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

Batina, P.; Arnold, Françoise; Bédouet, Laurent; Robreau, Georges; Talbot, F.; Malcoste, R.

doi:10.1111/j.1365-2672.1997.tb02871.x

Cited by 7 publications

(2 citation statements)

References 13 publications

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“…The prerequisite for all these techniques was that the samples were treated so that bacteria remained sufficiently intact to keep their DNA intracellular. In one version of these methods, the bacteria were visualized with a monoclonal antibody conjugated to alkaline phosphatase (6). However, none of these studies tested mastitic milk samples, which may contain solid particles that render filtration of the sample difficult.…”

Section: Discussionmentioning

confidence: 99%

Rapid Method for Detection of Gram-Positive and -Negative Bacteria in Milk from Cows with Moderate or Severe Clinical Mastitis

et al. 2001

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A rapid method for demonstration of gram-positive and gram-negative bacteria in milk is described. The technique is based on dilution of the sample in a medium, followed by filtration through a porous polysulfone membrane with a pore size retaining and concentrating bacteria from the sample. The bacteria concentrated on the surface of the membrane are stained with a cationic dye (toluidine blue) that can be visualized by the naked eye. After staining, the membrane is treated with ethanol-acetic acid (pH 2.8 to 3.0), which causes decolorization of gram-negative bacteria, whereas gram-positive bacteria retain the stain. The method does not require heat fixation, electrical power, microscopic examination, or specially trained personnel. The time needed to perform the test is approximately 5 min. The technique was applied to artificially infected milk and milk from cows with moderate or severe clinical mastitis for detection and differentiation of bacteria. The sensitivity of the filtration method was 92 and 100% for gram-positive and gram-negative bacteria, respectively, compared with traditional bacteriological culture of milk samples. The detection limit was 5 ؋ 10 6 CFU/ml for Staphylococcus aureus and 1 ؋ 10 6 CFU/ml for Escherichia coli in spiked milk samples. The overall specificity of the method was 86%. This diagnostic method can provide on-site guidance to the veterinarian to optimize use of antibiotics in mastitis therapy.Gram staining of bacteria segregates bacteria into two categories based on cell wall composition. The cell wall of grampositive bacteria consists of a cytoplasmic membrane, many polymeric layers of peptidoglycan connected by amino acid bridges, and a variable outer layer called the capsule (12). Gram-negative bacteria possess a bilayered outer membrane, a thin peptidoglycan layer, and a bilayered plasma membrane (7).The Gram stain procedure, developed by Hans Christian Gram in 1884, relies on the differential cell wall staining properties of gram-positive and -negative bacteria. It remains today as a fundamental step in bacterial classification. The reaction is based on the retention of a dye (crystal violet complexed with iodine) within the cell wall of bacteria. The dye is retained in gram-positive organisms following an alcohol wash. The gramnegative bacteria, which lose the dye following the alcohol wash, may subsequently be counterstained with carbolfuchsin or safranin.Alternative bacterial staining methods have been developed using fluorescein-labeled wheat germ agglutinin (22) or rhodamine 123 (a lipophilic cationic dye) (1, 9, 21). Furthermore, a staining technique for unfixed organisms in suspension has been developed (14) employing two fluorescent nucleic acid binding dyes, hexidium iodide and SYTO 13. These alternative staining methods are more expensive than traditional Gram staining and require expensive instruments such as epifluorescence microscopes or flow cytometers.A method of membrane filtration of bacteria in raw milk, followed by staining with a fluorescent dye, e.g....

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Section: Discussionmentioning

confidence: 99%

Rapid Method for Detection of Gram-Positive and -Negative Bacteria in Milk from Cows with Moderate or Severe Clinical Mastitis

et al. 2001

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“…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).…”

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

Bédouet

Arnold

Robreau

et al. 1998

Microbiology and Immunology

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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

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Influence of Membrane Separation Processes on Food Bioactives

Razola-Díaz

Ochando-Pulido²,

Guerra‐Hernández³

et al. 2022

Food Bioactive Ingredients

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

Cited by 7 publications

References 13 publications

Rapid Method for Detection of Gram-Positive and -Negative Bacteria in Milk from Cows with Moderate or Severe Clinical Mastitis

Rapid Method for Detection of Gram-Positive and -Negative Bacteria in Milk from Cows with Moderate or Severe Clinical Mastitis

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

Influence of Membrane Separation Processes on Food Bioactives

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