DOSAGE METHOD

Abstract

The invention relates to a method for quantifying a target analyte in a biological sample in liquid medium, comprising the steps (a.) bringing the biological sample into contact with first magnetic particles bearing a first receptor specific to a first binding site of the target analyte so as to form first complexes by the binding of first magnetic particles with the target analyte, this contact being accompanied, when an interfering analyte is present in the sample, by the formation of interfering complexes by the non-specific binding of said interfering analyte to the first magnetic particles; (b.) applying a first magnetic field, and maintaining it, so as to locally bring together all the complexes formed in step a., and where appropriate to agglomerate interfering complexes together to form interfering aggregates; (c.(a) cancellation of the first magnetic field and addition to the liquid medium of second magnetic particles carrying a second receptor specific to a second binding site of the target analyte; (d) measurement of a first quantity representative of the amount of interfering aggregates in the liquid medium; (e) application of a second magnetic field so as to form second complexes by the binding of the first complexes with second magnetic particles; and (f) measurement of a second quantity representative of the combined amount of interfering aggregates and the amount of second complexes in the liquid medium so as to determine the amount of second complexes formed in step (e) as a function of the first quantity, and thus deduce the amount of target analyte present in the biological sample. Abstract figure: no figure.

Description

Title of the invention: DOSING METHOD

[0001] The invention relates to a method for dosing a target analyte in a liquid sample, and to a dosing device arranged to carry out this method.

[0002] More generally, the invention relates to the field of quantifying molecules of interest soluble in a liquid medium. In particular, it relates to the field of immunological quantification of proteins, for example in a whole blood sample.

[0003] The prior art provides methods for assaying that use magnetic particles for the capture and / or extraction of analytes present in a liquid sample. To this end, the magnetic particles carry one or more receptors specific to the target analyte, which allows them to bind to the analyte to form complexes. The complexes can then be captured by applying a magnetic field.

[0004] The applications of magnetic particle-based assays are vast. Some are aimed at applications in oncology; see, for example, Xu, Hengyi, et al., "Antibody conjugated magnetic iron oxide nanoparticles for cancer cell separation in fresh whole blood," Biomaterials 32.36 (2011): 9758-9765. Others are aimed at applications in nucleic acid extraction, as described in particular in documents US9976136 or Tan, Siun Chee, and Beow Chin Yiap, "DNA, RNA, and protein extraction: the past and the present," BioMed Research International, 2009.

[0005] Applications for nucleic acid extraction generally involve a plurality of successive steps, including cell lysis to release the nucleic acids, contacting the lysed solution with magnetic particles to capture the nucleic acids, applying a magnetic field (also called "magnetization"), and washing. This is followed by elution of the nucleic acids (generally by a change in pH) and removal of the magnetic particles. The isolated nucleic acids can then be processed and quantified.

[0006] However, cell lysis followed by washing leads to sample dilution and consequently a decrease in capture performance. Furthermore, once captured, the nucleic acids must be separated from the magnetic particles for processing, which increases the number of steps.

[0007] Other applications, such as in diagnostics or oncology, aim to measure analytes directly in the blood. The prior art thus offers immunoassay methods that attempt to measure proteins in a whole blood sample. Notable examples include US6030845, US6855562, and US7326579, as well as Applicant's document EP2810042. However, these methods also rely on a first step of blood lysis to destroy the cells. Lysis is followed by an immunoagglutination assay on the lysed sample. While this lysis step allows for agglutination measurement under good conditions, it involves a relatively significant dilution of the sample, which negatively impacts the limit of detection.

[0008] Other immunoassay methods using magnetic particles exist. In particular, there are methods described as homogeneous and those described as heterogeneous.

[0009] Homogeneous methods have the advantage of eliminating the washing step. The documents Baudry, Jean, et al., "Acceleration of the recognition rate between grafted ligands and receptors with magnetic forces", Proceedings of the National Academy of Sciences 103.44 (2006): 16076-16078; Ranzoni, Andrea, et al. "One-step homogeneous magnetic nanoparticle immunoassay for biomarker detection directly in bloodplasma" Acs Nano 6.4 (2012): 3134-3141; and Aurich, Konstanze, et al. "Determination of the magneto-optical relaxation of magnetic nanoparticles as a homogeneous immunoassay", Analytical Chemistry 79.2 (2007): 580-586, describe homogeneous tests. However, the absence of a washing step makes these assay methods difficult to use in complex media such as blood. Indeed, the presence of cells, particularly in blood, significantly interferes with the measurements.

[0010] Heterogeneous methods require a greater number of steps, which generally makes them time-consuming to implement. They are also complex to implement. The document Kourilov, Vitaly, and Michael Steinitz, "Magnetic-bead enzyme-linked immunosorbent assay verified adsorption of ligand and epitope accessibility" Analytical biochemistry 311.2 (2002): 166-170 describes a heterogeneous method.

[0011] Regardless of the assay method, there is a risk of measuring a non-specific signal. A non-specific signal is measured, in particular, when at least one interfering analyte competes with the target analyte to bind to the magnetic particles or to the receptor specific to the target analyte.

[0012] The nature of the interfering analyte can vary. For example, it may be a protein whose three-dimensional structure is similar to that of the target analyte, or a molecule carrying an electrical charge. More generally, an interfering analyte can be any molecule exhibiting a non-negligible affinity for magnetic particles or for receptors grafted onto their surface. Consequently, the signal measured during the assay is never zero. Existing methods therefore aim to reduce the probability of a non-specific signal. However, existing methods generally do not allow differentiation between a specific and a non-specific signal.

[0013] All of this means that methods for quantifying complex samples, and in particular The results of whole blood sample assays are unsatisfactory. The presence of cells in the sample, the need to lyse these cells followed by at least one washing step, and the risk of a non-specific signal can degrade the method's performance.

[0014] The present invention improves the situation.

[0015] Thus, the present invention relates to a method for assaying a target analyte in a biological sample in liquid medium, comprising the following steps: a. bringing the biological sample into contact with first magnetic particles carrying a first receptor specific to a first attachment site of the target analyte so as to form first complexes by the binding of first magnetic particles with the target analyte, this contact being accompanied, when an interfering analyte is present in the sample, by the formation of interfering complexes by the non-specific binding of said interfering analyte to the first magnetic particles; b. the application of a first magnetic field, and its maintenance, so as to locally bring together all the complexes formed in step a., and where appropriate to agglomerate interfering complexes to form interfering aggregates; c. cancellation of the magnetic field applied in step b. and the addition in the liquid medium of second magnetic particles carrying a second receptor specific to a second attachment site of the target analyte; d. the measurement of a first quantity representative of the quantity of interfering aggregates in the liquid medium, to identify the presence or absence of said interfering aggregates; e. the application of a second magnetic field so as to form second complexes by the binding of the first complexes with second magnetic particles; and f. the measurement of a second quantity representative of the total quantity of interfering aggregates and the quantity of second complexes in the liquid medium so as to determine the quantity of second complexes formed in step e. as a function of the first quantity to deduce the quantity of target analytes present in the biological sample, and where applicable the quantity of interfering analytes.

[0016] The invention thus makes it possible to verify the presence or absence of an interfering analyte in the biological sample. Thus, when the biological sample contains both a target analyte and an interfering analyte, step a. comprises the formation of interfering complexes by the binding of the first magnetic particles with the interfering analyte; step b. thus comprises the formation of aggregates between the interfering complexes; step d. then provides for the quantitative measurement of the interfering aggregates. formed; and step f. provides for a calculation to determine the amount of target analyte present in the biological sample, as well as the amount of interfering analyte.

[0017] Furthermore, the method of the invention avoids excessive dilution and consequently ensures good analytical sensitivity. In addition, the invention reduces the number of steps required to simplify the entire test. This results in a drastically reduced time, not exceeding a maximum of approximately 15 minutes.

[0018] Obviously, when no interfering analyte is present in the sample In biological processes, interfering complexes, and therefore interfering aggregates, do not form. It follows that the quantity measured at step d is zero or does not exceed a predefined threshold value.

[0019] The method of the invention thus makes it possible not only to identify the presence of an interfering analyte in the biological sample, but also to quantify it. In parallel, the method makes it possible to deduce the quantity of the target analyte in the biological sample.

[0020] Step f. may include calculating the difference between the second quantity measured in this step f. and the first quantity measured in step c. in order to determine the quantity of target analyte.

[0021] In the main embodiment, the cancellation of the first magnetic field in step c is followed or accompanied by the addition of second magnetic particles, and then the measurement in step d of the first quantity representative of the amount of interfering aggregates is carried out. In another embodiment, this measurement can be carried out directly after the cancellation of the magnetic field, that is, before the introduction of the second magnetic particles into the liquid medium (or reaction medium).

[0022] For the measurement in step f, i.e. the measurement of the second quantity representing the total quantity of interfering aggregates and the quantity of the second complexes, the second magnetic field is preferably canceled prior to said measurement. This increases the sensitivity of the measurement.

[0023] To ensure the specificity of the process of the invention, each first receptor is specific to a first binding site of the target analyte, and each second receptor is specific to a second binding site, different from the first, of the target analyte. The flexibility arising from two distinct binding sites allows the process of the invention to be rapidly adapted to various needs depending on the nature of the application implemented.

[0024] In a particular embodiment of the invention which aims at the extraction of magnetic particles from the liquid medium, the magnetic field applied in step b. is greater than or equal to 100 mT, and the holding of the magnetic field is less than or equal to 5 minutes, and preferably less than or equal to 3 minutes. In another particular mode, the application of the first magnetic field in step b. and the application of the second magnetic field in step e. comprises a magnetization at 8 mT for 1 second, followed by three successive sequences of the following magnetizations and cuts: 15 mT for 60 seconds, 0 mT for 28 seconds, 8 mT for 1 second, 0 mT for 1 second.

[0025] This allows the process of the invention to be carried out quickly, in particular in less than 15 minutes. Furthermore, the chosen magnetization and cutting sequence allows for increased precision of the process of the invention by promoting the formation of complexes between the analytes and the magnetic particles on the one hand, and by enabling a more precise reading on the other.

[0026] In a preferred embodiment, the measurements performed in steps d and f are chosen from the group consisting of a turbidimetry measurement, a nephelometry measurement, and a counting measurement (by image analysis and / or processing or by flow analysis, in particular). This makes it possible to obtain reliable results quickly.

[0027] In another embodiment, step a. involves adding a diluent so as to dilute the biological sample, the ratio between the sample and the diluent being greater than or equal to 1:10.

[0028] This dilution, in particular, reduces the viscosity of the medium to facilitate the capture of the molecule to be measured, or in other words, the formation of the first complexes. Furthermore, the diluent may contain agents that reduce the probability of aggregation of magnetic particles. For this purpose, nonionic surfactants, anionic surfactants, cationic surfactants, zwitterionic surfactants, proteins (albumin, casein, gelatin, antibodies, in particular) or polymers (such as polyvinylpyrrolidone, polyvinyl alcohol) may be used.

[0029] In a particular embodiment, the application of the first magnetic field in step b allows the extraction of the first complexes from the liquid medium. This is particularly useful for working with complex media (such as whole blood). Thus, a particular embodiment specifically provides that the biological sample is whole blood. An advantage of the invention is that the steps of the process can be directly applied to a blood sample, without requiring washing and / or dilution.

[0030] In another embodiment, step b. involves removing the liquid medium and adding a reaction buffer. The reaction buffer may comprise an anti-aggregation agent (nonionic surfactants, anionic surfactants, cationic surfactants, zwitterionic surfactants, proteins, or polymers) and / or comprise a cell-lysing agent (saponins, quaternary ammonium compounds, sodium dodecyl sulfate). in particular). Furthermore, at least one wash can be provided between the removal of the liquid medium and the addition of the reaction buffer. This wash further increases the specificity of the process of the invention.

[0031] Anti-aggregation agents allow the dissociation of any aggregates of magnetic particles that may have formed, while the cell-lysis agent allows the lysis of any cells that may not have been removed with the elimination of the liquid medium. It should be noted that, unlike prior art methods, the lysis agent does not require further dilution of the sample. In this embodiment, a more or less lengthy incubation period can then be provided to allow sufficient action of the agent(s) present in the reaction buffer.

[0032] Other advantages and features of the invention will become apparent from the detailed description below and the accompanying drawings, in which:

[0033] [Fig-1] shows a schematic diagram of the invention.

[0034] [Fig.2] shows a principle diagram of a first incubation stage of a biological sample with first magnetic particles according to the invention;

[0035] [Fig.3] shows a principle figure of a first magnetization step of the process of the invention;

[0036] [Fig.4] shows a principle figure of a second incubation stage of a biological sample with second magnetic particles according to the invention;

[0037] [Fig.5] shows a principle figure of a second magnetization stage of the process of the invention;

[0038] [Fig.6] shows a principle figure of a first incubation stage of a biological sample comprising molecules interfering with first magnetic particles according to the invention;

[0039] [Fig.7] shows a principle figure of a first magnetization step of the process of the invention applied to a biological sample containing interfering molecules;

[0040] [Fig.8] shows a principle figure of a second incubation stage of a biological sample comprising molecules interfering with second magnetic particles according to the invention;

[0041] [Fig.9] shows a graph of optical density as a function of prostate-specific antigen (PSA) concentration in a first embodiment;

[0042] [Fig. 10] shows a graph of optical density as a function of antibody (Ab) concentration of a second example embodiment;

[0043] [Fig. 11] shows a graph of optical density as a function of antibody (Ab) concentration of the second embodiment;

[0044] [Fig. 12] shows a graph of optical density as a function of anti-PCT antibody concentration (procalcitonin) of a third embodiment;

[0045] [Fig. 13] shows a graph of optical density as a function of prostate-specific antigen (PSA) concentration from a sixth example (not according to the invention); and

[0046] [Fig. 14] shows a graph of optical density as a function of prostate-specific antigen (PSA) concentration of a seventh example (according to the invention).

[0047] The figures, tables, and description below essentially contain elements of a certain nature. The figures and tables form an integral part of the description and may therefore not only serve to better understand the present invention but also contribute to its definition, if necessary.

[0048] Generally speaking, the present invention relates to the identification of an analyte in a liquid biological sample. The sample can be of any suitable type, such as, for example, a sample of bone marrow, cerebrospinal fluid, lymph, urine, or preferably a whole blood sample. The target analyte can be, for example, a protein, a nucleic acid, or any other molecule of interest present in the sample. The target analyte is present in a greater or lesser quantity in the sample. Thus, a sample may contain a target molecule present in a greater or lesser number of copies. In other words, the concentration of the target analyte can be greater or lesser in the sample. This also applies to any other non-target analyte potentially present in the sample.

[0049] The invention uses a technique for capturing the target analyte(s) with magnetic particles that have been surface-functionalized with ligands specific to each target analyte. These particles may be of the type described above. The diameter of the magnetic particles used is generally between 5 nm and 10,000 nm, preferably between 100 nm and 500 nm.

[0050] In the assay process of the invention, a first step a. comprises bringing the biological sample into contact with first magnetic particles

[0051] The [Fig. 1] shows a schematic diagram of the invention.

[0052] Thus, this diagram generally illustrates one embodiment of the implementation of the process of the invention. In a first step a, magnetic particles are mixed with a liquid biological sample, which may or may not contain a target analyte to be quantified in the presence of a buffer. The biological sample may also, but not necessarily, contain an interfering analyte.

[0053] Initially, the biological sample is brought into contact with initial magnetic particles. Each magnetic particle carries a specific receptor at a first binding site of the target analyte, thus forming initial complexes through the binding of these magnetic particles to the target analyte when the latter is present in the biological sample. This contact This process is accompanied, when an interfering analyte is present in the sample, by the formation of interfering complexes through the non-specific binding of the interfering analyte to the first magnetic particles. Logically, when no interfering analyte is present in the biological sample, no interfering complexes are formed.

[0054] Figure 2 shows a biological sample comprising a liquid medium containing molecules. Figure 2 shows first magnetic particles 10 or PMIs bearing specific receptors, referred to herein as first receptors 101. These first magnetic particles 10 are brought into contact with the molecule to be measured present in the biological sample. The molecule to be measured is referred to as the target analyte 20. The biological sample also contains other species or molecules 40, 50 which may be, for example, soluble molecules, cells, or particles (or interfering molecules 30 as will be seen later).

[0055] This contact in step a results in the molecule 20 being captured by the first receptors 101. For this purpose, the first receptors 101 are specific to the target analyte 20. For good specificity, monoclonal antibodies or antibody fragments are used as receptors. The binding between each receptor and each target analyte results in the formation of complexes, which are referred to here as first CL complexes. These complexes are thus formed by the first magnetic particles 10 associated with the target analytes 20. These complexes are dispersed in the liquid medium.

[0056] Generally, the contact in step a. can last about 10 minutes, preferably less than 5 minutes. Optionally, the liquid medium can be mixed or agitated during the incubation phase to increase the capture yield.

[0057] Step a. may involve the addition of a diluent to dilute the biological sample, or more particularly the liquid medium. Generally, a dilution not exceeding one-tenth of the initial concentration (i.e., 1Ox) will be chosen.

[0058] The process of the invention then includes a step b, in which a first magnetic field is applied. This allows all the complexes formed in step a to be locally brought together, and, where appropriate, interferent complexes to be agglomerated to form interferent aggregates. Locally bringing the complexes together means organizing them within the liquid medium. Depending on the applied magnetic field, the localization may vary.

[0059] It should be noted that a main effect of this step b., and more particularly of the application of the magnetic field, is indeed the formation of interfering aggregates resulting from the agglomeration of interfering complexes, when the latter formed at step a. of contact between the biological sample and the first magnetic particles.

[0060] In a particular embodiment of the invention, this step involves attracting the first magnetic particles towards a magnet. Consequently, the first complexes formed in step a, which are essentially composed of the association of the first magnetic particles with the target analyte, are attracted to the magnet. It follows that all the complexes formed in step a are gathered locally against the magnet, or against an environment close to the magnet. This makes it possible to isolate the first complexes and separate them from the liquid medium. In other words, in this particular embodiment, the first complexes are extracted from the liquid medium of the biological sample. Logically, the separation of the first complexes from the liquid medium will only be complete when the liquid medium is removed later in the process. In this embodiment, step b.It is possible to maintain the magnetic field for a certain time, for example 5 to 10 minutes, preferably less than 5 minutes. This contributes to the successful extraction of all the initial complexes formed in step a from the liquid medium.

[0061] Figure 3 shows the magnetic particles 10 attracted or organized according to the Bo field generated by a magnet. The first Cl complexes are assembled locally.

[0062] In a particular embodiment, the liquid medium of the biological sample is removed, followed by the addition of a reaction buffer. In other words, the liquid medium is replaced by a reaction buffer, also in liquid form. To do this, the liquid medium is first drained from the reaction vessel, and then the reaction buffer is poured into the vessel. In this embodiment, a permanent magnet is generally provided to allow the initial Cl complexes bearing the target analyte 20 to be assembled locally against it. The target analyte 20 molecules, bound to the receptors 101, are isolated and retained during the removal of the liquid medium from the biological sample. The reaction buffer is added to replace the liquid medium.

[0063] In this embodiment, one or more washes can be performed between the removal of the liquid medium and the addition of the reaction buffer. This washing increases the specificity of the process of the invention. During the wash(s), the magnetic field can be maintained or switched off. In the latter case, it is necessary to reapply the magnetic field for a given time after each wash in order to recapture the suspended magnetic particles.

[0064] In one embodiment, the reaction buffer may include one or more anti-aggregation agents and / or one or more cell-lysis agents. The anti-aggregation agents allow the dissociation of any aggregates or clusters of magnetic particles formed in step a and / or step b. This increases the precision of the process of the invention. The cell-lysis agent allows the lysis of potential cells that would not have been removed with the elimination of the liquid medium described above. It should be noted that, unlike prior art methods, the lysis agent does not require further dilution of the sample.

[0065] Step c. of the process of the invention involves canceling the magnetic field Bo. When the field is cut off, the particles and complexes disperse in the liquid medium, as well as any aggregates that may have formed.

[0066] Figure 4 shows the resuspended Cl complexes. Step c further involves adding to the liquid medium second magnetic particles 11, or PM2, bearing a second receptor 111 specific to the target analyte 20. At this stage, the second receptors 111 of the second magnetic particles 11 do not bind, or bind very little, to the target analyte 20. If necessary, stirring of the reaction medium may be provided.

[0067] It should be noted that the target analytes 20 generally have a three-dimensional shape, allowing for the selection of diverse, specific receptors 101, 111. Thus, each first receptor 101 is specific to a first binding site of the target analyte 20, and each second receptor 111 is specific to a second binding site of the target analyte, different from the first. This increases the specificity of the method of the invention. This flexibility, arising from two binding sites, also allows the method of the invention to be rapidly adapted to various needs, particularly according to the nature of the application. The receptors can thus be diversified depending on the target analyte to be measured.

[0068] The method of the invention then includes a step d, in which a first quantity representative of the amount of interfering aggregates in the liquid medium is measured to identify the presence or absence of said interfering aggregates. When the measured quantity is zero or below a predefined threshold value, there are no (or extremely few) interfering aggregates in the liquid medium. In this case, it can be assumed that there are no (or extremely few) interfering analytes in the biological sample. When the measured quantity is non-zero or exceeds the predefined threshold value, there are interfering aggregates in the liquid medium, and therefore an interfering analyte in the biological sample.

[0069] The measurement can in particular be carried out by turbidimetry, by nephelometry or by counting (by image analysis and / or processing or by flow in particular).

[0070] The process of the invention then includes a step e, in which a second magnetic field is applied so as to form second complexes by the binding of the first complexes Cl to the second magnetic particles 11, or more precisely to the receptors 111 carried by the second magnetic particles. In terms of detectability, these second complexes are comparable to interfering clusters, namely that they can be detected relatively easily by measurements optical density in particular.

[0071] Figure 5 shows the second complexes 61 generated by the application of the second magnetic field B1. The target analyte 20 is sandwiched between the first and second receptors carried by the first 10 and second 11 magnetic particles, respectively. In general, this step e aims to generate chains or clusters of magnetic particles. The "sandwich" complexes or molecules, formed by the first 10 and second 11 magnetic particles bordering the target analyte 20, are detectable by various techniques, including optical density measurement.

[0072] Documents WO 2009 / 034271, FR2919390, and FR2959820 describe assay processes in which a series of cycles of applying and removing a magnetic field are applied to the liquid reaction medium to induce the formation of magnetic particle chains or clusters during the reaction between the magnetic particles and a target analyte. Each application of the field results in an increase in the number of analyte / magnetic particle bonds, and each removal of the field causes the unbound magnetic particles to disperse in the liquid medium. During the process, the number and / or size of the bonded particle chains or clusters increases. This proportionally increases the turbidity of the reaction medium. Measurements of the optical density of the medium after each removal of the magnetic field thus allow the concentration of the target analyte in the sample to be calculated.

[0073] Other types of magnetic pulse sequences are possible. Examples are described in the publication Fast Magnetic Field-Enhanced Linear Colloidal Agglutination Immunoassay, Daynès et al., Anal. Chem. 2015, 87, 7583-7587.

[0074] In the present invention, the first magnetic field Bo applied during step b. may be identical or different from the second magnetic field Bl applied in step e.

[0075] It can be advantageous to apply magnetic fields with different properties during these two steps. This is the case in the embodiment where the first complexes are extracted from the liquid medium. Under these conditions, to allow optimal capture of the first magnetic particles 10, the field B0 of step b must combine a high intensity (typically greater than 100 mT) with a significant gradient in the magnetization direction. Conversely, it is preferable to apply a field B1 of moderate intensity (typically less than 50 mT) and with a low gradient for step e. The field B1 of step e can also be applied in the form of several pulses, separated by relaxation periods. The duration and intensity of these pulses can be variable.

[0076] More generally, the application of the second magnetic field Bl involves Preferably, a plurality of magnetic pulses, separated by rest periods. In one embodiment, this step e. provides for a magnetization at 8 mT for 1 second, followed by three successive sequences of magnetizations and cutoffs: 15 mT for 60 seconds, 0 mT for 28 seconds, 8 mT for 1 second, 0 mT for 1 second.

[0077] 1 s, 8 mT + 3x[60 s, 15 mT + 28 s, OmT + 1 s, 8 mT + 1 s, 0 mT].

[0078] The various 8 mT pulses for 1 second essentially allow for a more precise reading of the aggregation state of the PMI particles in the presence of interfering analyte. The pulse / rest series of 15 mT for 60 seconds, followed by rest (0 mT) for 28 seconds, followed by 8 mT for 1 second, followed by rest (0 mT) for 1 second, essentially allows for the formation of complexes.

[0079] The second complexes 61 form aggregates that are detectable by optical density measurement or by counting, for example. More generally, the second complexes can be detected because the magnetic particles are aggregated together due to the binding of the target analyte to the two receptors (sandwich), whereas the signal of a first complex does not differ significantly from that of a free magnetic particle, given the difference in size (and / or optical index and / or magnetic moment) between a magnetic particle and a target analyte molecule.

[0080] Thus, step f of the process of the invention aims to determine the quantity of second complexes formed in order to deduce the quantity of target analytes present in the biological sample. To this end, step f involves measuring all the aggregates, that is, all the second complexes and, where applicable, the interfering aggregates. Ultimately, this measurement makes it possible to determine the quantity of second complexes 61 formed in step e, and in particular as a function of the measurement of interfering aggregates alone carried out previously in step d. The result of the measurement then makes it possible to calculate the quantity of target analyte 20 present in the biological sample.

[0081] The invention has the advantage of identifying not only the target analyte, but also an interfering analyte, or even several interfering analytes.

[0082] Reference is now made to figures 6, 7 and 8 to describe in more detail the steps of the process with the presence of an interfering analyte.

[0083] Step a. (see [Fig.6]) in this case includes not only the formation of the first Cl complexes, but also the formation of interfering Cint complexes by the binding between the first magnetic particles 10 and an interfering analyte 30. The interfering Cint complexes are then dispersed in the liquid medium of the biological sample.

[0084] More generally, in step a, the magnetic particles 10 carrying re Specific receptors 101 are brought into contact with the molecule to be measured 20, in the presence of other species 30, 40, which may be soluble molecules, cells, particles, or other. One of these species (molecules 30 in [Fig. 6]) binds non-specifically to the first magnetic particles 10. Molecule 20 is nevertheless captured by the receptors 101.

[0085] During step b (see [Fig. 7]), the first magnetic particles 10 are attracted or locally organized within the liquid medium according to the magnetic field B0 generated by the magnet. In the particular embodiment of the invention described above with reference to the extraction of the magnetic particles, the liquid medium is replaced by the reaction buffer. In this specific case, a permanent magnet (not shown in the drawings) is used to attract the magnetic particles.

[0086] More generally, the molecules 20 (target analyte) bound to the receptors 101, as well as the molecules 30 bound non-specifically to the magnetic particles 10, are locally assembled within the liquid medium. This local assembly at a given location within the liquid medium of the first Cl complexes and the interfering Cint complexes results in the formation of non-specific aggregates 60 (or interfering aggregates 60) between the first Cl complexes and the interfering Cint complexes. These non-specific aggregates can also form through the agglomeration of the interfering Cint complexes with each other. More generally, the non-specific aggregates form in essentially the same way as the complexes of interest, that is, between a Cint complex and a neighboring magnetic particle. This particle may be bound to an analyte molecule (Cl), to an interfering molecule (Cint), or be a free PMI particle.These non-specific aggregates 60 are detectable in liquid media, for example by turbidimetry measurement (see [Fig.8]).

[0087] Once the aggregates have formed, step d. allows for their quantitative measurement in the liquid medium.

[0088] As explained above, step f uses the measurement taken during step d to calculate the quantity of specific aggregates formed by the second complexes. For this purpose, the calculation takes into account both the measurement taken in step d and that taken in step f. The quantity of target analyte present in the biological sample can thus be deduced.

[0089] Measuring the state of aggregation during step d limits the risk of interference that could lead to false positive results. In the presence of a molecule that can cause such interference, step b can lead to the formation of non-specific aggregates of first magnetic particles (FMPs). Without step d, these aggregates would only be detected during step f, without it being possible to distinguish the specific aggregates, caused by the presence of the molecule to be measured (target analyte), from the non-specific aggregates. caused by the presence of another molecule (interfering analyte). By applying step d, it is possible, for example, to determine an initial aggregation threshold beyond which no results will be obtained due to interference, thus avoiding erroneous results. An advantage of the invention is that the aggregation measurements in steps d and f can be combined to calculate an estimate of the specific aggregation, excluding non-specific aggregation.

[0090] Optionally, it is also possible to stop the process of the invention after step d. If the quantity of non-specific aggregates measured is too high and / or exceeds a predefined sensitivity threshold, the process of the invention can be interrupted after carrying out the measurement in step d. It is then possible to restart the process of the invention by selecting other receptors specific to the target analyte.

[0091] In a particular embodiment of the invention, the process described above is therefore stopped after step d, namely after the measurement of the first quantity representative of the amount of interfering aggregates in the liquid medium. For this purpose, a threshold quantity can be set beyond which the results are not sufficiently sensitive. This avoids the need to perform subsequent steps, which become unnecessary or at least unusable. Unnecessary additional costs and wasted time can thus be avoided.

[0092] EXAMPLES OF IMPLEMENTATION

[0093] A first series of experiments (examples 1 to 3) is carried out in order to demonstrate the feasibility and sensitivity of the invention.

[0094] EXAMPLE 1

[0095] A PSA (prostate-specific antigen) assay is performed. Magnetic particles (Carboxyl Adembeads 200nm, Ademtech) are functionalized with anti-PSA antibodies. One batch of particles is functionalized with a first clone (P4, reference 7820-0370, available from Bio-Rad). Referring to the general description, these are the first PMI magnetic particles. A second batch is functionalized with a second clone recognizing a different epitope (214, reference 7820-0217, available from Bio-Rad). Referring to the general description, these are the second PM2 magnetic particles. The samples consist of purified PSA (reference P3338, available from Sigma-Aldrich) at 100 nM diluted in horse serum (reference H1270, available from Sigma-Aldrich) at various concentrations. The target concentration of PSA in the samples is therefore known.

[0096] The analysis of the samples is carried out as follows:

[0097] 71.5 pL of sample are brought into contact with 1.5 pL of functionalized particles PMI (P4) at 1% for 2 min;

[0098] The medium is magnetized for 3 min on a permanent magnet, and the supernatant is removed;

[0099] The particles are taken up by 73.5 pL of HEPES buffer 50 mM, pH 7.5; F108 0.8%; NaCl 800 mM; NaN3 0.09% and 1.5 pL of PM2 (214) functionalized particles at 1% are added.

[0100] The reaction medium thus formed is subjected to the following magnetic field sequence:

[0101] 1 s, 8 mT + 3x[60 s, 15 mT + 28 s, OmT + 1 s, 8 mT + 1 s, 0 mT],

[0102] The light intensity through the reaction medium illuminated by an RC-LED at 650 nm is measured and the optical density difference before and after application of the last 8 mT intensity pulse (Dodiff3) is used as the signal of interest.

[0103] Control measurements are performed with the same samples and the same particles, but without the extraction step (step c. in reference to the general description). The reaction medium, consisting of 57 pL of HEPES 50 mM buffer, pH 7.5; F108 0.8%; NaCl 800 mM; NaN3 0.09%, 15 pL of sample and 1.5 pL of each batch of functionalized particles, is subjected to the same magnetic field cycle as described above.

[0104] The results shown in Table 1 below are obtained as a function of the concentration of PSA in the samples tested.

[0105] [Tables 1] [PSA] (nM) 0 0.5 1 2 4 Dodiff3 (extraction) 10.27 22.49 34.35 53.45 59.01 Dodiff3 (control) 8.87 14.62 22.14 35.42 52.12

[0106] Table 1.

[0107] Fig. 9 shows the results and corresponding curves of Dodiff3 as a function of PSA concentration.

[0108] It is observed that the method using the process according to the invention makes it possible to obtain a signal increasing with the concentration of PSA in the sample, and with an activity superior to the control tests.

[0109] EXAMPLE 2

[0110] Tests to simulate the presence of an interfering molecule in the sample of interest are carried out. Magnetic particles (Carboxyl Adembeads 200 nm, from Ademtech) are functionalized with anti-PSA antibodies. One batch of PMI particles is functionalized with a first clone (P4, reference 7820-0370, available from Bio-Rad), and a second batch of PM2 is functionalized with a second clone recognizing a different epitope (214, reference 7820-0217, available from Bio-Rad). The samples consist of a 9 g / L NaCl solution to which is added an anti-mouse IgG antibody (M8642, available from Sigma-Aldrich) capable of binding non-specifically to anti-PSA antibodies grafted onto the particles.

[0111] The analysis of the samples is carried out as follows:

[0112] 71.5 pL of sample are brought into contact with 1.5 pL of functionalized particles PMI (P4) at 1% for 2 min;

[0113] The medium is magnetized for 3 min on a permanent magnet, and the supernatant is removed;

[0114] The particles are resuspended in 73.5 pL of HEPES 50 mM buffer, pH 7.5; F108 0.8%; NaCl 800 mM; NaN3 0.09% and 1.5 pL of 1% PM2 (214) functionalized particles are added.

[0115] The reaction medium thus formed is subjected to the following magnetic field sequence:

[0116] 1 s, 8 mT + 3x[60 s, 15 mT + 28 s, OmT + 1 s, 8 mT + 1 s, 0 mT],

[0117] The light intensity through the reaction medium illuminated by an RC-LED at The 650 nm wavelength is measured, and the difference in optical density before and after application of the last 8 mT pulse (Dodiff3) is used as the signal of interest. This same signal can be measured during the application of the first 8 mT pulse (Dodiff0) to determine the aggregation of the medium before application of the magnetic field.

[0118] Control measurements are performed with the same samples and the same particles, but without an extraction step. The reaction medium, consisting of 57 pL of HEPES 50 mM buffer, pH 7.5; F108 0.8%; NaCl 800 mM; NaN3 0.09%, 15 pL of sample and 1.5 pL of each batch of functionalized particles, is subjected to the same magnetic field cycle as described above.

[0119] The results shown in Table 2 below are obtained, depending on the antibody concentration in the reaction media, with regard to the measurement before agglutination:

[0120] [Tables2] [Ac] (nM) 0 2.2 4.4 8.8 Dodiffo (extraction) 2.72 13.02 15.3 6.08 Dodiffo (control) 1.41 1.84 2.12 1.41

[0121] Table 2.

[0122] Figure 10 shows the results and corresponding curves of the Dodiff3 in function of the concentration of Ac before aggregation.

[0123] While the signal before field application does not depend on the concentration of antibodies interfering with the control measurement, it is observed that the addition of this antibody leads to an increase in the aggregation signal before field application due to the magnetization step, which makes it possible to consider alarming these measurements.

[0124] The following results are obtained, as shown in Table 3 below, depending on the antibody concentration in the reaction media, with regard to the measurement after agglutination:

[0125] [Tables3] [Ac] (nM) 0 2.2 4.4 8.8 Dodiff3 (extraction) 4.1 17.29 18.57 10.01 Dodiff3 (control) 3.95 27.47 65.23 90.46

[0126] Table 3.

[0127] Fig. 11 shows the results and corresponding curves of Dodiff3 as a function of Ac concentration after aggregation.

[0128] In both cases, the aggregation signal is increased in the presence of the interfering antibody. Thus, without the initial measurement performed during extraction, it would be impossible to determine whether the sample contains PSA or an interfering molecule.

[0129] EXAMPLE 3

[0130] Tests to verify the applicability of the process of the invention on a sample of whole blood are carried out.

[0131] Magnetic particles (Carboxyl Adembeads 200nm, available from Ademtech) are functionalized with anti-PCT antibodies (procalcitonin). One batch of PMI particles is functionalized with a first clone (E86813M, available from Meridian Life Sciences), and a second batch of PM2 particles is functionalized with a second clone recognizing a different epitope (E01342M, available from Meridian Life Sciences). The samples consist of recombinant PCT at 400 nM diluted in human blood at different concentrations. The target concentration of PCT in the samples is therefore known.

[0132] The analysis of the samples is carried out as follows:

[0133] 71.5 pL of sample are brought into contact with 1.5 pL of functionalized particles E86813M at 1% and 71.5 μL of 50 mM HEPES buffer, pH 7.5; F108 0.8%; 800 mM NaCl; DTT lOmM; NaN3 0.09% for 2 min;

[0134] The medium is magnetized for 3 min on a permanent magnet, and the supernatant is removed;

[0135] The particles are resuspended in 73.5 pL of 50 mM HEPES buffer, pH 7.5; F108 0.8%; NaCl 800 mM; DTT 10mM; NaN3 0.09% and 1.5 pL of E01342M functionalized particles at 1% are added.

[0136] The reaction medium thus formed is subjected to the following magnetic field sequence:

[0137] 1 s, 8 mT + 3x[60 s, 15 mT + 28 s, OmT + 1 s, 8 mT + 1 s, 0 mT],

[0138] The light intensity through the reaction medium illuminated by an RC-LED at 650nm is measured and the optical density difference before and after application of the last 8 mT intensity pulse (Dodiff3) is used as the signal of interest.

[0139] The same tests are carried out on plasma samples taken from blood samples. These are centrifuged for 10 min at 1500g to extract the plasma. The hematocrit of the blood sample being 40.4%, it is then possible to calculate the expected plasma concentration of PCT based on the expected concentration of PCT in the blood.

[0140] The results shown in Tables 4 (blood) and 5 (plasma) are obtained below:

[0141] [Tables4] [PCT]blood (nM) 0 0.0625 0.13 0.26 1.04 Dodiff3 6.83 22.82 37.1 48.62 79.75

[0142] Table 4.

[0143] [Tables5] [PCT]piasma (nM) 0 0.106 0.22 0.5 0.88 1.76 Dodiff3 8.19 39.02 52.99 72.81 79.74 81.91

[0144] Table 5.

[0145] Figure 12 shows these results in a graph.

[0146] It is observed that the signal measured as a function of PCT concentration in the sample is independent of the nature of the sample, whether blood or plasma. This demonstrates the possibility of applying the described method to a whole blood sample.

[0147]

[0148] A second series of experiments (examples 4 to 7) is carried out in order to demonstrate the effect of an interfering antibody on the aggregation of magnetic particles.

[0149] To this end, aggregation measurements are performed in the presence of different concentrations of anti-mouse antibodies. This is done to simulate or mimic the effect of an interfering antibody.

[0150] For this series of experiments, Adembeads 200 nm particles functionalized either by the P4 antibody (reference 7820-0370, available from Bio-Rad) or by the 214 antibody (reference 7820-0217, available from Bio-Rad) are used.

[0151] EXAMPLE 4: Measurements without PS A

[0152] In this example, the first and second magnetic particles (PMI and PM2) are introduced into the liquid medium simultaneously. Consequently, this is not an example according to the invention.

[0153] Initially, a standard cycle is performed. The prepared samples include:

[0154] - 57 μL of 50 mM HEPES buffer, pH 7.5; F108 0.8%; 800 mM NaCl;

[0155] - 15 pL of anti-mouse antibody (M8642, Sigma-Aldrich) at the desired concentration in a 9 g / L NaCl solution;

[0156] - 3 pL of P4 particles 1% w / v (weight per volume, or w / v i.e. weight per unit of volume); and

[0157] - 3 pL of particles 214 1% w / v.

[0158] The following cycle (Cagg) is then applied to the samples:

[0159] 1 s, 8 mT + 3x[60 s, 15 mT + 28 s, OmT + 1 s, 8 mT + 1 s, 0 mT],

[0160] The difference in optical density before and can be measured for each sample after the first pulse of intensity 8 mT (Dodiff0) as an indicator of the initial level of particle aggregation. To measure the final level of aggregation, one can measure the difference in optical density before and after the last pulse of intensity 8 mT (Dodiff3) or the difference in optical density between the end and the beginning of the Cagg cycle (ADO).

[0161] The results shown in Table 6 are obtained as a function of the antibody (Ab) concentration in the reaction medium:

[0162] [Tableauxô] [Ac] (nM) 0 1 2 3 4 5 ADO (mDO) 2.86 25.62 51.46 74.37 79.03 101.08 Dodiffo (mDO) 2.71 2.23 2.58 2.28 2.58 2.78 Dodiff3 (mDO) 6.08 23.89 42.62 56.59 60.09 71.79

[0163] Table 6.

[0164] It is observed that the chosen antibody causes interference since the Dodiff3 or the ADO levels increase with the concentration of introduced antibodies. The Dodiff0 measurement, however, does not vary significantly with antibody concentration, therefore this interference is not cause for alarm.

[0165] EXAMPLE 5: Measurements without PSA

[0166] The prepared samples include:

[0167] - 57 μL of 50 mM HEPES buffer, pH 7.5; F108 0.8%; 800 mM NaCl;

[0168] - 15 pL of anti-mouse antibody (M8642, Sigma-Aldrich) at the desired concentration in a 9 g / L NaCl solution; and

[0169] - 3 pL of P4 particles 1% w / v.

[0170] The Cagg cycle (detailed above) is applied to this sample, then 3 pL of 214 1% w / v particles are added to the medium and the Cagg cycle is applied again.

[0171] The same indicators as before can be measured on the first cycle, applied only to P4 particles (Dodiffo1, Dodiff3', ADO1), or on the second cycle, applied to all particles (Dodiffo2, Dodiff32, ADO2). The results are shown in Table 7 below:

[0172] [Tables7] [Ac] (nM) 0 1 2 3 4 5 ADO1 (mDO) 1.39 13.96 25.84 31.87 35.36 45.75 Dodiffo1 (mDO) 1 0.99 1.09 0.89 0.77 0.96 DodilTï1 (mDO) 1.82 12.7 22.15 25.67 28.19 34.69 ADO2 (mDO) 2.22 19.65 40.27 50.02 54.94 61.28 Dodiffo2 (mDO) 3.21 13.72 22.83 26.53 28.59 34.63 Dodiff32 (mDO) 4.87 28.54 51.43 56.41 63.23 74.97

[0173] Table 7.

[0174] The ADO2 or Dodiff32 indicators increase with the concentration of the introduced antibody. However, with this protocol, the ADO1 or Dodiff3* values ​​can be used to detect the presence of aggregates prior to the application of the second cycle, due to interference in the sample. Thus, in this example, applying a threshold chosen at 5 mDO allows the detection of samples containing the antibody in- Interferent.

[0175] EXAMPLE 6: PSA dose-response curves

[0176] In this example, the first and second magnetic particles (PM1 and PM2) are introduced into the liquid medium simultaneously. Consequently, this is not an example according to the invention.

[0177] Initially, a dose-response curve is performed without interfering antibodies.

[0178] The samples include:

[0179] - 57 μL of 50 mM HEPES buffer, pH 7.5; F108 0.8%; 800 mM NaCl;

[0180] - 15 pL of PSA (P3338, Sigma-Aldrich) at the desired concentration in a NaCl solution 9 g / L;

[0181] - 3 pL of P4 particles 1% w / v; and

[0182] - 3 pL of particles 214 1% w / v.

[0183] After application of the Cagg cycle, the results shown in Table 8 below are obtained:

[0184] [Tables8] [PSA] (nM) 0 1 3 5 10 ADO (mDO) 2.86 6.45 14.27 20.83 33.28 Dodiffo (mDO) 2.71 2.23 2.18 2.56 2.11 Dodiff3 (mDO) 6.08 9.11 17.32 24.24 33.5

[0185] Table 8.

[0186] Next, a dose-response curve is performed by adding an anti-mouse antibody.

[0187] For this purpose, the samples include:

[0188] - 57 μL of 50 mM HEPES buffer, pH 7.5; F108 0.8%; 800 mM NaCl;

[0189] - 15 pL of PSA (P3338, Sigma-Aldrich) at the desired concentration in a 9 g / L NaCl solution + 5 nM anti-mouse antibody;

[0190] - 3 pL of P4 particles 1% w / v; and

[0191] - 3 pL of particles 214 1% w / v.

[0192] We therefore have an antibody concentration of 1 nM in the reaction medium. After application of the Cagg cycle, we obtain the results shown in Table 9 below:

[0193] [Tables9] [PSA] (nM) 0 1 3 5 10 ADO (mDO) 25.62 30.98 38.59 42.72 49.9 Dodiffo (mDO) 2.23 2.48 2.17 2.35 2.07 Dodiff3 (mDO) 23.89 30.63 33.57 39.42 45.52

[0194] Table 9

[0195] Figure 13 shows these results in a graph.

[0196] It is observed that, in the absence or presence of anti-mouse antibodies, the Dodiffo value does not vary significantly and therefore does not allow for the prediction of the presence of an interfering molecule. However, this molecule leads to an overestimation of Dodiff3 or ADO values. Thus, by plotting the ADO values ​​measured in the presence of the antibody onto the dose-response curve in the absence of antibodies, used as a standard curve, one would end up with an overestimation of PSA titers. For example, the sample containing no PSA would be titrated at approximately 5 nM, and the sample containing InM of PSA would be titrated at more than 8 nM.

[0197] Example 7: PSA dose-response curves

[0198] Initially, a dose-response curve is performed without interfering antibodies.

[0199] The samples include:

[0200] - 57 μL of 50 mM HEPES buffer, pH 7.5; F108 0.8%; 800 mM NaCl;

[0201] - 15 pL of PSA (M8642, Sigma-Aldrich) at the desired concentration in a 9 g / L NaCl solution; and

[0202] - 3 pL of P4 particles 1% w / v.

[0203] The Cagg cycle is applied to this sample, then 3 pL of 214 1% w / v particles are added to the medium and the Cagg cycle is applied again.

[0204] The results shown in Table 10 below are obtained as a function of the amount of PSA present in the reaction medium:

[0205] [TableauxlO] [Ac] (nM) 0 1 3 5 10 ADO1 (mDO) 1.39 0.93 1.07 1.23 1.89 Dodiffo1 (mDO) 1 0.72 0.86 0.9 0.77 DodifC1 (mDO) 1.82 1.78 2.19 2.58 3.21 ADO2 (mDO) 2.22 11.51 22.98 33.79 51.34 Dodiffo2 (mDO) 3.21 2.98 3.6 3.44 4.57 Dodiff32 (mDO) 4.87 14.03 25.83 34.35 47.64

[0206] Table 10.

[0207] It is observed that while the values ​​of Dodiff3 or ADO increase with the amount of PSA after the second agglutination cycle, as expected, they do not vary significantly after the first agglutination cycle, since specific aggregates only form between particles carrying the P4 antibody and particles carrying the 214 antibody. These samples will therefore not be alarmed by maintaining the hypothesis of a threshold at 5 mDO as described above.

[0208] A dose-response curve is then performed by adding an anti-mouse antibody.

[0209] The samples include:

[0210] - 57 μL of 50 mM HEPES buffer, pH 7.5; F108 0.8%; 800 mM NaCl;

[0211] - 15 pL of PSA (M8642, Sigma-Aldrich) at the desired concentration in a 9 g / L NaCl solution + 5 nM anti-mouse antibody; and

[0212] - 3 pL of P4 particles 1% w / v.

[0213] The Cagg cycle is applied to this sample, then 3 pL of 214 1% w / v particles are added to the medium and the Cagg cycle is applied again.

[0214] The results shown in Table 11 below are obtained as a function of the amount of PSA present in the reaction medium:

[0215] [Tables 11] [Ac] (nM) 0 1 3 5 10 ADO1 (mDO) 13.96 14.92 13.9 11.83 11.63 Dodiffo1 (mDO) 0.99 0.89 0.87 0.8 0.72 DodifC1 (mDO) 12.7 13.95 13.88 11.82 12.09 ADO2 (mDO) 19.65 28.29 40.83 48.06 58.91 Dodiffo2 (mDO) 13.72 15 14.69 12.69 13.06 Dodiff32 (mDO) 28.54 36.01 44.95 47.54 56.29

[0216] Table 11

[0217] The values ​​of Dodiff32 or ADO2 are higher, at a given PSA concentration, than in the case without interfering antibodies. However, unlike the standard measurement, here the measurement of Dodiff32 or ADO1 allows the presence of the interfering molecule to be detected. It is therefore possible to apply an alarm to avoid reporting an erroneous result.

[0218] Fig. 14 shows the differences in optical density (mDO) of Dodil'L1 respectively with and without interfering antibody.

[0219] The process of the invention can be carried out in a device of a known type, if necessary adapted by means of the knowledge of a person skilled in the art.

[0220] .

Claims

1. Claims Method for measuring a target analyte (20) in a biological sample in a liquid medium, comprising the following steps: a. bringing the biological sample into contact with first magnetic particles (10) carrying a first receptor (101) specific to a first attachment site of the target analyte (20) so as to form first complexes by the binding of first magnetic particles (10) with the target analyte (20), this contacting being accompanied, when an interfering analyte (30) is present in the sample, by the formation of interfering complexes by the non-specific binding of said interfering analyte (30) to the first magnetic particles (10); b. the application of a first magnetic field, and its maintenance, so as to locally bring together all of the complexes formed in step a., and where appropriate to agglomerate interfering complexes together to form interfering aggregates; c. canceling the first magnetic field applied in step b. and adding into the liquid medium second magnetic particles (11) carrying a second receptor (111) specific to a second attachment site of the target analyte (20); d. the measurement of a first quantity representative of the quantity of interfering aggregates in the liquid medium, to identify the presence or absence of said interfering aggregates; e. applying a second magnetic field so as to form second complexes (61) by binding the first complexes with second magnetic particles (11); and f. measuring a second quantity representative of the total quantity of interfering aggregates and the quantity of second complexes (61) in the liquid medium so as to determine the quantity of second complexes (61) formed in step e. as a function of the first quantity to deduce therefrom the quantity of target analyte (20) present in the biological sample, and where appropriate the quantity of interfering analyte (30).

2. A dosing method according to claim 1, wherein step f. comprises calculating the difference between the second quantity measured in this step f. and the first quantity measured in step d.

3. A method according to any preceding claim, wherein steps c. and d. are performed substantially simultaneously.

4. Method according to one of the preceding claims, in which step d. is carried out after the cancellation of the first magnetic field, before the addition of second magnetic particles to the liquid medium.

5. Method according to one of the preceding claims, in which the maintenance of the magnetic field applied in step b. is less than or equal to a duration of 5 minutes, preferably less than or equal to a duration of 3 minutes.

6. Method according to one of the preceding claims, in which the application of the second magnetic field comprises a magnetization at 8 mT for 1 second, followed by three successive sequences of magnetizations and subsequent cuts: 15 mT for 60 seconds, 0 mT for 28 seconds, 8 mT for 1 second, 0 mT for 1 second.

7. Method according to one of the preceding claims, in which the measurement of the first and / or the second quantity is chosen from the group consisting of a measurement by turbidimetry, a measurement by ne-phelometry and a measurement by counting.

8. A method according to any preceding claim, wherein step a. comprises adding a diluent so as to dilute the biological sample, the ratio of sample to diluent being greater than or equal to 1:10 (volume).

9. Method according to one of the preceding claims, in which step b. comprises the extraction of the first complexes from the liquid medium.

10. Method according to one of the preceding claims, in which step b. comprises a sub-step b1. comprising the removal of the liquid medium followed by the addition of a reaction buffer, and preferably at least one wash between the removal of the liquid medium and the addition of the reaction buffer.

11. The method of claim 10, wherein the reaction buffer comprises at least one anti-aggregation agent and / or one lysis agent. cellular.

12. A method according to any preceding claim, wherein the sample is a whole blood sample.

13. Method according to one of the preceding claims, in which the interfering aggregates are identified when the first quantity exceeds a predefined threshold quantity.

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