographic systems, which are not commonly in laboratory use.

Amino Acids↗
PubMed1988

Antigenic and allergenic determinants of ovalbumin. II. The reactivity of the NH2 terminal decapeptide.

The decapeptide 1-10 of hen's egg ovalbumin (OA), deduced from the known amino acid sequence (Gly-Ser-Ile-Gly-Ala-Ala-Ser-Met-Glu-Phe), was synthesized by Merrifield solid phase peptide synthesis with a yield of greater than 70%. The completeness of the insertion of amino acids during synthesis was monitored by the amino acid compositions of the peptide-resin, prior to coupling of the preceding residue. The linearity of the synthesis was supported by dansyl Edman degradation and detection of three NH2 terminal residues 5-dimethylamino-naphtheline-1-sulphonyl (DNS)-Gly, -Ser, -Ile, respectively. The peptide was purified by gel filtration chromatography and analytical reversed-phase high-performance liquid chromatography (HPLC). The homogeneity of the preparation was calculated both from the amino acid analysis and by integrating the peaks of HPLC to be greater than 83%. The antigenicity of the purified (P2) peptide could be detected by precipitation inhibition with the nephelometric technique. The decapeptide could also specifically react with functional structures on reaginic IgE molecule from the sera of individuals allergic to eggs, inhibiting its further binding to ovalbumin. An in-vivo experiment using direct skin test on two patients allergic to eggs showed no activity, rendering further testing unnecessary. The results suggest that the decapeptide of the NH2 terminal segment of OA encompasses an Ig-binding haptenic epitope.

Allergens↗
PubMed1988

Immuno-electronmicroscopic identification and localization of the antigenic proteins of tree pollen grains.

The localization of antigenic proteins on ultrathin sections of pollen grains represents an interesting approach to understanding the release mechanisms of these antigens when the pollen grains come in contact with various physiological fluids. Using different rabbit antibodies we have demonstrated the locations of these antigens in the various structures of pollen grains. We further demonstrated the cross-reactivities between alder (Alnus incana), birch (Betula verrucosa) and hazel (Corylus avellana) pollen allergens. Ultrathin sections of the pollen grains were prepared and allowed to react with two individually raised rabbit antibodies, (Ab-BV and Ab-ALK), against birch pollen. The sites of the Ag/Ab complex on the sections were labelled by protein A/gold, and identified in a transmission electron microscope. The two birch antibodies showed either quantitative or qualitative differences regarding their binding to various structures on the pollen sections. Using Ab-BV, the antigen-binding sites were located in the apertural region of the pollen grain and in the cytoplasm, while almost no gold labelling could be seen on the pollen surface. With the other antibodies (Ab-ALK), we could visualize the antigen-binding locations on the surface material of the pollen grains, particularly in the exine part of the wall and in the cytoplasm. A few gold particles could also be seen in the apertural region of the pollen. In hazel and alder pollen the exine part of the wall was the most densely labelled, whereas the cytoplasm and the aperture bound smaller numbers of gold particles. Cross-incubations: birch pollen incubated with antibodies against hazel (Ab-CA), or alder (Ab-AI), showed various intensities of gold labelling for each of the three species. Statistically, the differences in the number of gold particles bound per micron 2 grain section between birch, hazel and alder, were highly significant. The cross-reactivities between these antigens from the three pollen species were further tested using house-produced rabbit antisera against antigens of the three species by means of electrophoretic and autoradiographic techniques (CIE and CRIE). The three antibodies could precipitate the major IgE-binding antigen from all three pollen species.

Animals↗
PubMed1988

A comparison of the antigenic and allergenic components of birch and alder pollens in Scandinavia and Australia.

Allergens in birch (Betula) pollens from B. pendula grown in Australia and Norway, B. davurica and B. populofolia and from alder (Alnus incana) were identified by electroblotting, following separation by SDS-PAGE, transfer to nitrocellulose membranes and incubation with sera from birch pollen-allergic subjects. Of 42 antigenic components detected by protein staining in the pollen extract from B. pendula grown in Norway, 17 bound IgE. The allergenic components included those already reported in the literature at MWs of 40, 29, 25, 17 and 10-12 kd, as well as previously undescribed components at MWs of 90, 79, 60, 50, 38, 35, 31, 27, 23, 16, 15 and 14 kd. The major IgE-binding components were located in the low MW region 10-17 kd for all species of birch and alder pollen proteins studied. Results reported here provide the first evidence of birch and alder pollen allergies in Australia. Extensive heterogeneity was observed amongst the sera from birch pollen-allergic subjects in both Norway and Australia. Cross-reactivity appears to exist among the proteins present in pollen from the various birch species and from alder.

Australia↗
PubMed1988

Allergic response to birch and alder pollen allergens influenced by geographical location of allergic subjects.

A detailed analysis was made of the reactivity patterns of birch pollen-allergic subjects from Norway and Australia to the various IgE-binding components of pollens from several different birch and alder species. Typically, with each of the pollens examined, the Norwegian subjects exhibited a specific limited response pattern in which only a few of the 18 possible allergenic bands were recognized. A single, major IgE-binding band of approximate molecular weight 16-17 kD was recognized in each pollen by all the Norwegian patients used in the study. In contrast to this situation, there was no 'typical' recognition pattern amongst the Australian patients. Although recognition of the 16- to 17-kD components occurred in some cases, it was not dramatically superior to any other single component. Recognition of higher molecular weight components occurred more frequently than with the Norwegian group. Reasons for the differences in the response patterns of the Australian and Norwegian groups are discussed with reference to the exposure of the populations to different allergenic sources and genetic variation.

Allergens↗
PubMed1988

Comparative studies on tree pollen allergens. XIV. Characterization of the birch (Betula verrucosa) and hazel (Corylus avellana) pollen extracts by horizontal 2-D SDS-PAGE combined with electrophoretic transfer and IgE immunoautoradiography.

The crude aqueous extracts of birch (Betula verrucosa) and hazel (Corylus avellana) pollen were characterized by horizontal isoelectric focusing (IEF) and one-dimensional and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1-D and 2-D SDS/PAGE). Isoelectric focusing reference patterns obtained in pH 3.5 to 9.5 gradient gels contained 31 and 41 bands while the 2-D patterns consisted of 51 and 72 protein spots, respectively. Electrophoretic transfer to nitrocellulose membranes and subsequent 125I-immunoautoradiography demonstrated IgE binding in the region between pH 4.8 and 5.4 for both extracts. The main allergenic reactivities of this pH region were distributed by SDS/PAGE to the molecular weights of 29.5, 17, and 12.5 kilodaltons for birch and 15.5 and 12 kilodaltons for hazel. The high resolving power and the independence of rabbit antibodies were considered the major advantages of the described 2-D methodology as compared with conventional crossed immunoelectrophoretic and radioimmunoelectrophoretic techniques.

Animals↗
PubMed1987

Comparative studies on tree pollen allergens. XVII. Immunochemical analysis of the international standardization extracts of birch (Betula verrucosa) pollen as compared with a local partially purified extract.

Six different birch pollen extracts were analyzed by 20 laboratories for the standardization of birch (Betula verrucosa) pollen extracts used for diagnosis and specific therapy of patients with birch pollen allergy. The extracts were collected and delivered by the International Union of Immunological Societies, Allergen Standardization Subcommittee. One of the extracts, designated M, was proposed as an international standard (IS)-candidate of birch pollen extracts. The protein content of the IS candidate M was found to be 1.12 mg/mL, more than 2-fold higher than any of the other extracts analyzed. This preparation was among the extracts containing the highest number of protein components, as shown by isoelectric focusing, 28 lines, and by 11 precipitates in crossed immunoelectrophoresis. The allergenic reactivities were tested by crossed radioimmunoelectrophoresis (CRIE) and by radioallergosorbent test (RAST)-inhibition. In CRIE, the proposed IS (M) showed similar affinity for binding patients' IgE as the other extracts, as judged by the autoradiographic illustrations. Except for extract L, the values of RAST-inhibition for the rest were very similar. An IS extract should qualify for the criteria suggested for an optimal allergen preparation, containing minimal amounts of non-allergenic antigens and providing quantitatively and qualitatively all the allergenic proteins. The appropriateness of this selection seems unjustified in view of the above criteria.

Allergens↗
PubMed1986

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.

Allergens↗
PubMed1986

Comparative studies on tree pollen allergens. XIII. Partial characterization of the alder (Alnus incana) pollen extract by two-dimensional IEF/SDS-PAG electrophoresis combined with electrophoretic transfer and immunoautoradiography.

Horizontal isoelectric focusing (IEF) and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2-D SDS-PAGE) combined with electrophoretic transfer (ET) and immunoautoradiography (IAR) were used to characterize the crude alder (Alnus incana) pollen extract (AI crude) and fraction AI 34. IEF resolved AI crude and AI 34 into 34 and 39 distinct protein bands, respectively, when stained with Coomassie brilliant blue. The band patterns were similar and the majority of bands were located in the pH region 4.0-7.0. After ET and IAR, the distribution of antigenic reactivities was similar to that of the protein stain. Allergenic reactivity was detected in the pH region 4.6-5.2 only, where two bands at pH 4.9 demonstrated dominating IgE-binding properties. The 2-D reference maps of AI crude and AI 34 consisted of 40 and 45 protein spots, respectively. The autoradiogram of AI crude demonstrated 35 spots capable of binding rabbit IgG. The main IgE-binding zone was located at pH 4.6-5.2 and 20 kilodaltons with an area of weaker autoradiographic signals at pH 4.6-5.2 and 40 kilodaltons. ET from polyacrylamide gels to nitrocellulose (NC) membranes was quantitative and IAR gave significant radiostaining of spots containing 0.01 and 0.1 microgram of AI crude in the IgG and IgE systems, respectively. For the characterization of the alder pollen extract, horizontal 2-D SDS-PAGE combined with ET and IAR provided a powerful supplement to conventional crossed immuno- and radioimmunoelectrophoretic techniques (CIE/CRIE).

Animals↗
PubMed1986

Comparative studies on tree pollen allergens. XI. Trials on the regulation of IgE response in mice using modified birch pollen allergens.

The major allergen of birch pollen, BV45, was isolated and conjugated to 2-o-methoxy polyethylene glycol-4,6-dichloro-5-triazine (mPEG). The molecular-weight variant of 6,000 daltons of the activated mPEG was used in an antigen-specific regulation of IgE response. In these studies 200 genetically high IgE responders (CBA/Ca) female mice were intradermally immunized by the purified BV45, with the use of various adjuvants. Four different sets of experiments were made. In the first experiment 50 mice were immunized on days 1 and 15 with BV45. This was followed by another booster dose of the native BV45 or the modified mPEG-BV45 in Freund's incomplete adjuvants (FIA) on day 72. In the 2nd experiment, a similar procedure was performed except that booster doses were injected on day 135. Subsequently on day 136, BV45/FIA was intradermally injected. In the 3rd and 4th sets of experiments, 2 groups of 50 mice were daily immunized by intraperitoneal injections of 10 successive doses of BV45. On day 33 a dose of BV45 or mPEG-BV45 respectively, was intraperitoneally injected in each mouse. This injection was followed by other 10 successive doses of BV45. In experiment 3 no adjuvants were used, and in experiment 4 aluminium hydroxide gel was used as adjuvant. The mice immune response was assessed by analyses of the serum IgG and IgE levels. In all experiments higher IgG concentrations were shown for the immunized mice as compared to the non-treated control animals. Administration of booster doses of BV45 or mPEG-BV45 induced increases in the IgG levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Allergens↗
PubMed1986

Comparative studies on tree pollen allergens. X. Further purification and N-terminal amino acid sequence analyses of the major allergen of birch pollen (Betula verrucosa).

The previously isolated major allergen of birch pollen (fraction BV45), Int. Archs Allergy appl. Immun. 68: 70-78 (1982), was further purified by recycling chromatography. The purified preparation was run on a high-performance liquid chromatography (HPLC) TSK-G-2000 gel filtration chromatography column and, finally, on paper high-volt electrophoresis. The protein recovered met the homogeneity criteria required for performing the N-terminal sequence analysis. The allergenic and antigenic reactivities of the HPLC-purified protein, designated BV45B, was examined. A single homogeneous precipitation line in crossed immunoelectrophoresis (CIE) was shown. Specific IgE-inhibition tests and immuno-autoradiographic prints indicated that this allergen could bind reaginic IgE specificially and with good affinity. The homogeneity of BV45B was examined by isoelectric focusing (IEF). Several minor bands of pI differences of less than 0.1 units were visible, demonstrating the existence of some molecular variants of this protein. The N-terminal sequence analysis of the molecule was performed, and the following four amino acids were tentatively shown by sequential cleavage: NH2-Ala-Gly-Ile-Val-. The demonstration of one dominant N-terminal 1-dimethyl-amino-5-naphthalene sulphonyl (DNS)-amino acid by polyamide thin-layer chromatography at each sequence step confirmed that the N-terminal residue of the protein was not blocked; the heterogeneity shown by the IEF system was merely due to the presence of several homologous polymorphic proteins with identical N-terminal amino acid, the adequacy of the purification repertoire used.

Absorption↗