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Abstract
BackgroundDifferent adiponectin isoforms appear to be differentially involved in the pathogenesis of various diseases. The purpose of this study was to generate monoclonal antibodies (mAbs) specific to different adiponectin isoforms and investigate whether these mAbs have potential as therapeutic agents for such diseases.MethodsHybridoma cells producing monoclonal antibodies were generated and screened using enzyme-linked immunosorbent assay and Western blotting for the production of mAbs recognizing human adiponectin isoforms.ResultsThe mAb from hybridoma clone KH7–41 recognized both the middle molecular weight (MMW) (hexamer) and low molecular weight (LMW) (trimer) isoforms of adiponectin in human serum, whereas the KH7–33 mAb detected only MMW (hexamer) adiponectin. The KH4–8 clone recognized both the high molecular weight (HMW) (multimer) and MMW adiponectin isoforms. However, in mouse and rat sera, the abovementioned antibodies recognized only the MMW isomer. These mAbs also recognized adiponectin in various human tissues, such as lung, kidney, and adipose tissues, although the three mAbs had different staining intensities. The mAb from clone KH4–8 effectively inhibited increases in interleukin-6 (IL-6) and IL-8 expression in recombinant adiponectin-stimulated human osteoblasts and human umbilical vein endothelial cells. Also, the mAbs KH7–33 and KH4–8 significantly ameliorated rheumatic symptoms in a collagen-induced arthritis mouse model. This result suggests that these mAb treatments may ameliorate adiponectin-mediated inflammatory response.ConclusionsmAbs against human adiponectin isomers can potentially be developed as therapeutic antibodies to target specific detrimental isoforms of adiponectin while maintaining the functions of beneficial isoforms.
Highlights
Different adiponectin isoforms appear to be differentially involved in the pathogenesis of various diseases
Screening hybridoma clones producing Monoclonal antibody (mAb) against adiponectin isomers and mAb purification from culture supernatants Each hybridoma clone generated against recombinant adiponectin was screened by using enzyme-linked immunosorbent assay (ELISA) performed on its culture supernatant
15 mg of mAb was obtained from 500 mL of supernatant. mAb purity was determined by Coomassie staining after PAGE
Summary
Different adiponectin isoforms appear to be differentially involved in the pathogenesis of various diseases. Adipose tissue produces a variety of adipokines (leptin, adiponectin, resistin, and visfatin) as well as pro- and anti-inflammatory cytokines (tumor necrosis factor-alpha (TNF-α), interleukin-4 [IL-4] and IL-6, and others) [1]. We recently suggested that adiponectin may contribute to synovitis and joint destruction in RA by stimulating the expression of vascular endothelial growth factor (VEGF) and MMP-1 and MMP-13 in fibroblast-like synoviocytes (FLSs) to a greater extent than do pro-inflammatory mediators [14]. At physiological concentrations, adiponectin has been suggested to be more important than IL-1β in stimulating the production of mediators that drive synovitis and joint destruction in endothelial cells and osteoblasts [15]. We demonstrated that adiponectin in combination with IL-1β may have synergistic effects on the production of pro-inflammatory mediators during arthritic joint inflammation [16]. A recombinant adiponectin monomer produced in Escherichia coli was used in most of the above studies
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