PARTIAL ANALYSIS OF THE FLAGELLAR ANTIGENIC DETERMINANT RECOGNIZED BYA MONOCLONAL-ANTIBODY TO CLOSTRIDIUM-TYROBUTYRICUM

In order to count Clostridium tyrobutyricum spores in milk after membr ane filtration, murine 21E7-B12 monoclonal antibody was produced, Elut ion of the monoclonal antibody from this antigen, the flagellar filame nt protein, by carbohydrate ligands was used to study the epitope stru cture, A competitive elution of an anti-dextran monoclonal antibody by carbohydrate ligands served as a control in order to validate the imm unological tool applied to flagellin epitope study, The carbohydrate m oiety of flagellin contained D-glucose and N-acetyl-glucosamine in a m olar ratio of 11:1 as determined by gas-liquid chromatography and 2 lo w-abundancy unidentified compounds, In ELISA, D-glucose and N-acetyl-g lucosamine did not dissociate the antibody-flagellin complex contrary to maltose, maltotriose, maltotetraose and maltopentaose. The efficien cy of elution increased from the dimer to the pentamer and became nil for maltohexaose and maltoheptaose, The fact that the hexamer and hept amer could not react with the 21E7-B12 monoclonal antibody could be ex plained by a drastic conformational change, The overall stretched malt opentaose switch to a helical-shaped maltoheptaose which could not fit the 21E7-B12 monoclonal antibody antigen-combining site, Thus, flagel lin epitope may contain alpha(1-->4) linked glucose residues plus eith er N-actyl-glucosamine or an unidentified compound that maintain it in an extended shape.