Chemistry: analytical and immunological testing – Biospecific ligand binding assay – Utilizing isolate of tissue or organ as binding agent
Reexamination Certificate
1999-04-29
2002-02-12
Smith, Lynette R. F. (Department: 1645)
Chemistry: analytical and immunological testing
Biospecific ligand binding assay
Utilizing isolate of tissue or organ as binding agent
C435S007320, C435S252400, C435S007100, C424S093480
Reexamination Certificate
active
06346422
ABSTRACT:
The present invention relates to a method of electing nonpathogenic bacterial strains useful for he prevention or treatment of infections mediated by issue adhesion of pathogenic bacterial strains.
In numerous bacterial infections, it is possible to observe an early phase during which the pathogenic agent adheres to particular sites of the host organism.
This adhesion makes it possible, in particular, for pathogenic bacteria to become established at sites of infection, such as the mucous membranes, which are continually washed by their own secretions and which may be subject to movements such as peristaltism. The adhesion can also help a pathogenic bacterium in its competition with the host's microflora.
The adhesion of a pathogenic bacterium to a tissue is frequently stereospecific and can only take place if the tissue carries a very specific type of receptor. The interaction between a bacterial lectin and a tissue sugar is a typical example of stereospecific interaction. These lectin molecules are often carried by filamentous appendages called fimbriae.
Fimbriae are very fine protein filaments found mainly, and very commonly, in Gram-negative bacteria. They may be distributed over the entire surface of the cell or they may be more localized. A fimbria consists of a repeating linear protein subunit. These subunits are often rich in nonpolar amino acids, such that cells carrying fimbriae tend to have more hydrophobic surfaces than those of cells lacking them. In Gram-negative bacteria, many types of fimbria bring about the adhesion of cells to each other and of cells to any surface. Each type of fimbria indeed possesses its lectin and adheres only to a specific receptor composed of glycoprotein epitopes.
Adhesion through specific types of fimbria is in particular essential for the virulence of enterotoxinogenic
Escherichia coli
strains (ETEC strains). These strains adhere to the duodenal mucosa and produce enterotoxins responsible, for example, for diarrhoeas.
The authors of the present invention have now developed a method for preventing tissue binding of pathogenic bacteria, the said method using nonpathogenic bacterial strains selected for their capacity to bind to the receptor sites for pathogenic bacteria with an affinity superior or equal to the latter.
The present invention relates more particularly to a method of selecting nonpathogenic bacterial strains capable of binding to the receptor sites for tissue adhesion of pathogenic bacterial strains, comprising the steps consisting in:
a) bringing the said receptors into contact in vitro with a bacterial strain to be tested;
b) adding an antibody directed against the said receptors, the said antibody being optionally labelled in a detectable manner;
c) in the case where the said antibody is itself not labelled in a detectable manner, adding an agent for detecting the antibody;
d) detecting the presence of the complex formed between the said antibody and the said isolated receptors.
The detection of the complex formed between the said antibody and the said receptors can therefore be carried out directly, by labelling the said antibody in a detectable manner, or indirectly, by using an agent for detecting the antibody.
The expression “antibody labelled in a detectable manner” is understood to mean that the antibody is conjugated or coupled with a labelling group.
The labelling group may be of various types such as, for example, the radioisotope (such as
1251
I,
3
H), enzymatic (in particular by means of alkaline phosphatase, horseradish peroxidase or &bgr;-galactosidase), fluorescent, (in particular by means of fluorescein or rhodamine) or particulate (in articular by means of latex or colloidal gold) type. These labelling groups are well known to persons skilled in the art.
The conjugation or coupling of the various markers with the antibody is carried out by conventional methods (coupling with glutaraldehyde, carbodiimide, maleic anhydride, succinic anhydride, heterobifunctional agents and the like).
“Agent for detecting the antibody” designates any means which make it possible to indirectly detect the said antibody. The said detection agent may be in particular a second antibody directed against the said antibody to be detected (or first antibody), the said second antibody being labelled in a detectable manner.
According to the invention, various methods of detection may be used: radioimmunological assay (RIA), immunoenzymatic assay (EIA, ELISA), fluoroimmunological assay (FIA), immunochemiluminescent assay (CLIA), immunoagglutination assay (IA), immunonephelometry, and the like.
The detection of the complex formed between the said first antibody and the said receptor may also be carried out by a Davidoff-type immunocytochemical method which combines the double peroxidase-anti-peroxidase technique with that of the avidin-biotin-peroxidase complex.
According to a preferred embodiment of the method of the invention, the bacterial strain to be tested is brought into contact with the said receptors in a decreasing bacterial concentration. The objective is to search, among various bacterial strains tested, for the one capable of exerting the greatest inhibition of the binding of the antibody to the receptor, for the highest possible dilution of bacteria with reference to the profile for the negative and positive controls.
The antibodies directed against the receptors for tissue adhesion of pathogenic bacteria strains may be mono- or polyclonal antibodies or fragments thereof, chimeric or immunoconjugated antibodies.
Polyclonal antibodies may be obtained from the serum of an animal immunized with the said receptor purified according to the customary procedures.
Preferably, however, monoclonal antibodies are used which may be obtained according to the conventional method of hybridoma culture described by Kohler and Milstein, from preparations of purified receptors for tissue adhesion.
The antibodies may be chimeric antibodies, humanized antibodies, Fab and F(ab′)2 fragments. They may also be provided in the form of immunoconjugates or of labelled antibodies.
Among the bacterial strains whose pathogenicity is mediated by tissue adhesion, there may be mentioned in particular the enterotoxinogenic
E. coli
strains, one of the most widespread being the strain identified by its ETEC/0147: K88ac serotype (Erickson et al., Infection and Immunity, 1992, 60:983-988).
The brush-like borders of the enterocytes of sensitive piglets express a receptor which allows massive binding of the strain of this pathogen. The binding of the bacterium is immediately followed by a cascade of intramembrane molecular reactions in the enterocyte which disrupts the ion flows causing a loss of water which, generalized to the whole intestine, finishes by irreversibly dehydrating the piglet to death. In another example, the fimbriae of
E. coli
K99 bind to N-glycolylneuraminyllactosyl ceramides but not to N-acetylated derivatives of sialic acid. The N-glycolyl bonds are in particular present in animals and in particular bovines which are greatly affected by
E. coli
K99.
Among the bacterial strains whose pathogenicity is mediated by tissue adhesion, there may also be mentioned Salmonellae. In these bacteria, the fimbriae are essential for initiating colonization, in particular in the caecum of poultry, a step preceding infection. The adhesion of pili is also essential for
Neisseria gonorrhoeae
to exert its virulence. In addition, the bacteria
Streptococcus mutans, S. sobrinus, S. cricetus
and
S. rattus
, which are the bacteria most frequently encountered in dental caries, are also included. These bacteria exhibit the characteristic feature of converting sucrose to glycans by excreting glycosyltransferases. These enzymes are very important for adhesion because the glycan thus formed binds to the enamel and the gum with food particles and turns out to be a very effective receptor for the lectins carried by these Streptococci. Once bound, the Streptococci multiply and cause dental lesions. In the case of the pathogen
Campylobacter jejuni
,
Butty Pascal Jean-Luc
Jeyasingham Marina
Ceva Sante Animale
Jacobson & Holman PLLC
Portner Ginny Allen
Smith Lynette R. F.
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