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

The normal procedure for bacterial colony detection requires a nitrocellulose transfer step after membrane filtration and culture to prevent the development of a high background during the immunodetection. In this paper, we describe a modification of the basic protocol that omits the transfer step a...

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Bibliographic Details
Published in:Journal of applied microbiology 1997-05, Vol.82 (5), p.619-624
Main Authors: BATINA, P, ARNOLD, F, BEDOUET, L, ROBREAU, G, TALBOT, F, MALCOSTE, R
Format: Article
Language:English
Subjects:
Animals
Antibodies, Bacterial - analysis
Antibodies, Bacterial - immunology
Antibodies, Monoclonal - analysis
Antibodies, Monoclonal - immunology
Antigens, Bacterial - immunology
Bacteriological methods and techniques used in bacteriology
Bacteriological Techniques
Bacteriology
Biological and medical sciences
Clostridium - immunology
Clostridium - isolation & purification
Clostridium tyrobutyricum
Collodion
Filtration
Food industries
Fundamental and applied biological sciences. Psychology
Immunoenzyme Techniques
Membranes, Artificial
Microbiology
Milk - microbiology
Milk and cheese industries. Ice creams
Spores, Bacterial
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Summary:The normal procedure for bacterial colony detection requires a nitrocellulose transfer step after membrane filtration and culture to prevent the development of a high background during the immunodetection. In this paper, we describe a modification of the basic protocol that omits the transfer step and reduces the risk of background. Previous observations indicated that interactions between milk components (principally cream) and membrane are responsible for the high non-specific staining observed. Experiments were performed to remove lipid components or to block the membrane binding sites before milk filtration. Samples of milks of different origin (collected at different times of the year) and different membranes were tested. The results obtained showed that removing lipids did not significantly improve the test but, on the contrary, led to an antigen diffusion. Incubation of the membrane in 0.1% (w/v) of Tween 20 in phosphate-buffered saline before milk filtration prevented non-specific binding, and allowed performance of the detection without any noticeable background.
ISSN:1364-5072
1365-2672
DOI:10.1111/j.1365-2672.1997.tb02871.x