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  • 1
    Electronic Resource
    Electronic Resource
    DNA immunization for the production of monoclonal antibodies to Blo t 11, a paramyosin homolog from Blomia tropicalis (2004)
    Oxford, UK : Munksgaard International Publishers
    Allergy 59 (2004), S. 0 
    Details
    ISSN: 1398-9995
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Background:  Blo t 11 is a high molecular weight allergen from Blomia tropicalis with significant immunoglobulin (Ig)E binding frequency. Native and recombinant Blo t 11 are susceptible to degradation and the isolation and expression of the allergen is problematic thus obtaining sufficient amounts of purified Blo t 11 for antibody production is limiting. DNA-based immunization is an attractive alternative strategy that bypasses antigen purification for antibody production.Objectives:  To use a DNA-based immunization protocol for the production and characterization of Blo t 11 monoclonal antibodies (mAbs).Methods:  The 2625 bp cDNA coding for Blo t 11 was cloned into a mammalian expression vector and immunized intramuscularly with electroporation into mice. Monoclonal antibodies to Blo t 11 were generated using a methylcellulose-based hybridoma cloning kit. These mAbs were utilized for native Blo t 11 isolation and the development of sandwich enzyme-linked immunosorbent assay (ELISA).Results:  Six mAbs recognizing the native and recombinant Blo t 11 were generated and characterized. Native Blo t 11 was affinity purified from Bt extract and its identity was confirmed by matrix assisted laser desorption/ionization – time of flight mass spectrometry. The native Blo t 11 showed IgE reactivity with 67% of mite allergic sera. A two-site ELISA developed showed a detection limit of 100 pg/ml of Blo t 11.Conclusion:  A DNA-based immunization protocol was successfully used to generate Blo t 11 mAbs with a spectrum of distinct epitopes located throughout the whole molecule, and they are useful for immunoaffinity purification and immunoassays.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1398-9995.2003.00409.x
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  • 2
    Electronic Resource
    Electronic Resource
    Generation of monoclonal antibodies against Blo t 3 using DNA immunization with in vivo electroporation (2003)
    Oxford, UK : Blackwell Science Ltd
    Clinical & experimental allergy 33 (2003), S. 0 
    Details
    ISSN: 1365-2222
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Background House dust mite allergy is closely associated with allergic diseases. Blomia tropicalis mite species is an important clinical species in the tropics. The cDNA clone encoding Blo t 3, a group 3 allergen from B. tropicalis, has been isolated in our laboratory.Objective This study was designed to generate Blo t 3-specific monoclonal antibodies (mAbs) for the detection, characterization and purification of this allergen.Methods Mice were immunized intramuscularly with naked plasmid DNA encoding Blo t 3 gene with in vivo electroporation. Hybridomas were generated by the fusion of the splenocytes to X63-Ag8.653 myeloma cells. Purified native Blo t 3 was obtained by mAb immuno-affinity purification and the allergenicity of native Blo t 3 was determined by human IgE enzyme-linked immunosorbent assay (ELISA).Results A panel of class-switched and high-affinity mAb recognizing a wide spectrum of Blo t 3 epitopes have been generated. These mAbs are useful for western immunoblot assay, sandwich ELISA and affinity purification of native Blo t 3. Allergenicity of native Blo t 3 protein was examined with 44 mite-allergic sera and approximately 57% of the tested sera had positive serum IgE reactivity to the native Blo t 3.Conclusions These results demonstrated that intramuscular injection of naked DNA encoding Blo t 3 gene combined with in vivo electroporation is an effective and simple method to raise monoclonal antibodies that can be used for characterization and purification of Blo t 3.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2222.2003.01648.x
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  • 3
    Electronic Resource
    Electronic Resource
    Identification of shared and unique immunoglobulin E epitopes of the highly conserved tropomyosins in Blomia tropicalis and Dermatophagoides pteronyssinus (2002)
    Oxford, UK : Blackwell Science, Ltd
    Clinical & experimental allergy 32 (2002), S. 0 
    Details
    ISSN: 1365-2222
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Background Tropomyosin belongs to a class of highly conserved proteins in invertebrates and vertebrates. The invertebrate tropomyosins are allergenic in man with high IgE cross-reactivity and have been therefore referred to as pan-allergens.Objectives This study aimed to clone and identify the IgE epitopes of tropomyosin from Blomia tropicalis (Blo t 10) mite. Cross-reactivity between the IgE epitopes of Blo t 10 and Der p 10 was also evaluated.Methods Blo t 10 was isolated using mouse anti-Der p 10 antibodies. Allergenicity of the cloned Blo t 10 was confirmed by skin prick test (SPT) and enzyme-linked immunosorbent assay (ELISA). Dose-dependent inhibition assay was performed to determine the degree of IgE cross-reactivity between Blo t 10 and Der p 10. Overlapping polymerase chain reaction-derived cDNA were generated and expressed as glutathione-S-transferase (GST) recombinant proteins in Escherichia coli and used to identify shared and unique IgE epitopes of Blo t 10 and Der p 10.Results The cloned Blo t 10 shared up to 96% amino acid identity to tropomyosin of other mites. SPT and ELISA IgE-immunoassay showed recombinant Blo t 10 sensitization rates of between 20% and 29% in atopic subjects. Results of SPT and dose-dependent inhibition assays showed that some allergic individuals had unique IgE epitopes for Blo t 10. IgE epitope mapping of Blo t 10 revealed that the epitopes were mainly located at N- and C-termini of the molecule. The results of ELISA inhibition assays of overlapping recombinant fragments indicated that the unique IgE epitopes of Blo t 10 were located at the C-terminal.Conclusion Although Blo t 10 and Der p 10 are highly conserved (shared 95% amino acids identity) and significantly cross-reactive, unique IgE epitopes do exist. The results suggest the potential deficiency of using only one of these highly conserved allergens as diagnostic or therapeutic reagents.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2745.2002.01449.x
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  • 4
    Electronic Resource
    Electronic Resource
    Peptide mapping of immunoglobulin E and immunoglobulin G immunodominant epitopes of an allergenic Blomia tropicalis paramyosin, Blo t 11 (2003)
    Oxford, UK : Blackwell Science Ltd
    Clinical & experimental allergy 33 (2003), S. 0 
    Details
    ISSN: 1365-2222
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Background The identification of immunodominant peptides containing the IgE and IgG epitopes on allergen molecules is an important step in understanding the interaction of the allergen with the immune system and, thus, essential for the development of effective immunotherapeutic and diagnostic reagents. The present study aimed to map the IgE and IgG immunodominant peptides of Blomia tropicalis (Bt) allergen Blo t 11, a high molecular weight allergen homologous to paramyosin, exhibiting important allergenic activity.Methods Eleven overlapping fragments of Blo t 11 cDNA gene were expressed as glutathione s-transferase (GST) fusion peptides, which were affinity-purified using the glutathione-Sepharose column. Human IgE and IgG immunodominant peptides were determined by dot blot immunoassay using crude Bt extract-positive sera from asthmatic patients. Evaluation of allergenicity, specific hIgG subclass analysis, and cross- and self-inhibition studies were determined by enzyme-linked immunosorbent assay.Results Blo t 11 contains multiple IgE and IgG immunodominant peptides scattered throughout the molecule. The dominant IgE and IgG peptides were mapped at amino acid positions 336–557 and 698–875, respectively. An immunodominant peptide (fD) registered a higher percentage of IgE and IgG reactivity compared to the rFL-Blo t 11. Significant serum levels of Blo t 11- and fD-specific IgG1, IgG2 and IgG4, but not IgG3 were detected in the Bt extract-positive sera tested. Cross-inhibition study revealed the rFL-Blo t 11 was significantly inhibited by fD.Conclusion The IgE and IgG immunodominant peptides of Blo t 11 have been mapped. Our data suggest that utilization of Blo t 11 fragment(s) or chimeric fusion fragments containing IgE and IgG epitopes could be a better alternative in the development of diagnostic and therapeutic reagents for mite allergy.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2222.2003.01623.x
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  • 5
    Electronic Resource
    Electronic Resource
    Immunoglobulin E reactivity of native Blo t 5, a major allergen of Blomia tropicalis (2004)
    Oxford, UK : Blackwell Science Ltd
    Clinical & experimental allergy 34 (2004), S. 0 
    Details
    ISSN: 1365-2222
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Background Blo t 5 is a major allergen of Blomia tropicalis and its complementary DNA (cDNA) has been expressed in both prokaryotic and eukaryotic expression systems. Although the recombinant Blo t 5 has been well characterized, relatively less is known about its native counterparts and the allergenicity comparison of the native and recombinant Blo t 5 allergens has not been reported.Objective The aims of this study are to characterize the native counterpart of Blo t 5, and compare the allergenicity of native and recombinant Blo t 5 by in vivo and in vitro assays.Methods Native Blo t 5 were purified by immuno-affinity chromatography and characterized by proteomic means. The allergenicity of the allergen was evaluated by skin prick tests, human IgE ELISA, ELISA inhibition and histamine release assays.Results Native Blo t 5 consists of at least five distinct isoforms, ranging from pI 3 to 5.5. Allergenicity assessment of recombinant and native Blo t 5 based on skin reaction, IgE-binding capacity and histamine release in allergic individuals indicated that there was a good correlation between both forms of Blo t 5 in general. However, data from IgE ELISA inhibition assay revealed the presence of additional unique IgE epitopes in native Blo t 5.Conclusions At least five distinct isoforms of Blo t 5 have been identified. Comparative assessment of native and recombinant Blo t 5 revealed that the allergenicity of these two forms was similar but not completely identical suggesting that the various isoforms of native Blo t 5 may exhibit additional unique IgE epitopes.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2222.2004.02107.x
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  • 6
    Electronic Resource
    Electronic Resource
    Production of monoclonal antibodies for immunoaffinity purification and quantitation of Blo t 1 allergen in mite and dust extracts (2004)
    Oxford, UK : Blackwell Science Ltd
    Clinical & experimental allergy 34 (2004), S. 0 
    Details
    ISSN: 1365-2222
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Background Blo t 1 is a cysteine protease-like allergen from Blomia tropicalis. Recombinant Blo t 1 binds up to 90% of IgE from allergic patients and shows limited cross-reactivity to Der p 1. The generation of monoclonal antibodies (mAbs) against Blo t 1 is important for the detection, isolation and characterization of the native form of the allergen.Methods Mice were immunized intramuscularly with naked plasmid DNA encoding Blo t 1 gene with in vivo electroporation and boosted intraperitoneally with recombinant Blo t 1. mAbs against Blo t 1 were generated using a methylcellulose-based hybridoma cloning kit. The native Blo t 1 was isolated by mAb affinity purification and its allergenicity was determined by ELISA. A two-site ELISA for Blo t 1 was developed using the mAbs generated.Results A DNA-based immunization protocol induced high titre Blo t 1-specific antibodies in mice. Six stable hybridoma clones secreting mAbs recognizing the native and recombinant Blo t 1 were generated. The native Blo t 1 was affinity-purified from a B. tropicalis extract and its allergenicity was determined at 63% using a panel of Singaporean and Malaysian mite allergic patients' sera. A two-site ELISA was developed, which showed a detection limit of 10 ng/mL of Blot t 1.Conclusion Six Blo t 1 mAbs were successfully generated by DNA immunization. These mAbs are useful for nBlo t 1 immunoaffinity isolation and quantitative immunoassays for Blo t 1 in mite and environmental dust extracts.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2222.2004.1922.x
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  • 7
    Electronic Resource
    Electronic Resource
    Quantification of Blo t 5 in mite and dust extracts by two-site ELISA (2005)
    Oxford, UK : Munksgaard International Publishers
    Allergy 60 (2005), S. 0 
    Details
    ISSN: 1398-9995
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Background:  Blomia tropicalis is an important mite species in the tropical and sub-tropical regions of the world. Blo t 5 is the major allergen with up to 70% sensitization rates in B. tropicalis allergic populations.Methods:  Mice were immunized intramuscularly with naked plasmid DNA encoding Blo t 5 gene with in vivo electroporation. Blo t 5 monoclonal antibodies were generated using methylcellulose-based hybridoma kit. Monoclonal antibody (mAb) 4A7 was characterized by two-dimensional electrophoresis immunoblotting. A specific quantitative two-site enzyme-linked immunosorbent assay (ELISA) was developed with mAb 4A7 and guinea pigs Blo t 5 polyclonal antibody as capture and detection antibodies, respectively. This system was tested with Blo t 5 in crude extracts and dust samples.Results:  A high-affinity mAb 4A7 recognizing several isoforms of Blo t 5 has been generated. Monoclonal antibody 4A7 is useful for immunoblotting and two-site ELISA. The two-site ELISA developed has a high sensitivity, with a detection limit of 10 pg/ml. The assay is species-specific and recognized the same epitopes on both native and recombinant Blo t 5. The assay developed is able to detect Blo t 5 in commercial diagnostic and therapeutic B. tropicalis extract. Blo t 5 quantification in dust samples showed that Blo t 5 is present in a high quantity in Singapore dust.Conclusions:  A highly sensitive and specific two-site ELISA has been developed. The assay system developed is useful for the quantification of Blo t 5 in mite and environmental dust extracts.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1398-9995.2004.00597.x
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  • 8
    Electronic Resource
    Electronic Resource
    Cloning of a group 3 allergen from Blomia tropicalis mites (2003)
    Oxford, UK : Munksgaard International Publishers
    Allergy 58 (2003), S. 0 
    Details
    ISSN: 1398-9995
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Background:  Blomia tropicalis is an important mite species in the tropics and subtropical regions of the world. It is well established that the allergen from this species of mite is one of the triggering factors for allergic asthma. The isolation and characterization of allergens in this mite species is desired to provide sensitive and specific reagents for diagnostic as well as therapeutic purposes.Methods:  The SMART (Clontech Laboratories, Palo Alto, CA, USA) rapid amplification of complementary DNA ends (RACE cDNA amplification) method was used to isolate the putative Blo t 3 gene. Polymerase chain reactions (PCR) were performed in the presence of specific gene primers to obtain the full-length gene, and were confirmed by DNA sequencing. The putative gene was cloned into E. coli expression vector GST-4T-1 and expressed as a fusion protein with glutathione-S-transferase (GST). The allergenicity of the GST-Blo t 3 recombinant protein was evaluated by human IgE enzyme-linked immunoassay (ELISA) and skin pricks tests.Results:  The full length Blo t 3 gene had 1138 base pairs, including a 105-bp long 5′ nontranslated region, an ATG start codon at positions 106–108, and a stop codon TAA at positions 904–906, with an open reading frame coding for a polypeptide of 266 amino acids. Protein analysis revealed that it was a serine protease that had a prepro-mature structure that shared high sequence homology with group 3 dust mite allergens. The predicted molecular weight of the matured protein was approximately 23.8 kD with a theoretical pI of 8.87. The frequency of IgE reactivity of the recombinant protein showed up to 50% of IgE reactivity with mite allergic subjects but IgE titer was generally low.Conclusion: We had isolated and fully characterized the cDNA encoding an important B. tropicalis allergen that was highly homologous to Group 3 dust mite allergens and we proposed that it should be designated as Blo t 3. Its clinical importance was implicated by the high frequency of IgE reactivity with allergic sera.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1034/j.1398-9995.2003.00033.x
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  • 9
    Electronic Resource
    Electronic Resource
    Comparative allergenicity studies of native and recombinant Blomia tropicalis Paramyosin 
(Blo t 11) (2003)
    Oxford, UK : Munksgaard International Publishers
    Allergy 58 (2003), S. 0 
    Details
    ISSN: 1398-9995
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Background: The complementary DNA (cDNA) encoding for Blo t 11, a 102 kD allergen from Blomia tropicalis (Bt) was isolated, expressed and characterized previously. This study aimed to isolate the native Blo t 11 allergen and compare its allergenicity with the recombinant forms.Methods: Native Blo t 11 (nBlo t 11) was isolated from crude Bt extract by immuno-affinity chromatography, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot, and verified by MALDI-TOF MS. Recombinant full-length Blo t 11 (rFL-Blo t 11) and its immunodominant peptide (fD) were expressed as glutathione S-transferase (GST)-fusion proteins in Escherichia coli. Immunoglobulin E (IgE) reactivity of the Blo t 11 allergens were determined by enzyme-linked immunosorbent assay (ELISA) and skin prick test. The inhibition capacity of the nBlo t 11 against fD and vice versa was determined by absorption studies.Results: Affinity purified nBlo t 11 was susceptible to degradation with the major degraded product resolved at ∼66 kD. The nBlo t 11 was confirmed by immunoblot analysis and MALDI-TOF MS that generated 13 peptides with complete identity to the deduced amino acid sequence of Blo t 11. Comparative in vitro and in vivo allergenicity tests and the cross inhibition studies between the native and recombinant Blo t 11 showed that recombinant fD, but not the rFL-Blo t 11, has comparable IgE reactivity with the native counterpart.Conclusions: This comparative study confirmed that the recombinant peptide fD contains the main immunodominant region of Blo t 11. This recombinant peptide, instead of the full-length protein, is a good candidate for diagnostic and therapeutics development for mite allergy.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1034/j.1398-9995.2003.00106.x
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  • 10
    Electronic Resource
    Electronic Resource
    Loss of S-phase-dependent radioresistance in irs-1 cells exposed to X-rays
    Amsterdam : Elsevier
    Mutation Research/DNA Repair 314 (1994), S. 77-85 
    Details
    ISSN: 0921-8777
    Keywords: Cell cycle, radiosenstivity in ; Cell killing, radiation-induced ; Radiation-induced cell killing ; Radiosensitivity, in the cell cycle ; irs-1 cells
    Source: Elsevier Journal Backfiles on ScienceDirect 1907 - 2002
    Topics: Biology , Medicine
    Type of Medium: Electronic Resource
    URL: http://linkinghub.elsevier.com/retrieve/pii/0921-8777(94)90063-9
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