Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-08-08
2002-11-19
Graser, Jennifer E. (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S973000, C435S975000, C435S007200, C436S518000, C436S526000, C436S531000, C436S534000, C436S523000, C436S820000, C530S387100, C530S387500, C530S388400, C530S391100, C424S130100, C424S131100, C424S150100, C424S165100
Reexamination Certificate
active
06482602
ABSTRACT:
The present invention relates to a reagent for the detection of
Staphylococcus aureus
by agglutination.
Various reagents are already known for the detection of
Staphylococcus aureus
. These reagents are based on the search for either protein A of Staphylococcus or the affinity factor for fibrinogen, or both simultaneously.
Protein A is an antigen of protein nature, an external component of the wall of the majority of the strains of
Staphylococcus aureus
of human origin (85 to 95). By a non-immunological process, protein A binds the Fc fragment of the immunoglobulins, leaving the Fab part free.
If strains of
Staphylococcus aureus
possessing protein A and sheep red blood cells or latex particles sensitized, for example, with rabbit anti-sheep red blood cells serum are placed together, an agglutination visible to the naked eye is observed within a few minutes.
The affinity factor for fibrinogen which is attached to the surface of
Staphylococcus aureus
reacts directly with fibrinogen. This affinity factor for fibrinogen can be determined within a few seconds by placing 7 in contact the strain under study and sheep red blood cells (passive hemagglutination) or latex particles, one or other being coated with fibrinogen.
Various published studies show that a certain number of strains of
Staphylococcus aureus
are not identified by these reagents. Their percentage varies from 1 to 5% when these studies are carried out on all of the
Staphylococci aureus
isolated. But this percentage of failure is larger when only the strains resistant to oxacillin (or meticillin, are taken into consideration and this percentage attains 25% in a recent study carried out with 73 strains of
Staphylococcus aureus
resistant to oxacillin (see P. J. Ruane et al., J. Clin. Microbiol. 24, 490, 1986).
In a study carried out under the direction of one of the inventors on 183 strains of
Staphylococcus aureus
isolated from patients in 5 hospitals in Paris and the Paris region, it has been found that 7 strains (4%) are not agglutinated by any of the three reagents used (Staphyslide®, Staphaurex® and Pastorex® Staph). If only the 50 strains resistant to oxacillin are considered, it is found that 6 strains (12%) are falsely negative with the commercial reagents. Hence, these results are in agreement with those described in the literature.
Two hypotheses may be envisaged to explain the fact that some strains of
Staphylococcus aureus
are not agglutinated by the commercial reagents:
1) These strains produce protein A in an amount too small for there to be a reaction between this protein and the latex particles adequate to lead to bacterial agglutination.
2) Protein A is produced in normal amounts, but this antigen as well as the affinity factor for fibrinogen are masked by one or more other antigens and are thus inaccessible to the latex particles.
In a study carried out under the direction of one of the inventors, it has been shown that the strains non-agglutinated by the latex particles produce as much protein A as the other strains. This result thus makes it possible to eliminate the first hypothesis. It was then shown, by determining the capsular polysaccharide by means of an immunoenzymatic reaction utilizing monoclonal antibodies, that all of the strains non-agglutinated by the latex particles sensitized by fibrinogen and against protein A possess the capsular polysaccharide. Finally, the antigens exposed at the surface of the staphylococcus and, consequently, capable of reacting with the latex particles were studied by immunofluorescence. This study focused, on the one hand, on protein A by utilizing the Fc fragment of human immunoglobulin and pepsinized F(ab′)
2
fragments of anti-human Fc sheep immunoglobulins labelled with fluorescein) and, on the other, on the capsular polysaccharide (by utilizing mouse monoclonal antibodies of the M isotype specific for the capsular polysaccharide and F(ab′)
2
fragments of anti-mouse M immunoglobulin goat immunoglobulins labelled with rhodamine. This study clearly showed that protein A is not exposed or then only in very small amount at the surface of the bacteria which are not agglutinated by the latex, and that the surface of these bacteria is, on the other hand, totally masked by the capsular polysaccharide. As a control, it was verified that the strains which are agglutinated by the latex particles do indeed display protein A at their surface.
Thus, this study shows that the capsular polysaccharide synthesized by some strains of
Staphylococcus aureus
masks all of the bacterium, masks the antigens capable of being recognized by the commercial reagents and thus prevents the identification of these strains as belonging to the
Staphylococcus aureus
species by the fact of their agglutination by the commercial reagents.
The result of this study and, in particular the fact that the strains of
Staphylococcus aureus
which do not exhibit protein A at the surface are capsulated strains, the capsular polysaccharides of which mask the protein A has made it possible to design a reagent for the detection of the strains of
Staphylococcus aureus
which possesses a greater reliability than the known reagents.
A subject of the present invention is thus a reagent for the detection by agglutination of
Staphylococcus aureus
of the type comprising particles in suspension to which are bound fibrinogen and antibodies recognized by affinity by the protein A of staphylococcus, characterized in that it contains particles in suspension to which are bound at least one antibody recognizing specifically a capsular polysaccharide of
Staphylococcus aureus.
Another subject of the present invention is a procedure for the detection by agglutination of
Staphylococcus aureus
in a sample, which consists of mixing the sample with a reagent according to the invention and of observing whether an agglutination occurs.
At present 11 types of capsular polysaccharides have been identified by essentially immunological methods. See in this connection: W. W. Karakawa et al., Capsular polysaccharides of
Staphylococcus aureus
p. 285-293. In J. B. Robbins, J. C. Hill, and J. C. Sadoff (ed.) Seminars in infectious disease. vol. 4. Bacterial vaccines. Thieme Stratton. Inc. New York; W. W. Karakawa et al. J. Clin. Microbiol. 22: 445-447, 1985; Sompolinsky et al., J. Clin. Microbiol. 22: 828-834, 1985.
The purification and the biochemical and immunological characterization of the capsular polysaccharide of type 8 were carried out in 1984 (J. M. Fournier et al., Infect. Immun. 45: 87-93) and those of type 5 in 1987 (J. M. Fournier et al., Ann. Inst. Pasteur/Microbiol. 138: 561-567).
Specific monoclonal antibodies of the capsular polysaccharides 5 and 8 have been described (H. K. Hochkeppel et al., J. Clin. Microbiol. 25: 526-530, 1987, and M. J. Nelles et al., Infect. Immun. 49: 14-10, 1985).
Furthermore, epidemiological studies carried out on a large number of strains of
Staphylococcus aureus
isolated from patients have shown that 70 to 80% of these strains possess one or other of the capsular polysaccharides 5 and 8 (for example R. D. Arbeit et al., Diagn. Miticrobiol. Infect. Dis. 2: 85-91, 1984).
Also in the present invention the antibodies recognizing a capsular polysaccharide of
Staphylococcus aureus
are advantageously constituted by at least antibodies recognizing a capsular polysaccharide of type 5 or 8 and preferably simultaneously by antibodies recognizing a capsular polysaccharide of type 5 and antibodies recognizing a capsular polysaccharide of type 8.
But it is obvious that the most reliable diagnostic reagent contains a set of antibodies recognizing the different types of capsular polysaccharides.
In the reagent according to the invention, the different antibodies and fibrinogen may be bound to only one suspension of particles or be bound to different suspensions of particles (in a proportion of one or lore types of component per suspension of particles) which are then mixed to constitute the reagent.
The particles in suspension used in the reagent according to the
Boutonnier Alain
Fournier Jean-Michel
Finnegan, Henderon, Farabow, Garrett & Dunner, L.L.P.
Graser Jennifer E.
Institut Pasteur
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