Richard Dickason - Academia.edu (original) (raw)
Papers by Richard Dickason
JOURNAL OF THE GEORGIAN GEOPHYSICAL SOCIETY, 2015
The cytokine interleukin-5 (IL-5) specifically induces the maturation and activation of eosinophi... more The cytokine interleukin-5 (IL-5) specifically induces the maturation and activation of eosinophils, which are important in normal host defense and allergic disease. Crucial to the development of specific modulators of IL-5 is a comprehensive understanding of the interaction of IL-5 with its receptor complex, which consists of alpha and beta subunits. IL-5 is a unique member of the short chain subfamily of helical bundle cytokines. IL-5 is a glycosylated, disulfide-linked, interdigitating homodimer, which contains two canonical helical bundle motifs. This distinguishes IL-5 from the other subfamily members, which fold unimolecularly into a single helical bundle. I undertook a systematic approach to identify functional domains on IL-5 and solve the enigma surrounding its unique dimeric configuration. A library of neutralizing monoclonal antibodies (mAb) was generated and characterized. Two noncompetitive anti-IL-5 mAb were used to develop a highly sensitive sandwich ELISA capable of quantitating IL-5 in biological fluids. The anti-IL-5 mAb were also used to map functional domains on IL-5. These results predicted regions which engaged the IL-5 receptor. Each functional domain was defined twice on the IL-5 dimer. However, the functional significance of each domain pair could not be established. To determine whether a single helical bundle of IL-5 was independently functional or if IL-5 required both helical bundles for bioactivity, I created a novel monomer, which folded unimolecularly into a single IL-5 helical bundle motif. Generation of this cytokine, designated mono5, relied on the hypothesis that the shortened loop 3 of IL-5 physically restricts unimolecular completion of a single helical bundle. Structural and functional analyses demonstrated that mono5 was indeed a monomer that possessed full biological activity comparable to native IL-5 at saturating concentrations. Although slight tertiary structural differences were identified, the full functional capacity of mono5 demonstrated for the first time that, all structural features necessary for IL-5 biological activity are contained within a single helical bundle motif. The creation of biologically active mono5 revolutionizes the understanding of IL-5 functional structure. Furthermore, my loop 3 hypothesis is applicable to the entire family of helical bundle cytokines and may lead to the creation of other novel proteins
Journal of Immunological Methods, 1995
A recently dcvelqxd E. co/i thioredoxin 0i-x) gene fusion expression system has circumvented the ... more A recently dcvelqxd E. co/i thioredoxin 0i-x) gene fusion expression system has circumvented the diffxulties aswciated with iociesion bady fommtion. Although ample quautities of soluble recombinant protein can be expressed using this system. uo universal means of quantifying or purifying the fusion product exists. To facilitate the study of Tnr fusion proteins, anti-E. coil Tn monocloual antibodies (mAb) were generated. Two distinct Tnr epitupes were defined by competitive ELI%. Etoth mAb were capable of detecting Trx fusion proteins by sandwich ELISA, and by immunoblot analysis under reducing and nun-reducing conditions. In addition, these mAb enabled purification of Trx fusion proteins by immunoprecipitation, as well as affinity chromatography. This report provides the first dcwxiption of anti-Trx antibodies. Thhese reagents rcpweut a major advance in the isalation and analysis of prokatyote expressed recombinaut Trx fusion proteins.
Journal of Allergy and Clinical Immunology, 1996
Journal of Allergy and Clinical Immunology, 1996
Journal of Allergy and Clinical Immunology, 1996
Journal of Allergy and Clinical Immunology, 1997
Background: The late-phase allergic reaction is an eosinophilic inflammatory response that begins... more Background: The late-phase allergic reaction is an eosinophilic inflammatory response that begins several hours after allergen exposure, may persist for 24 hours, and is an important pathogenic mechanism in allergic disease. Objective: Cultured naive human mast cells were used to investigate whether mast cells are a direct source of the eosinophil-promoting cytokines IL-5, IL-3, and granulocyte-macrophage colony-stimulating factor (GM-CSF). Methods: Naive human mast cells were derived from bone marrow mononuclear cells cultured in the presence of stemcell factor. Cytokine message and protein productiolTin response to high-affinity IgE receptor ligation of cultured mast cells were measured by semiquantitative polymerase chain reaction and ELISA, respectively. Results: IL-5, IL-3, and GM-CSF messenger RNA increased within 2 hours of mast cell activation, with IL-5 and GM-CSF message remaining elevated for 24 hours, whereas IL-3 mRNA rapidly declined. IL-5 and GM-CSF protein were measurable 4 to 6 hours after stimulation and peaked by 24 and 12 hours, respectively. IL-3 protein was not detectable. Conclusion: These findings demonstrate that naive mast cells do not constitutively produce IL-5 or GM-CSF protein but are a major source of these eosinophilotropic cytokines on high-affinity IgE receptor ligation. (J Allergy Clin Immunol 1997;99:508-14.) High-affinity IgE receptor (FceRI) ligation results in mast cell activation, degranulation, and release of preformed and newly synthesized mediators?, 2 In vivo, this results in an immediate hypersensitivity reaction, which is often followed several hours later by a late-phase allergic reaction (LPAR) that may persist for more than 24 hours?-7 The LPAR is characterized by a leukocytic infiltrate, predominantly comprised of eosinophils and
Immunology, 1997
Interleukin-13 (IL-13) shares many, but not all, of the properties of the prototypic T-helper typ... more Interleukin-13 (IL-13) shares many, but not all, of the properties of the prototypic T-helper type 2 (Th2) cytokine IL-4, but its role in allergen-driven T-cell responses remains poorly defined. We hypothesized that allergen stimulation of peripheral blood T cells from patients with atopic disease compared with non-atopic controls results in elevated IL-13 synthesis in the context of a 'Th2-type' pattern. Freshly isolated peripheral blood mononuclear cells (PBMC) obtained from sensitized atopic patients with allergic disease, and non-atopic control subjects, were cultured with the allergens Phleum pratense (Timothy grass pollen) or Dermatophagoides pteronyssinus (house dust mite) and the non-allergenic recall antigen Mycobacterium tuberculosis purified protein derivative (PPD). Supernatant concentrations of IL-13, along with IL-5 and interferon-y (IFN-y) (Th2-and Thl-type cytokines, respectively) were determined by enzyme-linked immunosorbent assay (ELISA). Allergen-induced IL-13 and IL-5 production by T cells from patients with allergic disease was markedly elevated (P =00075 and P=0O0004, respectively) compared with non-atopic controls, whereas IFN-y production was not significantly different. In contrast to allergen, the prototypic Thl-type antigen M tuberculosis PPD induced an excess of IFN-y over IL-13 and IL-5 production, and absolute concentrations of cytokines were not affected by the presence or absence of atopic disease. Addition of exogenous recombinant IFN-y or IL-12, cytokines known to inhibit Th2-type responses, significantly inhibited allergen-driven production of both IL-13 and IL-5, but not T-cell proliferation, whereas exogenous IL-4 did not significantly affect production of IL-13 or IL-5. We conclude that allergen-specific T cells from atopic subjects secrete elevated quantities of IL-13 compared with non-atopic controls, in the context of a Th2-type pattern of cytokine production.
European Journal of Immunology, 1995
Specific eosinophil accumulation and activation within the asthmatic bronchial mucosa are thought... more Specific eosinophil accumulation and activation within the asthmatic bronchial mucosa are thought to occur at least partly through the actions of cytokines, including interleukin (IL)-5, IL-3 and granulocyte/macrophage colony-stimulating factor (GM-CSF). Although mRNA encoding some of these cytokines has been demonstrated in bronchoalveolar lavage (BAL) fluid cells and bronchial biopsies from asthmatics, it has yet to be established whether these cells produce the translated products and whether expression is associated with CD4+ T helper or CD8+ cytotoxic T cells. We addressed this problem by raising polyclonal CD4+ and CD8+ T cell lines from the BAL fluid of six atopic asthmatics, five atopic non-asthmatics and seven non-atopic non-asthmatic controls. BAL fluid cells obtained at fiberoptic bronchoscopy were depleted of adherent cells, and then T lymphocytes expanded by stimulation with monoclonal anti-CD3 antibody and recombinant human IL-2. When lymphocytes had expanded to sufficient numbers, CD4+ and CD8+ cells were separated by positive selection with magnetic beads coated with anti-CD4 or anti-CD8 monoclonal antibodies and further expanded. Cytokine secretion by standardized cell numbers was measured by enzyme-linked immunosorbent assays. BAL CD4+ T cell lines from the asthmatics secreted significantly elevated quantities of both IL-5 and GM-CSF as compared with lines from the atopic and non-atopic controls (p = 0.023-0.003). In contrast, IL-3 secretion did not significantly differ between the groups. In some subjects, CD8+ T cell lines also secreted significant quantities of these cytokines and there was a trend for IL-5 secretion by these cells to be higher in asthmatics than non-atopic controls (p = 0.035). These data are consistent with the hypothesis that activated T lymphocytes from asthmatics, particularly of the CD4+ subset, are predisposed to release elevated quantities of cytokines relevant to the accumulation and activation of eosinophils.
American Journal of Respiratory and Critical Care Medicine, 1998
Journal of Allergy and Clinical Immunology, 1997
Background: IL-5-producing allergen-specific T cells are thought to play a prominent role in the ... more Background: IL-5-producing allergen-specific T cells are thought to play a prominent role in the pathogenesis of allergic inflammation. We hypothesized that T cell allergen-driven IL-5 synthesis is elevated in patients with atopic disease as compared with that in atopic patients free of disease and nonatopic control subjects. Objectives: The purpose of this study was to compare IL-5 and interferon-~, (IFN-~) secretion and proliferation by peripheral blood T cells from sensitized atopic patients with asthma, rhinitis, and no symptoms and from nonatopic control subjects in response to the allergen Dermatophagoides pteronyssinus (Der p) and the control recall antigen Mycobacterium tuberculosis purified protein derivative (PPD). Methods: To measure allergen-induced IL-5 production and proliferation, we developed a short-term culture technique that required a single antigenic stimulation of freshly isolated peripheral blood mononuclear cells (PBMC). With this technique, we measured Der p-and PPD-induced IL-5 production and proliferation in PBMC from atopic patients with asthma who were allergic to Der p, atopic patients with rhinitis, atopic patients with no symptoms, and a group of nonatopic normal control subjects. In four experiments, CD4+ or CD8+ T cells were depleted from PBMC to confirm that IL-5 synthesis was T cell dependent. Results: T cell IL-5 production, but not IFN-T production, in response to Der p was elevated in atopic patients with asthma and atopic patients with rhinitis compared with findings in atopic patients with no symptoms or nonatopic control subjects. IL-5 production was abrogated by depletion of CD4+, but not CD8+, T ceils. In subjects with asthma, allergen-driven IL-5 production correlated with bronchial hyperreactivity. Allergen-induced proliferation was also
Cytokine, 1994
Interleukin 5 (IL-S) is a homodimeric cytokine arranged in a head-to-tail configuration covalentl... more Interleukin 5 (IL-S) is a homodimeric cytokine arranged in a head-to-tail configuration covalently linked by two disulfide bonds. IL-5 has pleiotropic effects on murine and human leukocytes and has been implicated in the pathogenesis of many inflammatory disorders. To facilitate the study of functionally relevant IL-5 domains involved in receptor binding and to develop a highly sensitive and specific ELISA capable of detecting IL-5 in biological fluids, a library of murine anti-human IL-5 (hIL-5) mAb was generated to baculovirus expressed recombinant hIL-5 (rhIL-5). Fifteen subclones of seven hybridomas were characterized. All mAb bound hIL-5, but not murine IL-5 (mIL-5), and neutralized hIL-5 biological activity in the BClr proliferation assay. By competitive ELISA, the mAb were divided into two binding groups. Utilizing comparative'analysis with TRFK-5, a rat anti-mIL-5 mAb crossreactive with hIL-5, at least three hIL-5 neutralizing epitopes were defined. By ELISA and Western analysis, each epitope was shown to be present as a conformationally identical pair on the hIL-5 dimer. Various combinations of mAb in sandwich ELISA were used to predict the relative proximity of each epitope pair. Utilizing mAb binding characteristics, highly sensitive and specific sandwich ELISA were developed with a minimum detection limit of 6.25 pg hILJ/ml (P < 0.05). Quantitation of hIL-5 in both serum and bronchoalveolar lavage (BAL) fluid demonstrated the utility of these anti-hIL-5 mAb for investigating the role of hIL-5 in inflammation. These mAb should also serve as useful reagents for epitope mapping of functional hIL-5 domains. IL-5 is a homodimeric cytokine secreted predominantly by activated Th2 lymphocytes.1-4 The homodimer is covalently linked by two disulfide bonds in a head-to-tail configuration.5-7 Dimer formation is essential for biological activity as monomeric IL-5 does not bind to its receptor.5,6,s'9 IL-5 is heterogeneously glycosylated with both N-and O-linked residues. Although these residues may provide some thermal stability,' they are not required for biological activity. ",il Murine IL-5 and hIL-5 share 73% amino
JOURNAL OF THE GEORGIAN GEOPHYSICAL SOCIETY, 2015
The cytokine interleukin-5 (IL-5) specifically induces the maturation and activation of eosinophi... more The cytokine interleukin-5 (IL-5) specifically induces the maturation and activation of eosinophils, which are important in normal host defense and allergic disease. Crucial to the development of specific modulators of IL-5 is a comprehensive understanding of the interaction of IL-5 with its receptor complex, which consists of alpha and beta subunits. IL-5 is a unique member of the short chain subfamily of helical bundle cytokines. IL-5 is a glycosylated, disulfide-linked, interdigitating homodimer, which contains two canonical helical bundle motifs. This distinguishes IL-5 from the other subfamily members, which fold unimolecularly into a single helical bundle. I undertook a systematic approach to identify functional domains on IL-5 and solve the enigma surrounding its unique dimeric configuration. A library of neutralizing monoclonal antibodies (mAb) was generated and characterized. Two noncompetitive anti-IL-5 mAb were used to develop a highly sensitive sandwich ELISA capable of quantitating IL-5 in biological fluids. The anti-IL-5 mAb were also used to map functional domains on IL-5. These results predicted regions which engaged the IL-5 receptor. Each functional domain was defined twice on the IL-5 dimer. However, the functional significance of each domain pair could not be established. To determine whether a single helical bundle of IL-5 was independently functional or if IL-5 required both helical bundles for bioactivity, I created a novel monomer, which folded unimolecularly into a single IL-5 helical bundle motif. Generation of this cytokine, designated mono5, relied on the hypothesis that the shortened loop 3 of IL-5 physically restricts unimolecular completion of a single helical bundle. Structural and functional analyses demonstrated that mono5 was indeed a monomer that possessed full biological activity comparable to native IL-5 at saturating concentrations. Although slight tertiary structural differences were identified, the full functional capacity of mono5 demonstrated for the first time that, all structural features necessary for IL-5 biological activity are contained within a single helical bundle motif. The creation of biologically active mono5 revolutionizes the understanding of IL-5 functional structure. Furthermore, my loop 3 hypothesis is applicable to the entire family of helical bundle cytokines and may lead to the creation of other novel proteins
Journal of Immunological Methods, 1995
A recently dcvelqxd E. co/i thioredoxin 0i-x) gene fusion expression system has circumvented the ... more A recently dcvelqxd E. co/i thioredoxin 0i-x) gene fusion expression system has circumvented the diffxulties aswciated with iociesion bady fommtion. Although ample quautities of soluble recombinant protein can be expressed using this system. uo universal means of quantifying or purifying the fusion product exists. To facilitate the study of Tnr fusion proteins, anti-E. coil Tn monocloual antibodies (mAb) were generated. Two distinct Tnr epitupes were defined by competitive ELI%. Etoth mAb were capable of detecting Trx fusion proteins by sandwich ELISA, and by immunoblot analysis under reducing and nun-reducing conditions. In addition, these mAb enabled purification of Trx fusion proteins by immunoprecipitation, as well as affinity chromatography. This report provides the first dcwxiption of anti-Trx antibodies. Thhese reagents rcpweut a major advance in the isalation and analysis of prokatyote expressed recombinaut Trx fusion proteins.
Journal of Allergy and Clinical Immunology, 1996
Journal of Allergy and Clinical Immunology, 1996
Journal of Allergy and Clinical Immunology, 1996
Journal of Allergy and Clinical Immunology, 1997
Background: The late-phase allergic reaction is an eosinophilic inflammatory response that begins... more Background: The late-phase allergic reaction is an eosinophilic inflammatory response that begins several hours after allergen exposure, may persist for 24 hours, and is an important pathogenic mechanism in allergic disease. Objective: Cultured naive human mast cells were used to investigate whether mast cells are a direct source of the eosinophil-promoting cytokines IL-5, IL-3, and granulocyte-macrophage colony-stimulating factor (GM-CSF). Methods: Naive human mast cells were derived from bone marrow mononuclear cells cultured in the presence of stemcell factor. Cytokine message and protein productiolTin response to high-affinity IgE receptor ligation of cultured mast cells were measured by semiquantitative polymerase chain reaction and ELISA, respectively. Results: IL-5, IL-3, and GM-CSF messenger RNA increased within 2 hours of mast cell activation, with IL-5 and GM-CSF message remaining elevated for 24 hours, whereas IL-3 mRNA rapidly declined. IL-5 and GM-CSF protein were measurable 4 to 6 hours after stimulation and peaked by 24 and 12 hours, respectively. IL-3 protein was not detectable. Conclusion: These findings demonstrate that naive mast cells do not constitutively produce IL-5 or GM-CSF protein but are a major source of these eosinophilotropic cytokines on high-affinity IgE receptor ligation. (J Allergy Clin Immunol 1997;99:508-14.) High-affinity IgE receptor (FceRI) ligation results in mast cell activation, degranulation, and release of preformed and newly synthesized mediators?, 2 In vivo, this results in an immediate hypersensitivity reaction, which is often followed several hours later by a late-phase allergic reaction (LPAR) that may persist for more than 24 hours?-7 The LPAR is characterized by a leukocytic infiltrate, predominantly comprised of eosinophils and
Immunology, 1997
Interleukin-13 (IL-13) shares many, but not all, of the properties of the prototypic T-helper typ... more Interleukin-13 (IL-13) shares many, but not all, of the properties of the prototypic T-helper type 2 (Th2) cytokine IL-4, but its role in allergen-driven T-cell responses remains poorly defined. We hypothesized that allergen stimulation of peripheral blood T cells from patients with atopic disease compared with non-atopic controls results in elevated IL-13 synthesis in the context of a 'Th2-type' pattern. Freshly isolated peripheral blood mononuclear cells (PBMC) obtained from sensitized atopic patients with allergic disease, and non-atopic control subjects, were cultured with the allergens Phleum pratense (Timothy grass pollen) or Dermatophagoides pteronyssinus (house dust mite) and the non-allergenic recall antigen Mycobacterium tuberculosis purified protein derivative (PPD). Supernatant concentrations of IL-13, along with IL-5 and interferon-y (IFN-y) (Th2-and Thl-type cytokines, respectively) were determined by enzyme-linked immunosorbent assay (ELISA). Allergen-induced IL-13 and IL-5 production by T cells from patients with allergic disease was markedly elevated (P =00075 and P=0O0004, respectively) compared with non-atopic controls, whereas IFN-y production was not significantly different. In contrast to allergen, the prototypic Thl-type antigen M tuberculosis PPD induced an excess of IFN-y over IL-13 and IL-5 production, and absolute concentrations of cytokines were not affected by the presence or absence of atopic disease. Addition of exogenous recombinant IFN-y or IL-12, cytokines known to inhibit Th2-type responses, significantly inhibited allergen-driven production of both IL-13 and IL-5, but not T-cell proliferation, whereas exogenous IL-4 did not significantly affect production of IL-13 or IL-5. We conclude that allergen-specific T cells from atopic subjects secrete elevated quantities of IL-13 compared with non-atopic controls, in the context of a Th2-type pattern of cytokine production.
European Journal of Immunology, 1995
Specific eosinophil accumulation and activation within the asthmatic bronchial mucosa are thought... more Specific eosinophil accumulation and activation within the asthmatic bronchial mucosa are thought to occur at least partly through the actions of cytokines, including interleukin (IL)-5, IL-3 and granulocyte/macrophage colony-stimulating factor (GM-CSF). Although mRNA encoding some of these cytokines has been demonstrated in bronchoalveolar lavage (BAL) fluid cells and bronchial biopsies from asthmatics, it has yet to be established whether these cells produce the translated products and whether expression is associated with CD4+ T helper or CD8+ cytotoxic T cells. We addressed this problem by raising polyclonal CD4+ and CD8+ T cell lines from the BAL fluid of six atopic asthmatics, five atopic non-asthmatics and seven non-atopic non-asthmatic controls. BAL fluid cells obtained at fiberoptic bronchoscopy were depleted of adherent cells, and then T lymphocytes expanded by stimulation with monoclonal anti-CD3 antibody and recombinant human IL-2. When lymphocytes had expanded to sufficient numbers, CD4+ and CD8+ cells were separated by positive selection with magnetic beads coated with anti-CD4 or anti-CD8 monoclonal antibodies and further expanded. Cytokine secretion by standardized cell numbers was measured by enzyme-linked immunosorbent assays. BAL CD4+ T cell lines from the asthmatics secreted significantly elevated quantities of both IL-5 and GM-CSF as compared with lines from the atopic and non-atopic controls (p = 0.023-0.003). In contrast, IL-3 secretion did not significantly differ between the groups. In some subjects, CD8+ T cell lines also secreted significant quantities of these cytokines and there was a trend for IL-5 secretion by these cells to be higher in asthmatics than non-atopic controls (p = 0.035). These data are consistent with the hypothesis that activated T lymphocytes from asthmatics, particularly of the CD4+ subset, are predisposed to release elevated quantities of cytokines relevant to the accumulation and activation of eosinophils.
American Journal of Respiratory and Critical Care Medicine, 1998
Journal of Allergy and Clinical Immunology, 1997
Background: IL-5-producing allergen-specific T cells are thought to play a prominent role in the ... more Background: IL-5-producing allergen-specific T cells are thought to play a prominent role in the pathogenesis of allergic inflammation. We hypothesized that T cell allergen-driven IL-5 synthesis is elevated in patients with atopic disease as compared with that in atopic patients free of disease and nonatopic control subjects. Objectives: The purpose of this study was to compare IL-5 and interferon-~, (IFN-~) secretion and proliferation by peripheral blood T cells from sensitized atopic patients with asthma, rhinitis, and no symptoms and from nonatopic control subjects in response to the allergen Dermatophagoides pteronyssinus (Der p) and the control recall antigen Mycobacterium tuberculosis purified protein derivative (PPD). Methods: To measure allergen-induced IL-5 production and proliferation, we developed a short-term culture technique that required a single antigenic stimulation of freshly isolated peripheral blood mononuclear cells (PBMC). With this technique, we measured Der p-and PPD-induced IL-5 production and proliferation in PBMC from atopic patients with asthma who were allergic to Der p, atopic patients with rhinitis, atopic patients with no symptoms, and a group of nonatopic normal control subjects. In four experiments, CD4+ or CD8+ T cells were depleted from PBMC to confirm that IL-5 synthesis was T cell dependent. Results: T cell IL-5 production, but not IFN-T production, in response to Der p was elevated in atopic patients with asthma and atopic patients with rhinitis compared with findings in atopic patients with no symptoms or nonatopic control subjects. IL-5 production was abrogated by depletion of CD4+, but not CD8+, T ceils. In subjects with asthma, allergen-driven IL-5 production correlated with bronchial hyperreactivity. Allergen-induced proliferation was also
Cytokine, 1994
Interleukin 5 (IL-S) is a homodimeric cytokine arranged in a head-to-tail configuration covalentl... more Interleukin 5 (IL-S) is a homodimeric cytokine arranged in a head-to-tail configuration covalently linked by two disulfide bonds. IL-5 has pleiotropic effects on murine and human leukocytes and has been implicated in the pathogenesis of many inflammatory disorders. To facilitate the study of functionally relevant IL-5 domains involved in receptor binding and to develop a highly sensitive and specific ELISA capable of detecting IL-5 in biological fluids, a library of murine anti-human IL-5 (hIL-5) mAb was generated to baculovirus expressed recombinant hIL-5 (rhIL-5). Fifteen subclones of seven hybridomas were characterized. All mAb bound hIL-5, but not murine IL-5 (mIL-5), and neutralized hIL-5 biological activity in the BClr proliferation assay. By competitive ELISA, the mAb were divided into two binding groups. Utilizing comparative'analysis with TRFK-5, a rat anti-mIL-5 mAb crossreactive with hIL-5, at least three hIL-5 neutralizing epitopes were defined. By ELISA and Western analysis, each epitope was shown to be present as a conformationally identical pair on the hIL-5 dimer. Various combinations of mAb in sandwich ELISA were used to predict the relative proximity of each epitope pair. Utilizing mAb binding characteristics, highly sensitive and specific sandwich ELISA were developed with a minimum detection limit of 6.25 pg hILJ/ml (P < 0.05). Quantitation of hIL-5 in both serum and bronchoalveolar lavage (BAL) fluid demonstrated the utility of these anti-hIL-5 mAb for investigating the role of hIL-5 in inflammation. These mAb should also serve as useful reagents for epitope mapping of functional hIL-5 domains. IL-5 is a homodimeric cytokine secreted predominantly by activated Th2 lymphocytes.1-4 The homodimer is covalently linked by two disulfide bonds in a head-to-tail configuration.5-7 Dimer formation is essential for biological activity as monomeric IL-5 does not bind to its receptor.5,6,s'9 IL-5 is heterogeneously glycosylated with both N-and O-linked residues. Although these residues may provide some thermal stability,' they are not required for biological activity. ",il Murine IL-5 and hIL-5 share 73% amino