[Diagnostic values of clinical biochemical analyses].
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Chemistry, Clinical↗
Biomedical subjects
S Elsayed
Publications and source records attributed to S Elsayed.
[Variations in clinical biochemical tests].
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Chemistry, Clinical↗
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↗
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↗
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↗
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↗
[Acute-phase proteins in the blood of pregnant women].
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Adult↗
Increased concentrations of aspartic acid in the cerebrospinal fluid of patients with epilepsy and trigeminal neuralgia: an effect of medication?
The concentrations of free amino acids in the cerebrospinal fluid (CSF) were quantitated in 14 patients with epilepsy, 7 patients with trigeminal neuralgia, 10 patients with various neurological diseases and in 30 apparently healthy individuals. In the CSF of patients with epilepsy, the mean concentrations of aspartic acid (Asp), glutamine (Gln), histidine and phosphoserine were significantly higher than in the healthy individuals. The mean concentration of aspartic acid was higher in the patients on antiepileptic drug (AED) therapy than in the patients without AED therapy. The patients with trigeminal neuralgia had significantly increased CSF-concentrations of aspartic acid, glutamine and phosphoserine as compared to healthy individuals. Some patients receiving AED showed increased concentration of gamma-amino-butyric-acid (GABA).
Adolescent↗
Comparative studies on tree pollen allergens. VIII. Immunological properties of the alder (Alnus incana) pollen extract.
The immunological properties of the aqueous crude alder pollen extract (AI crude) and gel filtration fractions AI 3, AI 4 and AI 34 (pool of fractions AI 3 and AI 4) were examined by immuno- and radioimmuno-electrophoretic techniques, RAST titration, RAST inhibition and skin prick tests (SPT). In CIE, the AI crude extract and AI 34 displayed reference precipitate patterns consisting of 27 and 24 visible Coomassie brilliant blue stained lines, respectively. The CRIE allergogram performed by incubation with 18 individual reaginic sera detected three IgE-binding antigens characterized by different IgE-binding properties. Antigen No. 7 (Ag 7) was demonstrated to be the major IgE antibody-binding antigen of alder pollen, while Ag 1 and Ag 11 were classified as intermediate allergens. The allergens of alder pollen were located in fractions AI 3 and AI 4 of the gel filtration chromatogram. Ag 7 was present in both fractions as demonstrated by FRIE with autoradiography (FRIEWA) on the gel filtration fractions and tandem-CRIE of AI 3 and AI 4. The CRIE allergogram, RAST, RAST inhibition and SPT demonstrated fraction AI 34 to be allergenically representative of the AI crude extract both qualitatively and quantitatively. Thus, fraction AI 34 was considered an optimal purified allergen extract of alder pollen, a suitable material for further biochemical characterization and trials on purification of the allergenic reactive antigens.
Animals↗
Comparative studies on tree pollen allergens. VII. Monomethoxy polyethylene glycol conjugation of the major allergen of birch pollen (Betula verrucosa).
The major allergen of birch pollen (BV45) was conjugated to 2-0-methoxy polyethylene glycol-4,6-dichloro-5-triazine (mPEG). Three molecular weight variants of 4,000, 6,000 and 20,000 daltons, respectively, of the activated PEG were used. The modified preparations were labelled by 125I and both the native and the radiolabelled protein conjugates were purified by gel filtration chromatography. The relative molecular weights of the purified two peaks were preliminarily estimated by high performance liquid chromatography (two populations of greater than or equal to 100,000 or 20,000 daltons). The amino acid composition of the acid hydrolysates of the three conjugated proteins indicated that 5 residues of lysine were modified by mPEG. Other charged amino acid side chains could also be bound to the activated PEG. The immunochemical properties of the copolymers were studied. The immunogenicity and antigenicity were examined by immunizing rabbits with 125I-mPEG 4,000 daltons BV45 and 125I-mPEG 20,000 daltons BV45 and subsequently crossed immunoelectrophoresis. The clearence of the radiolabelled protein showed normal pattern. A sharp decline in the radioactivity could be measured. At days 10-12, the remaining radioactivity was below 2%. Preliminary studies showed that the modified proteins were immunogenic in rabbits. The findings were demonstrated by crossed immunoelectrophoresis of the 125I-mPEG 4,000, 6,000 or 20,000 daltons BV45 and the corresponding autoradiographs. Apparently, the immunogenicity and antigenicity of the preparations were qualitatively unaltered. The allergenicity of the modified preparation was measured in vitro by RAST and RAST inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acids↗
Solid-phase synthesis of the non-calcium-binding loop of cod allergen M. Direct evidence of the reactivity of the amino-terminal segment.
The synthesis of the non-calcium-binding AB loop of the assembly 13-32 of cod Allergen M (Mr 2122.1) was accomplished by solid-phase peptide synthesis. This peptide and the previously synthesized ones [12, 13, 14] have significant amino acid sequence homology. The synthetic crude preparation was obtained at relatively high recovery and purity. Further purification on a Bio-Gel P-2 column and a reversed-phase high-performance liquid chromatography column improved the grade of homogeneity, as demonstrated by high-voltage electrophoresis, end-terminal analysis, and amino acid composition. The peptide could, although to a much weaker extent than the intact Allergen M, directly bind IgE antibodies from the sera of cod-allergic individuals. At identical molar concentrations, a ratio of 1:6 for the in vitro reactivity of the peptide relative to the intact Allergen M was obtained. A similar reactivity was shown in the in vivo system used. The peptide also reacted with rabbit anti-Allergen M antibodies in rocket immunoelectrophoresis. The peptide appears to function as a divalent molecule in its primary interaction with antibodies.
Allergens↗
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 structure was based on the extensive intramolecular amino acid homologies and the immunochemical cross-reactivities of the intact molecule and the two major isolated fragments. The immunological structure of Allergen M was studied by: 1. Modification of certain amino acids residues and study of the reactivity of the modified derivatives. 2. Examination of the immunochemical reactivity of a large number of overlapping peptides obtained by limited and selective tryptic hydrolyses. 3. Solid phase peptide synthesis (SPPS) of segments selected in regard to the reactivity of pre-examined native peptides. The immunological reactivity of the derivatives of Allergen M was assigned by: 1. Rocket line immunoelectrophoresis and quantitative precipitation using rabbit anti-Allergen M in precipitating antibody-mediated reactions and, 2. RAST/RAST-inhibition and PK test/PK-test inhibition using sera from individuals allergic to codfish in IgE-mediated reactions. The modification of Tyr-30 and Arg-75 in isolated and purified peptides indicated that the former was part of a reactive site whereas the latter did not contribute to the activity. Masking of Arg-residue or unchelating the two calcium ions from the native Allergen M, with the resulting perturbation of the tertiary structure, decreased the allergenicity by approximately 25%. Two major fragments of Allergen M were produced and purified: TM1 (residues 1-75) comprising domains AB and CD, and TM2 (residues 76-113) covering domain EF. Both were immunologically reactive; TM1 showing intermediate reactivity between Allergen M and TM2. A high degree of immunological cross reactivity was evident between TM1 and TM2. The finding was in concert with the high intramolecular amino acid homologies of Allergen M, and suggested that the reactive sites were repetitively distributed along the polypeptide chain. The immunological reactivity of several long-sequence overlapping peptides obtained by limited and selective trypsin hydrolysis of Allergen M was studied. The immunologically reactive sites were accordingly assigned to the following regions of the chain: 1. Residues 33-44 on the junction between the AB and CD domains.(ABSTRACT TRUNCATED AT 400 WORDS)
Amino Acid Sequence↗
Serum protein pattern in normal pregnancy with special reference to acute-phase reactants.
The concentrations of acute-phase protein reactants, total protein, albumin and globulin fractions were measured throughout normal pregnancy in 27 women. alpha 1-Antitrypsin and caeruloplasmin concentrations increased gradually to reach their highest levels in the third trimester. Orosomucoid and haptoglobin showed similar patterns: higher levels in the first and third trimester with a decline around 24 weeks gestation. C-Reactive protein showed levels similar to those of non-pregnant healthy individuals (less than 5 mg/l) throughout pregnancy. alpha 1-, alpha 2- and beta-Globulin concentrations increased from the first trimester towards term. gamma-Globulin concentration changed little during gestation. The data obtained provide reference ranges for serum proteins in healthy pregnancy.
Adult↗
Comparative studies on tree-pollen allergens. V. Immunochemical mapping of the antigens and allergens of birch pollen extract (Betula verrucosa).
A comparative electrophoretic and autoradiographic analysis of the different fractions of birch pollen crude extract was performed. This extract included a minimum of 17 reproducible and distinct antigens located in the gel filtration fractions BV2-BV4. Only one of these precipitates had the ability to bind IgE, as demonstrated by immunoelectrophoretic and autoradiographic techniques using several poly- and monospecific rabbit antibodies. The allergenicity of the different fractions was examined in vitro by RAST and RAST inhibition, and in vivo by passive cutaneous transfer and skin prick tests. The data suggested that Betula verrucosa pollen extract contains a group of isoallergens with related antigenicity but with variable molecular sizes. The findings have also confirmed the presence of identical antigenic properties of the previously isolated pI 5.18 and BV45 as deduced from their immunoelectrophoretic and autoradiographic studies.
Allergens↗
The immunological reactivity of the three homologous repetitive tetrapeptides in the region 41-64 of allergen M from cod.
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Allergens↗
Comparative studies on tree-pollen allergens. III. A purified low molecular weight major allergen from birch pollen (Betula verrucosa) isolated by gel permeation chromatography.
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Allergens↗
Comparative studies on tree pollen allergens. II. Isolation of alder (Alnus incana) pollen allergens: purification and some characteristics of the major allergen pI 4.78.
The aqueous extract of alder (Alnus incana) pollen was found to consist of a protein mixture amounting to 3% of the dry pollen weight. Separation by gel permeation chromatography on an AcA-54 column resulted in 6 fractions designated chronologically to their relative elution volumes (AI 1 to AI 6). The immunological reactivity of these fractions was demonstrated in both IgE and IgG test systems. Being both immunochemically and quantitatively dominant fraction AI 4 was selected for further purification by a semipreparative isoelectric focusing column. A well-resolved and apparently homogeneous component with a pI value of 4.78 was eluted from the IEF column. The homogeneity of this fraction was demonstrated by one sharp band in thin layer IEF, one single band in HVE, one distinct sharp peak in HPLC and a symmetrical precipitate in CIE. The molecular weight was estimated to 22,500 daltons. Fraction pI 4.78 was shown to be a major allergen of alder pollen, it elicited a 1.17 histamine wheal reaction in skin prick test at a concentration of 1 mg . ml-1 and showed a significantly high RAST inhibition in all of the reaginic sera used. Fraction pI 4.78 was found to be an adequate model allergen of alder pollen well suited for further immunochemical characterization.
Allergens↗
Comparative studies on tree pollen allergens. IV. Evaluation of two commercially available allergen extracts of alder (Alnus incana) and birch (Betula verrucosa) pollen.
Two commercially available extracts of alder and birch pollen usually used in clinical allergy were evaluated and correlated to two corresponding reference extracts 'S' produced at the authors' laboratory. The set of extracts produced by ALK, Denmark are referred to as 'A', while those produced by Nyco, Norway are referred to as 'N'. The results of the CIE analysis demonstrated that, upon using the same antibodies, both the morphology and the intensity of the precipitates of the different extracts differed considerably. In the alder pollen extract, the 'N' preparation contained more immunoprecipitate lines than the two others. Extract 'S' contained the least number of antigenic lines. Only two of the antigenic lines bound specific IgE in CRIE and were represented in variable concentrations in the three extracts. These two allergenic precipitation lines were demonstrated to have an immunological reaction of identity as shown by tandem-CIE and tandem-CRIE systems, respectively. In the birch pollen system variable numbers of reproducible antigenic lines of precipitation were demonstrated. Preparation 'A' contained more antigens than the two other extracts. This extract lacked a cathodic antigen with slow electrophoretic mobility represented in 'N' and 'S'. One dominant antigenic line was commonly present in the three extracts and could bind IgE antibodies in the CRIE system. A minor cathodic allergen was shown by CRIE in extracts 'S' and 'N', but not in extract 'A'. The RAST and RAST-inhibition titration experiments in all extracts showed qualitative and quantitative similarities. The amino acid compositions and the carbohydrate analyses showed that extracts 'S' and 'N' were similar while extract 'A' contained higher concentrations of certain amino acids and carbohydrates. In both alder and birch pollen, the reference extract 'S' contained lower concentrations of the nonallergenic antigens and of the low molecular weight contaminants.
Allergens↗