Partial analysis of the flagellar antigenic determinant recognized by a monoclonal antibody to Clostridium tyrobutyricum.

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 ration 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 over-all 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 alpha (1-->4) linked glucose residues plus either N-actyl-glucosamine or an unidentified compound that maintain it in an extended shape.

Biochemical and immunological analyses of the flagellin of Clostridium tyrobutyricum ATCC 25755.

The monoclonal antibody 21E7-B12 (IgG3) can be used in a direct method of Clostridium tyrobutyricum detection based on an immunoenzymatic assay. Immunoelectron microscopy demonstrated that the 21E7-B12 antibody recognized the surface-exposed epitopes on the flagellar filaments of C. tyrobutyricum. After flagellar extraction, the purified flagellin showed an apparent molecular mass of 46 kDa with an isoelectric point of 3.6. Sugar staining, mild periodate oxidation and beta-elimination experiments showed that the flagellin was glycosylated and that the 21E7-B12 epitope was located in the sugar moiety. Amino acid composition showed that the flagellar filament protein contained a high percentage of serine and threonine, while proline was absent. The first 23 residues of the N-terminal were determined and sequence homology with other flagellins was found.

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.

Production of monoclonal antibodies against the outer cell wall of Clostridium tyrobutyricum.

Several hybridoma cell lines producing murine monoclonal antibodies (mAbs) directed to the Clostridium tyrobutyricum outer cell wall have been established and characterized. Whole bacteria, crude extract of cell wall, and polysaccharide fraction of crude extract have been used as immunogens. The immunizations were performed either in vivo or in vitro after priming in vivo. Amongst the clones obtained, six hybridoma cell lines were selected. Four mAbs recognized only the immunizing strain (ATCC 25755), while two mAbs recognized all the C. tyrobutyricum tested strains. Three mAbs were IgM, one IgG3, and two IgG1 isotypes. The antigens (proteins or polysaccharides) recognized by these mAbs have been characterized by Western Blot. These mAbs could be used for an early detection of C. tyrobutyricum in milk.

Demonstration of a surface antigen of Clostridium tyrobutyricum by use of immunoblotting with a monoclonal antibody.

A monoclonal antibody, prepared against whole cells of Clostridium tyrobutyricum, recognized a surface antigen extracted by heat treatment or by hot phenol-water treatment. This antigen, after analysis by polyacrylamide gel electrophoresis and immunoblotting, has been shown to present a regularly-spaced ladder pattern similar to those shown by the lipopolysaccharide of many gram-negative bacteria. The proteinase K has been shown to have no effect on the recognition of this epitope by the monoclonal antibody. On the contrary, the inhibition of the antigen reactivity to the monoclonal antibody after a mild periodate oxidation suggests the involvement of a carbohydrate moiety in the epitope. Moreover, the SDS-PAGE analysis of phenol-water extracts has shown an additional compound, detected by silver staining but not recognized by the monoclonal antibody.

[An in vivo study of phytochrome in seeds of Pinus nigra Arn by differential spectrophotometry]. / Etude du phytochrome des graines de Pinus nigra Arn par spectrophotométrie bichromatique in vivo.

Phytochrome photoconversions Pr→Pfr and Pfr→Pr can be measured by differential spectrophotometry in "dry" seeds (6% water content) of Pinus nigra Arn. A red light irradiation given before imbibition induces germination when the seeds are subsequently wetted and kept in darkness.In continuous darkness the phytochrome content shows a drastic increase at the beginning of moistening.The detectable pigment is entirely in the Pr form. The normal Pfr→Pr dark reversion is observed. Pfr destruction does not take place.

[An in vivo study of detectable phytochrome in seeds of Cucurbita pepo L. in the course of different phases of germination]. / Etude du phytochrome detectable, in vivo dans les graines de Cucurbita pepo L. au cours des differentes phases de la germination.

In dry gourd seeds all the phytochrome is in the Pfr form. The increase of phytochrome content from the beginning of hydration involves two phases, A and B, in the embryonic axis as well as in the cotyledons. Cycloheximide does not prevent the appearance of Pr during phase A. We assume that Pr is gradually released from an inactive complex. On the other hand phase B is inhibited by cycloheximide; this could mean that a de novo synthesis of Pr occurs.Some experiments indicate that the phytochrome which is localized in the embryonic axis may be involved only in the germinating process.The phytochrome which is synthesized during phase B disappears when the seeds are irradiated with red light, while the original phytochrome does not.According to our data it seems necessary to lay down a new and precise definition of the germination process.

The influence of blue light on dark-germinating seeds of Nemophila insignis.

After inhibition of Nemophila insignis seeds by far-red (FR) light, a short exposure to blue (Bl) will not induce germination again but stimulation by red (R), with reversion by FR, can be observed. Germination is inhibited by long exposures to Bl (maxima at 455 and 475 nm). These radiations are absorbed either directly by phytochrome or through intermediary pigments such as flavoproteins.

[Spectrophotometric analysis of phytochrome in the embryo of Cucurbita pepo and Cucurbita maxima]. / Analyse spectrophotométrique du phytochrome dans l'embryon de Courge (Cucurbita pepo) et de Potiron (Cucurbita maxima).

Phytochrome detection and △ (△O.D.) measurements were made for the first time in a single embryo of pumpkin or gourd thanks to an apparatus adjusted to the differential spectrophotometer constructed by Dr. Spruit (Wageningen). Observed △ (△O.D.) values are comparable with usual values obtained by analysis of several seeds in a cuvette. The differential spectrum obtained has the same characteristics as the spectra of seedlings and seeds which can be found in the present literature. The time course of phytochrome appearance in a single pumpkin embryo showed the different phases previously described in lettuce seeds. Use of masks which delimit different areas on the embryo made it possible to locate changes in phytochrome concentration. Analysis of pieces cut off from embryos and packed into a cuvette showed the existence of a decreasing concentration gradient from the cotyledons to the vegetative axis.

[Role of phytochrome in seed germination of Nemophila insignis]. / Le rôle du phytochrome dans la germination des graines de Nemophila insignis (L.).

After a long exposure to far-red light (20 hrs, 715 mn) a short red irradiation does not induce germination again. After shorter exposure to far-red (10 hrs) the reversible red far-red reaction takes place. The germination in complete darkness is probably due to the presence of Pfr (1) in dry seeds.The photoinhibition of the germination by far-red light depends on the level of the photostationary state (P730/PTotal) and on the quantum flux density.