Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1997-01-31
2002-10-15
Zeman, Mary K. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S005000, C435S007920, C436S518000, C436S531000, C530S350000
Reexamination Certificate
active
06465191
ABSTRACT:
This application claims priority to application No. 94/09528, filed Aug. 1, 1994 in France and application No. 94/09529, filed Aug. 1, 1994 in France.
The present invention relates to a process for separating and/or detecting and/or quantifying (an) infectious compound(s) in a biological material and to a support for implementing the process.
According to the present invention, infectious compounds, generically referred to below in abbreviated form by “IC”, are understood to mean both compounds, in particular proteinaceous compounds, which are constituents of an infectious agent and structures which include infectious compounds. These structures are, in particular, either complete or incomplete, endogenous or exogenous infectious agents, their metabolites or else assemblies which contain constituent compounds of these infectious agents, which assemblies exhibit certain properties of said infectious agents, in particular the property of being detected by certain antibodies which are specific for infectious compounds; the IC's can also be compounds which are specifically induced in the organism by the previously defined IC's or by the expression of genes which are being expressed in an abnormal manner. IC's which may be mentioned, for example, are viruses, bacteria, fungi, mycoplasmas, parasites and abnormal animal cells. A viral infectious compound will be designated below by “VIC” while a non-viral infectious compound, that is to say an infectious compound of a type other than solely viral, will be designated by “nVIC”.
“Biological material” is understood here to be a biological tissue, a liquid or solid preparation or extract derived from biological tissue, or a natural medium which is capable of containing an infectious compound in the above-defined sense (for example, drainage water). The material can also be a mixture of at least two materials as defined above. Such a biological material can, therefore, be, in particular, either prepared from tissues, organs, stools or biological liquids from a patient who is suffering from an infection, for example a viral, a bacterial, a parasitic, a mycotic or a mycoplasma infection, or obtained from “in-vitro” cultures; such a biological material can also be a serum, plasma, urine, cerebrospinal fluid, synovial fluid, peritoneal fluid, pleural fluid, seminal fluid or ascitic fluid.
It is known that &bgr;2-glycoprotein I, abbreviated “&bgr;2GPI” below, is a plasma glycoprotein whose sequence has been demonstrated, in particular, in the papers by J. LOZIER et al., Proc. Natl. Acad. Sci. ISA [sic], Vol. 81, pages 3640-3644, July 1984 and T. KRISTENSEN et al., FEBS Letters, Vol. 289, 1991, pages 183-186. &bgr;2GPI is also termed apolipoprotein H. It has been established that this protein exhibits a polymorphism: the name &bgr;2GPI will be regarded below as being generic for all the forms.
It has been shown in International Application WO 94/18569 that some viral compounds bind specifically to one form of &bgr;2GPI, namely that which is described in French Patent Application 2 701 263, whether this form of &bgr;2GPI is in a pure state or contained in a protein composition; this form of &bgr;2GPI is isolated from the residue which is bound to the affinity chromatography column(s) which is/are employed in the process for purifying albumin from blood plasma which is described in FR-A-2 690 444; it has a molecular weight of 50,000±3000 daltons; in the context of the present patent application, this form of &bgr;2GPI has been designated in abbreviated form “&bgr;2′GPI”. A process has therefore been proposed in WO 94/18569 for detecting and/or assaying viral compounds, in which the viral compounds (VIC's) are bound using &bgr;2′GPI. In such a process, the &bgr;2′GPI is therefore added to the VIC's which are contained in a biological material in such a way as to separate the VIC's which have thus been captured in order then to detect them and/or assay them.
In a general manner, a direct or indirect association between at least one IC and one &bgr;2GPI will be termed here “(a) complex(es)”: in a general manner, these complexes will be referred to below by the notation “&bgr;2GPI/IC”. The process described in WO 94/18569 detects and/or assays VIC/&bgr;2′GPI complexes between VIC viral compounds and &bgr;2′GPI which is either in a pure state or contained in a protein composition, with the &bgr;2′GPI being added to the biological material which contains the VIC's to be detected and/or assayed.
The form or forms of &bgr;2GPI which is/are naturally present in the biological material prior to implementing the process described below, and not intentionally added as such, will be designated here (&bgr;2GPI)n. The form or forms of &bgr;2GPI which are added intentionally in order to form the previously defined IC/&bgr;2GPI complexes will be designated (&bgr;2′GPI)a.
According to the present invention, it was found, in a novel manner, that it was possible to separate, detect and/or quantify other IC/&bgr;2GPI complexes from a biological material than those described in WO 94/18569, namely:
(&bgr;2GPI)n/IC complexes where the IC part can be of the VIC viral type or the nVIC non-viral type and the (&bgr;2GPI)n part derives naturally from the biological material under study,
(&bgr;2GPI)a
VIC complexes where the (&bgr;2GPI)a part is added intentionally and prepared in different pure or mixed forms for this purpose, and the nVIC part derives from non-viral infectious compounds in the biological material under study.
The present invention relates, therefore, to a process for separating and/or detecting and/or quantifying infectious compounds (IC's) in a biological material, characterized in that a &bgr;2GPI/IC complex selected from the group formed by:
a) the (&bgr;2GPI)n/IC complexes
b) the (&bgr;2GPI)a
VIC complexes
is separated and/or detected and/or quantified.
In a general manner, the &bgr;2GPI, like the IC's, can, for some applications of the process, be of animal origin or produced by genetic and/or chemical engineering. The process can be applied both to man and to animals.
According to the invention, the &bgr;2GPI part of the complexes will be identified by its being recognized with the aid of (a) substance(s) which may bind preferentially, or which binds specifically, to this part, and the IC part will be identified by any suitable means.
The formation of the complexes can be direct or indirect and can be mediated or promoted by certain additives, which can be chemical, biochemical or biological, such as certain lipids or detergents, in particular phospholipids. The complexes can be formed during the preparation of the biological material and/or during one (of the) stages of the process.
As previously pointed out, the IC's comprise the VIC's and the nVIC's. The VIC's which may in particular be mentioned are those of the group formed by HIV1, HIV2, HBV, HSV and particles or proteins of viral origin; those nVIC's which may in particular be mentioned are those of the group formed by bacteria, parasites, fungi and mycoplasmas and, more specifically: in the case of bacteria: Borellia; and in the case of parasites: Leishmania, infantum in particular, Toxoplasma gondii and Entamoeba histolytica.
Advantageously, the &bgr;2GPI which is retained in the complex(es) may or may not, according to the embodiments of the process, have been labeled; this labeling, which may or may not take place beforehand, can be effected, for example, by means of an antibody, an enzyme, a radioactive product, a fluorescent product or a metallic agent.
According to the present invention, a poly-specific test which is able to detect different infectious agents can be carried out using, in particular, simultaneously or successively, a different detection method for each agent, for example an alkaline phosphatase-conjugated antibody against HIV2p26 and then a peroxidase-conjugated antibody against HBsAg.
In a first subset of embodiments of the present invention
Graafland Hubert
Rucheton Marcell
Stefas Elie
Veas Francisco
Institut Francais de Recherches Scientifiques Pour le Developpem
Nixon & Vanderhye
Zeman Mary K.
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