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 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.
Batina
P
P
Université de Bretagne Occidentale, Institut Universitaire de Technologie, Quimper, France.
Arnold
F
F
Bedouet
L
L
Robreau
G
G
Talbot
F
F
Malcoste
R
R
D000818:Animals; D000907:Antibodies, Bacterial / Q000032:analysis* / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000276:immunology; D000942:Antigens, Bacterial / Q000276:immunology; D001431:Bacteriological Techniques; D003013:Clostridium / Q000276:immunology / Q000302:isolation & purification*; D003101:Collodion; D005374:Filtration; D007124:Immunoenzyme Techniques; D008567:Membranes, Artificial; D008892:Milk / Q000382:microbiology*; D013171:Spores, Bacterial
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D008567:Membranes, Artificial; D003101:Collodion