Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1997-11-13
2002-01-22
Navarro, Mark (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S326000, C435S332000, C530S388200, C530S388400
Reexamination Certificate
active
06340571
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the field of the detection of
Staphylococcus aureus
bacteria, and in particular strains of
Staphylococcus aureus
which are resistant to methicillin.
DESCRIPTION OF RELATED ART
“Detection” is understood to mean collectively all the techniques which make it possible to identify, qualitatively and/or quantitatively, or to enrich, purify or separate a biological analyte, in this case
Staphylococcus aureus
bacteria.
In accordance with the document EP-A-0 323 331, a reagent for detecting
Staphylococcus aureus
bacteria, including the methicillin-resistant strains, is known, this reagent comprising:
fibrinogen,
an antibody recognized by
Staphylococcus aureus
protein A,
an antibody specifically recognizing the type 5 capsular serotype of
Staphylococcus aureus
or an antibody specifically recognizing the type 8 serotype of
Staphylococcus aureus
, and preferably a mixture of these two antibodies.
SUMMARY OF THE INVENTION
According to the present invention, a monoclonal or polyclonal antibody is provided which specifically recognizes an epitope common to
Staphylococcus aureus
strains of various capsular serotypes, particularly the methicillin-resistant strains. This antibody is selected from the type G immunoglobulins, the type M immunoglobulins and the type A immunoglobulins.
A particularly advantageous application of the antibody according to the invention consists in incorporating it into a reagent specific for the detection of
Staphylococcus aureus
, including the methicillin-resistant strains. Compared with the document EP-A-0 323 331, the sensitivity of the reagent of the invention is superior, because the capacity for recognition of the capsular serotypes is greater.
Thus, according to the invention, there are provided and described:
a monoclonal or polyclonal antibody specific for an epitope common to the
Staphylococcus aureus
strains of various capsular serotypes, particularly the methicillin-resistant strains, the said antibody being selected from the type G immunoglobulins, the type M immunoglobulins and the type A immunoglobulins; this antibody specifically recognizes at the same time at least two different capsular serotypes of the said
Staphylococcus aureus
strains; preferably, it is selected from the type G immunoglobulins and the type M immunoglobulins;
a monoclonal or polyclonal antibody as defined above which in particular recognizes at least the type 5 capsular serotype and the type 8 capsular serotype of the said
Staphylococcus aureus
strains,
a monoclonal antibody capable of being obtained according to a technique adapted from Galfre et al. (Nature, 266, 550-552, (1977)), using as initial cell line for the fusion, the myeloma line SP2/0-Ag14 (ATCC CRL 1581); the fusion is performed with spleen cells from mice of the BALB/C species and of the BALB/CBYJICO strain (marketed by the company IFFA Credo), immunized with a
Staphylococcus aureus
type 5 strain, according to conventional techniques; the clones obtained were screened by immunoenzymatic techniques (ELISA); the selected clones were reinjected by the intraperitoneal route into mice prepared beforehand for the production of ascitic fluid; each monoclonal antibody was used to sensitize latex particles and was tested for its specificity to recognize
Staphylococcus aureus
capsular polysaccharides; the monoclonal antibodies called P2G7A1E5 and P6D8D12E1 respectively were selected because of their specificity in recognizing an epitope common to various capsular serotypes, in particular of types 5 and 8;
a monoclonal antibody capable of being obtained, or as obtained from the hybridoma cell line deposited under the No. 96021514, on Feb. 15, 1996 with the ECACC;
a monoclonal antibody capable of being obtained, or as obtained from the hybridoma cell line deposited under the No. 96021513, on Feb. 15, 1996 with the ECACC;
a hybridoma cell line such as deposited under the No. 96021514, on Feb. 15, 1996 with the ECACC, or such as that deposited under the No. 96021513, on Feb. 15, 1996 with the ECACC, or any other derived hybridoma cell line, for example any progeny of this line; the cell lines are claimed as such, as well as any derived cell line, that is to say capable of producing antibodies exhibiting the same immunological characteristics as those described in the present invention;
a specific reagent for the detection of
Staphylococcus aureus
bacteria and particularly methicillin-resistant strains, comprising at least one antibody specifically recognizing at least one epitope common to the various capsular serotypes of the methicillin-resistant
Staphylococcus aureus
strains, the said reagent comprising at least one monoclonal or polyclonal antibody as defined above; the said antibody can be attached or coupled, or otherwise, to a support or a marker; monoclonal or polyclonal antibody is understood to mean the antibodies as defined above as well as any antibody exhibiting cross-specificity with the latter;
a specific reagent, particularly for the detection of
Staphylococcus aureus
bacteria and particularly of methicillin-resistant strains, comprising in addition fibrinogen or a compound based on fibrinogen, capable of being recognized by the
Staphylococcus aureus
affinity factor for fibrinogen; the fibrinogen or fibrinogen compound can be attached or coupled, or otherwise, to a support or to a marker;
a specific reagent, particularly for the detection of the bacteria
Staphylococcus aureus
and particularly for the methicillin-resistant strains, comprising in addition immunoglobulins or their Fc fragment recognized by
Staphylococcus aureus
protein A, attached or coupled, or otherwise, to a support or to a marker.
DESCRIPTION OF PREFERRED EMBODIMENTS
All sorts of support or marker can be envisaged according to the invention.
Particles in suspension can thus be used. These particles are in particular latex particles such as polystyrene beads or similar particles, preferably having a size of less than 2 &mgr;m. By way of example, there may be mentioned Estapor particles, marketed by the company RHONE-POULENC, such as:
polystyrene K080 particles having a diameter of 0.8 &mgr;m,
polystyrene K109 particles having a diameter of 0.8 &mgr;m,
polystyrene particles having carboxyl groups, PSI 480, having a diameter of 0.8 &mgr;m.
Magnetic gels, such as polyacrylamide and/or agarose gels containing magnetic particles can also be used. It is possible, in addition, to use gels such as Ultrogel and Magnogel (trademarks) from the company IBF.
The support used in a reagent according to the present invention may also be red blood cells, for example from sheep.
The support may be in the form of a plate, a cone, a strip, for example made of polystyrene or a styrene-based copolymer, a glass tube or the like.
The attachment of antibodies and of fibrinogen to particles in suspension, particularly of latex, may be achieved according to one of the following techniques:
by passive adsorption, it being possible for the attachment to be spontaneous, during incubation of the latex particles in a solution containing the antibodies and the fibrinogen; an incubation for example of about 2 hours at 20° C. is often sufficient;
by covalent bonding, it being possible for the attachment to be achieved by creating a covalent bond between the antibodies and the reactive groups present on some latex particles; it is possible for example to use a carbodiimide to create the covalent bond.
When the support consists of red blood cells, these may be sensitized beforehand with fibrinogen or any other appropriate molecule, according to conventional techniques.
The concentration of antibodies to be attached onto the latex particles, which should be determined for each antibody according to known methods, is usually between 0.1 &mgr;g and 100 &mgr;g of antibody proteins per millilitre of latex in solution.
In a reagent according to the invention, the antibodies and the fibrinogen may be attached onto a single suspension of particles, for example of latex, or alternatively may be att
Battail Nicole
Carret Gérard
Flandrois Jean-Pierre
Merlin Sylviane
Bio Merieux
Navarro Mark
Oliff & Berridge. PLC
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