Generation of high-affinity monoclonal antibodies of IgG class against native β-d-glucans from basidiomycete mushrooms

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.