Stabilization of recombinantly expressed proteins.
Explore the source record for details and available documents.
Amino Acid Sequence↗
Biomedical subjects
F Matthiesen
Publications and source records attributed to F Matthiesen.
HLA-DR and HLA-DP genotypes and immunoglobulin E responses to common major allergens.
In order to test for human histocompatibility leucocyte antigens (HLA) class II restriction of IgE responses, 431 subjects from 83 families were genotyped at the HLA-DR and HLA-DP loci and serotyped for IgE responses to six major allergens from common aero-allergen sources. A possible excess of HLA-DR1 was found in subjects who were responsive to Fel d I compared with those who were not (Odds Ratio (OR) = 2, P = 0.002), and a possible excess of HLA-DR4 was found in subjects responsive to Alt a I (OR = 1.9, P = 0.006). Increased sharing of HLA-DR/DP haplotypes was seen in sibling pairs responding to both allergens. Der p I, Der p II, Phl p V and Can f I were not associated with any definite excess of HLA-DR alleles. No significant correlations were seen with HLA-DP genotype and reactivity to any of the allergens. The results suggest class II HLA restriction is insufficient to account for individual differences in reactivity to common allergens.
Alleles↗
Predominance of the major allergen (Alt a I) in Alternaria sensitized patients.
Thirty-nine patients sensitized to Alternaria were evaluated using titrated skin-prick test (SPT), histamine release studies (HR), inhibition of RAST and immunoblotting studies. To determine the relevance of the major allergen, Alt a I, specific rabbit antibodies against Alt a I and Alt a B were used. The antibodies were preincubated at different concentrations: (i) with the Alternaria allergen dose required for maximum response in the HR assay (10 BU/ml) and (ii) with the Alternaria antigen coupled to RAST paper discs (1000 BU/disc). Dose dependent inhibition of histamine release (n = 30, mean = 80% +/- 4%, IC30 = 0.69 micrograms/ml) and of RAST (n = 7, IC30 = 4.4 micrograms/ml) was found in all patients sensitized to Alternaria as indicated by allergen induced HR. The greater the response to Alternaria in HR, the higher the antibody concentrations necessary for inhibition (P < 0.05). Immunoblot experiments (n = 25) using SDS-PAGE showed in all cases IgE- and IgG binding at approximately 28 kD, which is the size reported for the major allergen, Alt a I. In two cases, slight IgE binding at 45 and 66 kD was also found, while in two other patients, only IgE binding at 66 kD was seen. Our findings emphasize the major importance of Alt a I in patients sensitized to Alternaria.
Adolescent↗
Group V allergens in grass pollens: IV. Similarities in amino acid compositions and NH2-terminal sequences of the group V allergens from Lolium perenne, Poa pratensis and Dactylis glomerata.
Monoclonal antibodies (PpV4) raised against Phleum pratense group V allergen were used for immuno-affinity chromatography of cross-reacting group V allergens from related grass species. Fractions enriched in group V allergen were obtained from Lolium perenne, Poa pratense and Dactylis glomerata extracts. The major components in these fractions were found in the Mwr range 25-28 kD. IgE binding to these components was shown using a pool of grass allergic sera, by SDS-PAGE immunoblotting. These fractions were electroblotted from tricine SDS-PAGE gels onto a polyvinylidene-difluoride membrane and selected group V bands were directly cut out and used for amino acid analysis and NH2-terminal sequencing. Both the amino acid compositions and the NH2-terminal sequences obtained for each group V allergen were almost similar to each other and to the sequence and composition of the previously described allergen Phl p V from Phleum pratense. A common trait of the investigated allergens, is the very high contents of alanine (25-32%) and the presence of the modified amino acid, hydroxyproline.
Allergens↗
Characterization of the major allergen of Cynodon dactylon (Bermuda grass) pollen, Cyn d I.
An allergen from Cynodon dactylon (Bermuda grass) pollen, Cyn d I, has been purified by a combination of concanavalin A-Sepharose affinity chromatography, and carboxymethyl-Sepharose chromatography. The allergen constitutes the major allergenic component of the pollen extract as observed by immunoelectrophoretic techniques, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, an IgE-inhibition experiment, and skin testing. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Cyn d I is presented as a dominant 32 kd band and a minor 29 kd band, both binding IgE. Both bands are identified by monospecific rabbit antibodies (Abs) raised against Cyn d I. These Abs only weakly precipitate allergens from other grass species, indicating that Cyn d I possesses some unique immunochemical properties. Two of four purified murine monoclonal Abs raised against Cyn d I also bind to both bands of Cyn d I, indicating that the bands represent isoallergens with slightly different immunochemical properties. All four monoclonal Abs cross-react with pollen components from other grass species, especially Poa pratensis and Dactylis glomerata. The NH2-terminal sequence corresponding to approximately 10% of the complete sequence was determined, and it revealed high homology to the corresponding sequence of the major allergen of Lolium perenne, Lol p I. From the amino acid composition determination and immunoelectrophoretic comparison, the amount of Cyn d I in the source whole-pollen extract was estimated to be 15% wt/wt.
Allergens↗
Group V allergens in grass pollens. I. Purification and characterization of the group V allergen from Phleum pratense pollen, Phl p V.
An allergen from Phleum pratense (timothy) pollen, Phl p V, has been isolated by a combination of copper chelate affinity chromatography and ion exchange chromatography. Phl p V binds IgE from serum of grass-sensitized donors as revealed in immunoelectrophoretic techniques and in SDS-PAGE immunoblot, and luminescence immunoassay (LIA) inhibition experiments indicate that the allergen represents a significant part of the IgE binding capacity of the extract. In immunoelectrophoresis, Phl p V is revealed as a single precipitate. However, molecular weight studies show that Phl p V consists of at least two isoforms with similar immunochemical properties, but with different molecular size. After SDS-PAGE treatment purified Phl p V is identified as two IgE-binding components, Phl p Va and Phl p Vb, with molecular weights 33 and 29 kD. After HPLC gel filtration, Phl p Va and Phl p Vb are identified in the major 30-kD eluate. After Sephadex G75 gel filtration of whole pollen extract, Phl p V is identified in fractions corresponding to molecular weights 47 and 25 kD. The 47-kD fraction corresponds to Phl p Va/Phl p Vb as seen in SDS-PAGE, while the 25-kD component presumably corresponds to a degradation product present in whole pollen extract. The NH2-terminal sequence of Phl p V, corresponding to approximately 10% of the molecule, has been determined. The sequence shows minor variations in some residues and contains besides many alanine residues also hydroxyproline; the sequence reveals no homologies to any known NH2 terminal sequence of other proteins. The amino acid composition, revealing 26 mole % alanine and no cysteine, does not show any similarities to other known amino acid compositions of allergens. From the amino acid composition determination and an immunoelectrophoretic comparison, Phl p V is estimated to represent 6% (w/w) of the whole pollen extract.
Allergens↗
Group V allergens in grass pollens. II. Investigation of group V allergens in pollens from 10 grasses.
In an earlier study an allergen from Phleum pratense (timothy) pollen, Phl p V, has been isolated and physicochemically characterized. In this study Phl p V and immunochemically similar components from other grass pollens (group V allergens) have been investigated using immunoelectrophoretic techniques. To study the allergenic importance of the group V allergens, the allergenic compositions of 10 grass pollen extracts were investigated in crossed radioimmunoelectrophoresis (CRIE) using 20 sera from grass pollen-allergic donors. Group V allergens were identified using monospecific rabbit antibodies raised against Phl p V, anti-Phl p V, which react with other group V allergens usually producing dense precipitates in immunoelectrophoresis. In this way group V allergens were identified in eight extracts, and when present the precipitate corresponding to the group V allergen was the dominant IgE binding precipitate. All identified group V allergens bound IgE in at least 17 of the 20 investigated sera. Monospecific rabbit antibodies raised against the group I allergen of Lolium perenne (rye grass), anti-Lol p I, do not precipitate group V allergens, indicating that there are no immunochemical similarities between group I and group V allergens. In SDS-PAGE anti-Phl p V identifies IgE-binding components with molecular weights between 26 and 33 kD. In contrast, anti-Lol p I binds to components of slightly higher molecular weight. Apparently, the group V components are allergens that are physicochemically and immunochemically distinct from group I allergens.
Allergens↗
Hyposensitization therapy with whole pollen extract or purified allergens monitored by immunoblotting.
Patients allergic to grass pollen were hyposensitized with two major allergenic components or whole extract of timothy grass pollen. Specific IgE, IgG1, and IgG4 formed during immunotherapy were analyzed by immunoblotting. Similar antibody-binding patterns were observed in both patient groups. Inhibition experiments using allergenic components isolated by preparative sodium dodecyl sulphate-polyacrylamide gel electrophoresis indicated that all antigenic components of timothy grass pollen detected in immunoblot dispose of private and cross-reactive determinants for binding of human IgE. The worse clinical outcome of immunotherapy after hyposensitization with two cross-reactive components did, therefore, not correlate with a specific antibody formation pattern.
Allergens↗
An immunoelectrophoretic analysis of the allergens of Cynodon dactylon (Bermuda grass) pollen.
A crude extract of Cynodon dactylon (Bermuda grass) pollen was obtained by extraction, centrifugation, dialysis, and lyophilization. The corresponding polyspecific rabbit antibodies were obtained by immunization, bleeding, and purification and were used for a cross immunoelectrophoretic analysis of the crude extract. At least 52 antigens (Ags), of which 47 migrated toward the anode, and five, toward the cathode, were revealed. Crossed radioimmunoelectrophoretic analysis performed with sera from 32 patients allergic to Bermuda grass and a pool from 1000 normal individuals revealed specific IgE binding to 13 of these Ags. The approximate molecular weights (MWs) for 10 of these IgE-binding Ags were determined by a combination of gel filtration and immunochemical analysis. These Ags had apparent MWs greater than 14 kd. A combination of preparative sucrose gradient isoelectric focusing and immunochemical analysis was used to determine the approximate isoelectric point values of five of the IgE-binding Ags. Most precipitating Ags had isoelectric points between 4.5 and 5.5. Four of the Ags (Ag 24, MW, less than 65 kd; Ag 31, MW, 33 kd; Ag 33, MW, 20 kd; and Ag 34/35, MW, 32 kd) were classified as major allergens.
Adolescent↗
Standardization of allergen extracts.
In order to increase the precision of diagnosis and the safety of specific immunotherapy, standardization of allergenic extracts has increasingly been required. Because allergenic extracts are complex mixtures of potential allergenic components, it is appropriate to express the composition and potency relative to a standard. Several such standard reference extracts have now been established by international working groups (statued as subcommittees under IUIS and WHO) and are available from central control authorities. The international standards (IS) are available freeze-dried in ampoules and are meant as calibrators (not prototypes) for use in research, production and quality control. The potency of each ampoule is defined the arbitrary assignment of 100,000 IU, whatever potency determination method is used. In individual laboratories the internal reference should be assigned a potency relative to the IS by each of the commonly used methods. This should enable the collaboration of researchers, manufacturers, and control authorities and improve safety in practical immunotherapy.
Allergens↗