Generation of high-affinity monoclonal antibodies of IgG class against native β-d-glucans from basidiomycete mushrooms. Issue 2 (February 2016)

• Record Type: Journal Article
• Authors: C. Semedo, Magda; Karmali, Amin; Martins, Sónia; Fonseca, Luís
• Is Part Of: Process biochemistry. Volume 51:Issue 2(2016:Feb.)
• Page Start: 333
• Page End: 342
• Publication Date: 2016-02
• Subjects: BRM biological response modifiers -- EBG extracellular β-d-glucan -- ELISA enzyme-linked immunosorbent assay -- HAT hypoxanthine–aminopterin–thymidine -- IBG intracellular β-d-glucans -- IEF isoelectric focusing -- Mabs monoclonal antibodies -- PAGE polyacrylamide gel electrophoresis -- PBS phosphate-buffered saline -- PDA potato dextrose agar -- PEG polyethylene glycol -- PR-Mabs polyol-responsive MAbs -- SDS sodium dodecyl sulfate -- MES 2-(N-morpholino) ethanesulfonic acid; Hybridoma technology -- Mabs of IgG class -- Extracellular β-d-glucans from Pleurotus ostreatus -- Basidiomycete mushrooms -- Specificity and cross-reactivity -- Epitope analysis of Mabs; Biochemical engineering -- Periodicals; Biotechnology -- Periodicals; Biochemistry -- periodicals; Biotechnology -- periodicals; Chemical Engineering -- periodicals; Génie biochimique -- Périodiques; Biotechnologie -- Périodiques; Biochemical engineering; Biotechnology; Periodicals; 660.63
• DOI: 10.1016/j.procbio.2015.11.029
• Languages: English
• ISSNs: 1359-5113

Abstract

Graphical abstract: Highlights: Novel monoclonal antibodies (Mabs) were raised against β-glucans from Pleurotus ostreatus. Novel polyol-responsive Mabs were raised against native β-glucans from basidiomycete mushrooms. Mabs recognized a conformation-sensitive epitope on several β-d -glucan molecules. Mabs exhibited high-affinity constant ( K A ) for β-d -glucans from mushroom strains. Mabs can be used for the assay of β-d -glucans in several basidiomycete strains by enzyme-linked immunosorbent assay (ELISA). Abstract: β-d -glucans from basidiomycete strains are powerful immunomodulatory agents in several clinical conditions. Therefore, their assay, purification and characterization are of great interest to understand their structure-function relationship. Hybridoma cell fusion was used to raise monoclonal antibodies (Mabs) against extracellular β-d -glucans (EBGs) from Pleurotus ostreatus . Two of the hybridoma clones (1E6_1E8_B5 and 3E8_3B4) secreting Mabs against EBGs were selected. This hybridoma cell line secreted Mabs of the IgG class which were then purified by hydroxyapatite chromatography to apparent homogeneity on native and SDS-PAGE. Mabs secreted by 1E6_1E8_B5 clone were found to recognize a common epitope on several β-d -glucans from different basidiomycete strains. This Mab exhibited high affinity constant ( K A ) for β-d -glucans from several mushroom strains in the range of 3.20 × 10 9 ± 3.32 × 10 3 –1.51 × 10 13 ± 3.58 × 10 7 L/mol. Moreover, they reacted to some heat-treated β-d -glucans in a different mode when compared with the native forms; these data suggest that this Mab binds to a conformational epitope on the β-d -glucan molecule. The epitope-binding studies of Mabs obtained from 1E6_1E8_B5 and 3E8_3B4 revealed that the Mabs bind to the same epitope on some β-d -glucans and to different epitopes in other antigen molecules. Therefore, these Mabs can be used to assay for β-d -glucan from basidiomycete mushrooms.