Chemistry: analytical and immunological testing – Measurement includes change in volume or pressure
Reexamination Certificate
2000-02-15
2002-05-28
Le, Long V. (Department: 1641)
Chemistry: analytical and immunological testing
Measurement includes change in volume or pressure
C435S007100, C435S286500, C435S287200, C422S068100, C422S082130, C436S052000, C436S518000, C073S061640, C073S061670
Reexamination Certificate
active
06395557
ABSTRACT:
TECHNICAL FIELD
The subject matter of the present invention is a method for determining an analyte present in a solution. More precisely, it relates to a method with which it is possible to detect and/or quantify this analyte. The analyte may be a chemical or biological entity.
Therefore, the present invention finds numerous applications in the area of chemical or biological analysis, for example for the determination of antigens, antibodies, DNA or RNA sequences, bacteria, viruses, bacterial fragments, etc.
PRIOR ART
Numerous systems for chemical or biological analyses are based on the reaction of the analyte to be determined with a suitable compound to form a reaction product, which must subsequently be detected and/or quantified in order to determine the presence and concentration of this analyte. Generally, this detection is made via labellers such as radioactive or luminescent tracers, enzymes or others which may be carried by the added compound added to react with the analyte to be detected. The use of such tracers assumes the use of specific apparatus to ensure the detection and/or quantification of the analyte.
Other detection and quantification methods such as topographic methods for example using atomic force microscopy (AFM), magnetic methods, electric methods for example by measuring capacity variation, and optic methods may also be used as described in FR-A-2 758 884 (1).
These methods, however, also have the disadvantage of requiring specific apparatus which can at times be costly.
The present invention sets out precisely to provide a method of determining an analyte which avoids the use of such apparatus and ensures the detection and/or quantification of the analyte using very conventional components at affordable price.
DESCRIPTION OF THE DISCLOSURE
According to the invention, the method for determining an analyte in a solution comprises the following steps:
a) fixing and immobilizing said analyte on the inner surface of a conduit having a reduced cross section over all or part of it, and
b) determining the variation in load loss of a fluid circulating inside the conduit, due to the analyte which has been fixed and immobilized at least in the reduced cross section part of said conduit during step a).
The method of the invention is therefore based on the use of a conduit having a suitable cross section over all or part of it, in which, during step a), the analyte to be determined is fixed and immobilized.
The fixing and immobilization of the analyte is made at least over all or part of the reduced cross section of the conduit, but they may also be conducted over the entire conduit.
This fixing and immobilization may be made either by contacting the solution with the inner surface of the conduit (for example by dipping) or by circulating the solution in the conduit, this circulation possibly entailing at least one sequential stop.
During the subsequent step b), the variation in load loss of a fluid circulating in the conduit is determined, such variation resulting from fixation of the analyte and being related to the quantity of analyte fixed.
This quantification of the analyte may be conducted using pre-set plots or curves, or by experimentation, or by simulation on the basis of known geometric data on the conduit and on the analytes used (size and quantity). Therefore, the value of the load loss is related to the quantity of analyte immobilized in the conduit and/or the size of this analyte.
Through experimentation, it is possible to cross-refer the measurement obtained in the method of the invention to previously made measurements to calibrate the performance of the conduit.
With both these methods it is possible to plot graphs showing the relationship between load loss and the quantity of immobilized analyte. Therefore, via simple measurement of load loss, it is possible to quantify the level of analyte immobilized in the conduit during step a) of the method of the invention.
According to the invention, it is possible to determine the variation in load loss by measuring the difference in pressure, at constant flow rate between two points located either side of the reduced cross section of the conduit, of a fluid circulating in the conduit before carrying out step a) and after conducting step a) of the method, or else the difference in flow rate at constant pressure before and after step a).
The fluid used for this determination may be all or part of the initial solution (in this case, step a) and step b) are simultaneous or successive) or a different liquid to this solution.
In the prior art, measurement of load loss in a conduit with a circulating fluid was used to determine certain characteristics of the fluid, its viscosity for example, and consequently its content of a particular product able to modify its viscosity. In document U.S. Pat. No. 4,964,847 [2] for example, this measurement is used to estimate packed cell volumes in blood.
This type of determination method is different from the method of the invention in which detection and quantification of the analyte require its immobilization on the wall of a conduit having a suitable shape and having, in addition, the property of being able to fix the analyte to be determined.
This latter property may be conferred upon the conduit by fixing on its wall a ligand that is able to bind with the analyte.
In this case, the determination of the analyte involves a recognition reaction between the analyte and the ligand with formation of an analyte-ligand complex immobilized in the conduit.
In this embodiment of the method of the invention, prior to step a), the wall of the reduced cross section is coated over all or part with at least one ligand able to fix itself to the analyte.
In this case, the presence of the ligand can be detected by determining the variation in load loss through measurement of the difference in pressure at constant flow rate, or the difference in flow rate at constant pressure, between two points located either side of the reduced cross section of the conduit, of a fluid circulating inside the conduit before and after making the ligand coating.
According to one variant of embodiment of the invention, when the size of analyte to be determined, or of the complex formed through reaction of this analyte with a ligand, is too small to generate sufficient load loss in the reduced cross section part of the conduit, a physical or chemical reaction of the analyte is additionally performed with a support material to form an analyte-support conjugate of greater size than the analyte, this reaction taking place either when said analyte is free in the solution, before step a), or when the analyte is already fixed directly or indirectly onto the inner wall of the reduced cross section part of the conduit after step a) to form an analyte-support conjugate.
This reaction between the analyte and the support material may be conducted before adding the solution to the conduit, or before conducting step a), or after fixing the analyte on the wall of the reduced cross section part of the conduit, that is to say after step a) but before step b).
To promote the binding of the support material with the analyte, the latter generally comprises a ligand on its surface able to bind with the analyte.
According to the invention, by “analyte” is meant any chemical or biological entity, in particular any biological entity in free form. As an illustration of analytes, the examples which may be cited are cells, organelles, viruses and bacteria, antibodies, antibody fragments, antigens, haptenes, lectines, sugars, ribonucleic and deoxyribonucelic acids, proteins, in particular A or G, hormones, hormone receptors, biotin, avidin, streptavidin and in general any molecule or macromolecule that is natural or synthetic, or analogue or even resulting from an association of at least two molecules or macromolecules of the type of those previously defined. Examples of associations of molecules or macromolecules which may be cited are the analyte-support conjugates which will be defined below.
According to the invention, by
Fouillet Yves
Perrin Agnès
Theretz Alain
Burns Doane , Swecker, Mathis LLP
Commissariat a l'Energie Atomique
Counts Gary W.
Le Long V.
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