Monoclonal antibodies to group II Dermatophagoides spp. allergens: Murine immune response, epitope analysis, and development of a two-site ELISA (original) (raw)
Related papers
Journal of Allergy and Clinical Immunology, 1994
Background: Group H allergens are a major cause of sensitization in patients allergic to mites. To facilitate the antigenic analysis of group II allergens and to develop improved methods of allergen detection, we compared IgG anti-group H antibody responses in inbred mouse strains and raised a panel of monoclonal antibodies (mAbs). Methods: IgE antibody responses were compared by antigen-binding radioimmunoassay. Epitope specificity of the mAbs was analyzed by two-site binding assays and by cross-inhibition radioimmunoassays. Results: Comparison of polyclonal IgG antibody responses in five BALB congenic strains showed that H-2 a mice had poor responses, whereas H-2 b and H-2 k mice had strong, cross-reactive, IgG anti-group H responses. The specificities of nine anti-Der p H IgE mAbs raised in A/J mice were compared with specificities of seven m,4bs produced previously. Most mAbs (11 of 16) recognized common epitopes on Der p H and Der f 11: three were specific to Der p 11, and two showed high binding to Der f 11. Epitope analysis showed that the mAbs defined four cross-reactive, nonoverlapping sites on the group 11 allergens. Binding of several combinations of mAbs was compared, and a two-site ELISA for group 11 antigens was developed. Linear regression analysis showed an excellent correlation between results of this assay and group H radioimmunoassay of house dust samples (n = 40, r = O. 85, p < O. 001). Conclusions: There are multiple cross-reactive B-cell epitopes on group H allergens. The group H ELISA has several important applications, including assessment of environmental allergen exposure, monitoring of the efficacy of avoidance procedures, and standardization of commercial mite allergen extracts. (J ALLERGY CLIN IMMUNOL 1994;94:537-46.)
Clinical & Experimental Allergy, 1997
Background and Objective Chimeric mouse/human monoclonal IgGl and IgG4 antibodies were developed against the house dust mite allergen Der p 1. These chimeric IgG antibodies, hIgGI-Dp2 A and hIgG4-Dp2 A, have the same binding characteristics as the previously reported chimeric hIgE-Dp2 A and are composed of the heavy chain variable domains and light chains ot the original murine monoclonal antibody 2B12. whereas the heavy chain constant domains have been replaced by the human IgGl or IgG4 heavy chain. The expression level of hIgG 1-Dp2 A and hIgG4-Dp2 A was 1 and 3.5 ^g/mL, respectively. Methods and Results Since all IgG in these culture supernatants is allergen-specific, they are useful reference reagents and enable the calculation of the amount of allergen specific IgGl and lgG4 antibodies in absolute IgG amounts. The results obtained with two panels of sera from patients in immunotherapeutic treatment were evaluated and compared in Der p 2 IgE, IgGl and IgG4 RAST and with reversed lgG4 RAST using labelled purified Der p 2. Close agreement between the results for the two IgG4 assays was found. Conclusion With these chimeric reference reagents the quantities of isotype specific antiallergen antibodies can be calculated and compared.
The molecular basis of antigenic cross-reactivity between the group 2 mite allergens
Journal of Allergy and Clinical Immunology, 2001
Background: Mite group 2 allergens Der p 2, Der f 2, and Eur m 2 are 14-kDa proteins of unknown function that share 83% to 85% amino acid sequence identity. Isoforms of the allergens within each genus have been identified which differ by 3 or 4 amino acids, but little is known of the influence of group 2 polymorphisms on human IgE antibody binding. Objective: The purpose of this study was to investigate the importance of interspecies and isoform substitutions on murine mAb and IgE antibody binding and on the molecular structure of the group 2 allergens. Methods: Site-directed mutagenesis was used to incorporate the isoform amino acid substitutions onto the Der p 2.0101 sequence. Recombinant allergens were expressed and purified from Escherichia coli and used to evaluate antibody binding by enzyme-linked immunosorbent assay (ELISA). Molecular modeling of the tertiary structure was used to analyze structural differences between the various group 2 allergens.
Identification of immunodominant IgE epitopes of the major house dust mite allergen Der f 24
International Journal of Molecular Medicine, 2019
Previously, a ubiquinol-cytochrome c reductase binding protein (UQCRB) homolog was identified in the house dust mite (HdM) species Dermatophagoides farinae (der f) as a major allergen. In the present study, the immunodominant immunoglobulin E (IgE) epitope of the protein der f 24 was investigated. Analysis of the homologous amino acid (aa) sequences in der f and human UQcRB was performed. Four different recombinant der f 24 and hybrid proteins formed by integrating der f and human UQcRB sequences were expressed in Escherichia coli, purified using Ni-NTA resins, and IgE-binding activity was determined using IgE-western blotting and enzyme-linked immunosorbent assay (ELISA) experiments. IgE epitopes were further identified by IgE-dot blotting and IgE-ELISA with synthetic polypeptides and HdM-allergic sera. Three-dimensional (3d) structural modeling was used to analyze the position of the immunodominant IgE epitope. The amino acid sequence homology between der f 24 and the human UQcRB protein was determined to be 39.34%. IgE-ELISA and western blot analysis showed that all of the der f-human UQcRB hybrid proteins generated, except for the one lacking 59 residues of the N-terminal region of der f 24, were bound by allergic serum IgE. A synthetic polypeptide consisting of 32 residues of the N-terminal reacted with IgEs from HdM-allergic sera and could be used to generate high titer specific IgG or specific IgE antibodies in immunized mice. The 32-aa N-terminal region of der f 24 was localized to a structural protrusion, which may facilitate specific IgE-binding. These results indicate that the immunodominant IgE epitope of der f 24 is located mainly in a 32-residue region of the N-terminus. These findings may inform the mechanisms of HdM allergy sensitization and allergy immunotherapy development.
IgE cross-reactivity between house dust mite allergens and Ascaris lumbricoides antigens
Asia Pacific Allergy, 2012
Background: Common antigens between intestinal parasites and environmental allergens may play a role in the modulation of allergic immune responses. There is a growing interest in investigating cross-reactivity between common helminths and dust mites affecting humans, particularly in the tropics. Objective: This study examined the cross-reactivity between the human roundworm Ascaris lumbricoides (Al) and three house dust mite (HDM) species. Methods: Specific serum IgE levels to HDM species Blomia tropicalis (Bt), Dermatophagoides pteronyssinus (Dp), and Dermatophagoides farinae (Df ); and Al extracts among allergic (n=100) and ascariasis (n=60) subjects were measured through enzyme-linked immunosorbent assay (ELISA). IgE-reactive components of HDM and Al extracts were detected through Western-Blot Analysis. Crossreactivity between HDMs and Al was determined by ELISA inhibition using HDM and Al-specific sera from allergic (n=15) and ascariasis (n=15) subjects. The IgE-binding capacity of a recombinant paramyosin peptide (Blo t 11-fD) to allergic (n=50) and ascariasis (n=50) subjects' sera were likewise determined. Results: Among allergic subjects, 70% exhibited Al-specific positive IgE-reactivity, while 20-28% of ascariasis subjects demonstrated HDM-specific positive IgE-reactivity. Multiple IgE-reactive components of HDM allergens (14-240 kDa) and Al antigens (15-250 kDa) were detected, indicating multi-allergen sensitization among the subjects tested. Al antigens can inhibit up to 92% of HDM-specific IgE-reactivity among allergic subjects, while up to 54% of Al-specific IgE-reactivity among ascariasis subjects was inhibited by HDM allergens. Positive rBlo t 11-fD-specific IgE reactivity was observed in 80% of the allergic subjects and 46% of the ascariasis subjects. Conclusions: This study showed the presence of multiple cross-reactive antigens in HDM and Al extracts. Identification of these molecules may provide basis for designing novel diagnostic and therapeutic strategies. The potential role of paramyosin as a specific cross-reactive allergen present in HDMs and Al has been shown.
Analysis of cross-reactivity between group 1 allergens from mites
Puerto Rico health sciences journal, 2008
Mite allergen exposure can lead to sensitization in genetically predisposed individuals, and the development of asthma in previously sensitized individuals. The major allergens of mites belong to Dermatophagoides spp. and Blomia tropicalis (Bt). Various allergens of Bt have been cloned and sequenced. Some of them show homology sequence with purified allergens from Dermatophagoides pteronissynus (Dp). Recently, the allergen group 1 from Bt, Blo t 1, was cloned and sequenced at our laboratory. Recombinant Blo t 1 showed 35 % of identity and 50% of similarity with group 1 allergens as Der p 1 (from Dp), Der f 1 (from D. farinae) and Eur m 1 (from Euroglyphus maynei) at amino acid level. This would suggest that cross-reactivity between allergens of different mite species could exist. Here, we analyzed the crossreactivity between group 1 allergens from mites using recombinant proteins and monoclonal antibodies against them. ELISA inhibition assay showed that crossreactivity between homol...
Characterization and Immunobiology of House Dust Mite Allergens
International Archives of Allergy and Immunology, 2002
The examination of house dust mite extracts has indicated that over 30 different proteins can induce IgE antibody in patients allergic to the house dust mite. There are however dominant specificities especially the group 1 and 2 allergens which can account for much of the allergenicity of extracts. Of the 19 denominated allergens, the major IgE binding has been reported for the group 1, 2, 3, 9, 11, 14 and 15 allergens. The high-molecular-weight group 11, 14 and 15 allergens have only recently been described and although high IgE binding has been anticipated from immunoblotting, there is a need for considerable corroboration. Similarly, the study of the group 3 and 9 serine protease allergens has been incomplete. The group 4, 5, 7 and 8 allergens have shown intermediate IgE binding and the group 10 tropomyosins are of interest because of their potential cross-reactivity with allergen from disparate species. Although the progress with the production of recombinant group 1 allergens h...
Lack of human IgE cross-reactivity between mite allergens Blo t 1 and Der p 1
Allergy, 2003
It has been well established that mite species such as Dermatophagoides pteronyssinus, Dermatophagoides farinae and Euroglyphus maynei from the family Pyroglyphidae are important clinical species (1-4). In recent years, Blomia tropicalis mite classified under the family Glycyphagidae (5) is an important source of indoor allergens that associate with allergic asthma and rhinitis in the tropics (6-11). The first major B. tropicalis allergen cloned was Blo t 5 (12, 13). Subsequently, cDNA encoding for Blo t 3, Blo t 4, Blo t 6, Blo t 10, Blo t 11, Blo t 12, Blo t 13, and Blo t 19 were isolated by us and others . The estimated frequency of human IgE reactivity to Blo t 3, Blo t 5, Blo t 10, Blo t 11, Blo t 12, and Blo t 13 with mite allergic sera was about 51, 70, 20-29, 52, 50, and 11%, respectively.
PubMed, 2006
Several studies have shown that the presence of IgE antibodies to house dust mites (HDM), particularly Dermatophagoides pteronyssinus (Dpt), is an important risk factor for asthma. Allergen immunotherapy is indicated for patients with IgE antibodies to clinically relevant allergens. The aims of this study were to analyze the levels of specific serum IgE to Der p 1 and Der p 2 allergens in mite-sensitized atopic patients and to compare them with both in vivo (skin prick test) and in vitro (IgE-ELISA) sensitizations to Dpt crude extract. Forty-seven atopic patients with allergic rhinitis with or without intermittent or persistent mild asthma and positive skin prick test (SPT) to Dpt total extract were studied. Thirty age-matched healthy subjects with negative SPT to HDM were included as controls. Levels of total IgE and Dpt-, Der p 1- and Der p 2-specific IgE were measured by ELISAs in SPT-positive atopic patients and SPT-negative control subjects. Among 47 symptomatic atopic patients, 27 (57.4%) were double positive IgE to Der p 1 and Der p 2 allergens, 3 (6.4%) were single positive IgE to Der p 1, 4 (8.5%) were single positive IgE to Der p 2, and 13 (27.6%) were double negative IgE to both allergens. There was a significant correlation between Der p 1- and Der p 2-specific IgE levels, but not between Der p 1- or Der p 2-IgE levels and SPT results. The double negative IgE patients had the smallest skin test reactions although they showed high mean levels of total serum IgE. Therefore, the knowledge of specific IgE levels to Der p 1 and Der p 2 major allergens might support physicians for indication or follow-up in mite-sensitized patients under allergen-specific immunotherapy. These approaches might be important for obtaining improved safety and efficacy of the current clinical practice of allergen immunotherapy.
International Archives of Allergy and Immunology, 2016
sensitized to Der p 23, and 11 patients were negative for all HDM MeDALL chip components. Seven sera were available for further testing, and 3 of them showed IgE reactivity to dot-blotted nDer p 1, and 2 reacted with high-molecular weight components (>100 kDa) in nitrocellulose-blotted HDM extract when tested with 125 I-labeled anti-IgE in a RASTbased assay. The HDM extract-specific IgE levels of the 11 patients were <3.9 kU/l. Conclusions: Recombinant allergen-based IgE serology is of great value when conventional IgE diagnostics fails. Der p 23 is an important HDM allergen, especially when major allergens are negative. Therefore, it would be desirable to have Der p 23 commercially available. Further research concerning the prevalence and clinical significance of different HDM allergens is needed.