Monoclonal antibody detection of Clostridium microcolonies directly on membrane used for milk filtration

Batina

et al. 1998

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Biochemical and Immunological Analyses of the Flagellin of Clostridium tyrobutyricum ATCC 25755

Batina

et al. 1998

“…(21E7-B12) has been used in an immunoenzymatic assay to count C. tyrobutyricum cells in milk following membrane filtration (3). However, this MoAb detects not only the C. tyrobutyricum strains, but also, Clostridium sporogenes (strain PAO) and Clostridium butyricum (strain PA2).…”

mentioning

confidence: 99%

Cloning and Sequencing of the Central Region of the Flagellin Gene from the Gram‐Positive Bacterium Clostridium tyrobutyricum ATCC 25755

Batina

et al. 1999

The purpose of this study was to sequence the central part of the coding region of the Clostridium tyrobutyricum fiagellin gene to improve the immunoenzymatic counting of cells after milk filtration. The coding region was amplified by PCR, and the amplified products were cloned. A DNA sequence analysis of positive clones gave us 1,131 nucleotides with a partial calculated flagellin molecular mass of 40,143 Da. The flagellar filament protein sequence exhibited high levels of homology to sequences of flagellin protein from other bacteria in both N- and C-terminal parts, but little homology in the central domain. A PCR-restriction fragment length polymorphism analysis of amplified C. tyrobutyricum flagellin gene products confirmed the variability of the central domain. The flagellin mRNA was determined to be 1.1 kb in size, which suggests a monocistronic mRNA. Furthermore, the deduced protein flagellin contains eleven potential N-glycosylation sites and one sequence rich in serine, which could be modified by O-glycosylation.

“…One of these antibodies, 21E7-B12 MoAb (IgG3 isotype), detects all C. tyrobutyricum strains as well as C. sporogenes (strain 545) and C. butyricum (Strain PA2). To detect these Clostridium in milk after membrane filtration, an immunoenzymatic test was developed using 21E7-B12 MoAb (3).…”

mentioning

confidence: 99%

Partial Analysis of the Flagellar Antigenic Determinant Recognized by a Monoclonal Antibody to Clostridium tyrobutyricum

Robreau

et al. 1998

In order to count Clostridium tyrobutyricum spores in milk after membrane filtration, murine 21E7-B12 monoclonal antibody was produced. Elution of the monoclonal antibody from this antigen, the flagellar filament protein, by carbohydrate ligands was used to study the epitope structure. A competitive elution of an anti-dextran monoclonal antibody by carbohydrate ligands served as a control in order to validate the immunological tool applied to flagellin epitope study. The carbohydrate moiety of flagellin contained D-glucose and N-acetyl-glucosamine in a molar ratio of 11:1 as determined by gas-liquid chromatography and 2 low-abundancy unidentified compounds. In ELISA, D-glucose and N-acetyl-glucosamine did not dissociate the antibody-flagellin complex contrary to maltose, maltotriose, maltotetraose and maltopentaose.The efficiency of elution increased from the dimer to the pentamer and became nil for maltohexaose and maltoheptaose. The fact that the hexamer and heptamer could not react with the 21E7-B12 monoclonal antibody could be explained by a drastic conformational change. The overall stretched maltopentaose switch to a helical-shaped maltoheptaose which could not fit the 21E7-B12 monoclonal antibody antigen-combining site. Thus, flagellin epitope may contain a(14)linked glucose residues plus either N-actyl-glucosamine or an unidentified compound that maintain it in an extended shape.Key words: Clostridium tyrobutyricum, Flagellin, Antibody, ELISA, Maltodextrin, EpitopeThe anaerobic Gram-positive, spore-forming bacteriurn Clostridium tyrobutyricum is believed to be the main organism responsible for the late spoiling of Emmental or similar cheeses by the production of gas (2). In order to detect this bacterium in milk, we produced murine monoclonal antibodies (MoAb) raised against the outer cell wall (18). One of these antibodies, 21E7-B12 MoAb (IgG3 isotype), detects all C. tyrobutyricum strains as well as C. sporogenes (strain 545) and C. butyricum (Strain PA2). To detect these Clostridium in milk after membrane filtration, an immunoenzymatic test was developed using 21E7-B12 MoAb (3).In a former study, we showed that the 21E7-B12 MoAb epitope can be eluted from flagellin. The MoAb reactivity is abolished by periodate oxidation, suggesting the participation of carbohydrate in the antigenic determinant (Arnold et al, submitted for publication).The aim of this study is to elucidate the structure of the epitope using the reversible association of the 21E7-B12 MoAb to its determinant. Indeed, according to the key-lock model, parts of a ligand could compete with this full ligand for the binding site of a receptor. Thus, polysaccharide-associated lectins are eluted by monosaccharides (11) and interactions between antibodies and polysaccharides could be specifically disrupted by monosaccharides (16) and oligosaccharides (14,20). Monosaccharides bind to the combining area of antibody and