Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Reexamination Certificate
1999-09-17
2002-01-29
Le, Long V. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
C436S523000, C436S524000, C436S526000, C436S528000, C436S532000, C436S533000, C436S534000, C436S536000, C436S538000, C436S540000, C436S541000, C436S164000, C436S173000, C436S801000, C436S805000, C436S806000, C436S807000, C436S824000, C435S007100, C435S007200, C435S007920, C435S007930, C435S007940, C435S007950
Reexamination Certificate
active
06342396
ABSTRACT:
BACKGROUND
The present invention relates to a method for isolating at least one analyte from a liquid medium, in which the latter is distributed.
“Isolating” or “isolation” is generically understood to mean any technique which makes it possible to separate said analyte, but also to enrich or concentrate, in relation to said analyte, any liquid mixture containing it. However, it is also understood to mean, optionally conjointly with the preceding definition, any technique which makes it possible to determine the analyte, for the purpose of a detection and/or quantification thereof, in the liquid medium containing it.
“Analyte” is understood to mean any entity, in particular biological entity, to be isolated. Among the range of the analytes considered below by the present invention, there may be mentioned cells, organelles, viruses and bacteria, antibodies, antibody fragments, antigens, haptens, lectins, sugars, ribodeoxyribo-nucleic acids, proteins, in particular A or G, hormones, hormone receptors, biotin, avidin, streptavidin and in general all natural or synthetic molecules or macromolecules, or analogs, to be determined, that is to say detected and/or quantified.
In accordance with the document FR-A-2,679,660, a method is known for isolating at least one analyte, namely either an antigen which is free or bound to the surface of a cell, or an antibody directed against a cellular or tissue antigen, for example anti-erythrocyte antibody.
According to this method, at least one reagent is available which comprises, on the one hand, a free support, in discrete form, in this case relatively large-sized magnetic particles consisting of a polymer which encapsulates ferrite granules, and, on the other hand, exhibiting a receptor for the analyte, namely either an antibody, or an antigen or revealing substance, for example an anti-human immunoglobulin antibody.
Another support, for capture, is also available which consists of the wall of a container or well, whose accessible surface comprises at least one useful or active zone, of limited surface, exhibiting at least one other receptor for an entity to be captured, namely for said analyte or for said receptor. This other receptor is either an antibody, or an antigen, for example of the erythrocyte type.
According to this method, the accessible surface of said other support is brought into contact with the analyte, contained in a liquid sample, in order to bind the analyte to the accessible surface, and to assemble it in the form of a deposit on said surface, immobilized on said other support via said other receptor.
Next, for revealing purposes, the reagent is brought into contact with the immobilized analyte, in order to obtain with the latter a combination, also immobilized on said other support and capable of allowing the isolation or determination of the analyte.
A method as defined above is therefore carried out with a reagent consisting of a support in discrete form, to which at least one ligand forming a receptor for the analyte is bound.
Preferably, these particles are magnetic. They can be classified into two categories, namely particles of relatively large diameter, for example of the order of one or a few micron(s), and those of relatively small diameter, for example of the order of a few tens of nanometers, and in the colloidal state.
The magnetic particles of relatively large diameter, when they are placed in a magnetic field, move in the direction of the site where the field is highest and at a sufficient speed to be separated from their medium by this means.
By way of example, there may be mentioned the particles described in the document EP-A-0,125,995. They are obtained by precipitation of ferrous and ferric salts in basic medium, followed by a silanization reaction in methanol. Their final diameter is between 0.1 and 1.5 &mgr;m and their density is 2.5 g/cm
3
. Likewise, the particles described in the documents EP-A-0,106,873, EP-A-0,585,868 and U.S. Pat. No. 5,356,713 are obtained by various methods of polymerization, or alternatively those described in the document U.S. Pat. No. 4,297,337 use a porous glass matrix in which magnetic pigments are dispersed. Other patents also describe the use of small-sized particles, but deliberately aggregated in order to increase the magnetic mass as in the document U.S. Pat. No. 5,169,754. The article by P. A. Ris&phgr;en et al, Protein Expression and Purification, 6 (1995), 272-277 also describes magnetic gels.
Placed in a magnetic field, all these relatively large particles generate a movement in the direction of the side where the field is the most intense. A simple permanent magnet or equivalent assemblies as described for example in the document EP-A-0,317,286 or U.S. Pat. No. 565,665 may be used. These particles are commonly used for the separation of cells or of molecules, as well as in immunoassays as described in the document EP-A-0,528,708 but are not used as systems for detection and quantification.
Moreover, their diameter and their density are such that they sediment very rapidly under the effect of gravity, which makes them difficult to use because of the stirring stresses or lack of homogeneity of the solutions due to the creation of a concentration gradient.
In summary, the relatively large particles are difficult to use and are not very appropriate for the determination of an analyte.
Magnetic particles of relatively small diameter are practically not attracted by a simple permanent magnet within reasonable periods: these particles are in particular widely used for the magnetic separation of cells. These particles are known to persons skilled in the art by the name of “superparamagnetic” particles. “Superparamagnetic” particles are understood to mean particles whose diameter is too small to consist of several magnetic domains. They are characterized by a high magnetic susceptibility and a high magnetization at saturation, but a zero or very low magnetic stability. These particles are in particular widely used for the magnetic separation of cells. For example, those described in the document U.S. Pat. No. 4,230,685 are obtained by emulsifying a mixture of albumin, of protein A and of particles of Fe
3
O
4
15-20 nm in diameter and can immobilize antibodies via the protein A. The document U.S. Pat. No. 4,452,773 describes another type of particle, obtained by precipitation of ferrous and ferric salts in a basic medium, and in the presence of a polysaccharide. These particles can immobilize antibodies, oligonucleotides, lectins or other biomolecules, by coupling to the polysaccharide by means of known methods of grafting. Their use has often been repeated, as in the documents U.S. Pat. No. 5,543,289 or WO-A-88/00060, or they are used in specific applications such as those described in the documents FR-A-2,710,410 and FR-A-2,732,116. The document U.S. Pat. No. 4,795,698 describes a modification of the Molday procedure, by replacing, for example, the polysaccharide with another polymer of a protein nature. The proteins present at the surface of the particles can thus serve for the subsequent immobilization of antibodies by coupling methods known to persons skilled in the art.
These particles require the use of special assemblies which make it possible to locally increase the magnetic field gradient. This technique is known by persons skilled in the art by the name of HGMS (for High Gradient Magnetic Separation) and it is for example described by WO 96/09409. It uses a device discrete form and for this reason are not used for the separation, concentration or enrichment of an analyte.
Furthermore, after separation, the particles are rather considered as an inconvenience in the subsequent steps of any method. In the document U.S. Pat. No. 4,018,886, they are even removed deliberately. The document FR-A-2,710,410 uses the presence of small-sized superparamagnetic particles as detection components which make it possible to quantify a molecular recognition reaction. In this case, the principle used is an agglutination reaction resulting in the formation of an aggregate. Fur
Mandrand Bernard
Perrin Agnès
Theretz Alain
Bio Merieux
Do Pensee T.
Le Long V.
LandOfFree
Method for isolating, in particular for detecting or... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method for isolating, in particular for detecting or..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for isolating, in particular for detecting or... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2817339