EP1463946A2

EP1463946A2 - Method for obtaining a unique calibration system applied to multi-parametric doses of biological samples, immunological reagent prepared for that purpose, and dosage method

Info

Publication number: EP1463946A2
Application number: EP03709868A
Authority: EP
Inventors: Pascale Laroche
Original assignee: Biomedical Diagnostics SA
Current assignee: Biomedical Diagnostics SA
Priority date: 2002-01-11
Filing date: 2003-01-10
Publication date: 2004-10-06
Also published as: WO2003058244A3; FR2834796A1; AU2003214303A8; WO2003058244A2; US20050074902A1; CA2473027A1; JP2005514622A; AU2003214303A1; FR2834796B1

Abstract

The invention uses different categories of particles, each of which is sensitized by a specific ligand for one of the analytes to be dosed. An immunological reagent consisting of a mixture of each of said particle categories having been sensitized by a specific quantity of the respective ligand is prepared. The signals resulting from the interaction between said immunological reagent and the biological sample on one hand and a calibration standard on the other are then measured. A correction factor is subsequently determined which is applied to the different resulting signals in such a way that each analyte of the sample can be titrated in biological units.

Description

Method for obtaining a single calibration system applied to multi-parameter assays of biological samples, immunological reagent prepared for this purpose and assay method.

The present invention relates, in general, to the field of assays of analytes from biological samples and, more particularly to the calibration of such assay methods. Specifically, the present invention relates to a new process for obtaining a single calibration system, making it possible to simultaneously quantify several analytes, whatever their number and in the same reaction medium. By "single" calibration system is meant a single system regardless of the number of analytes sought simultaneously.

The quantification of the analytes included in a biological sample having become essential both in terms of research and of classical medicine, this has been the subject of numerous developments aimed not only at improving the reliability and reproducibility of the assays, but also to lower costs. The conventional techniques consist in assaying each analyte separately and, to do this, in carrying out for each of said analytes a proper calibration of the system used. This results in a large number of manipulations and a significant consumption of reagents, which results in a relatively high price.

If such a way of operating remains possible in the context of research where the quantity of parameters to be measured is limited and targeted, it poses a serious problem of cost in the context of medicine, where, in analysis laboratories, the 'It is sought to rapidly assay a large number of analytes. It has recently been described, in particular in international patent applications WO 99/36564 and WO 97/14028, a technique making it possible to quantify, in a precise and reproducible manner, several analytes in a single and same assay from the same sample. This technique is based on the use of different categories of fluorescent particles, each category of particles being sensitized by ligands having different properties, said ligands being adapted to bind directly or indirectly to the analytes and generally consisting of antibodies or antigens. The reaction between ligand and analytes is quantified by means of a fluorescent compound with excitation and emission wavelengths different from those of the fluorescent particles.

Thus, a simple reading by flow cytometry makes it possible to quantify the reaction associated with each category of particles and therefore to deduce from said quantification the amounts of the different analytes present in the same sample.

However, this quantification requires beforehand the construction of as many calibration systems as analytes to be assayed, that is to say to carry out X * Y assays reserved for calibration, X being the number of analytes to measure simultaneously (or the number of particles sensitized by ligands with different properties) and Y the number of calibration points. This method therefore involves not only an additional handling cost, but also a practical management problem, due to the size of the wells of the micro-titration plates, (the tests being generally carried out on ELISA micro-titration plates of 96 wells), as well as an increase in handling time. For example, for the simultaneous determination of 10 analytes, this system involves the constitution of 10 different calibration systems, i.e. for 100 samples tested, an additional consumption of reagents ranging from 10 to 40% depending on the number of calibration points, and an obvious problem of congestion.

The present invention aims to remedy the drawbacks of the prior art by proposing a new unique, rapid, simple and reproducible calibration method making it possible to reduce the consumption of reagents devoted to calibration, while also reducing the size of the plates. of titration. This object is achieved in the sense that the present invention relates to a calibration method, requiring only one calibration system, making it possible to simultaneously quantify several analytes in the same biological sample, a method which is based on the preparation and use of a specific immunological reagent.

More particularly, a first aspect of the present invention relates to a method for the preparation of an immunological reagent used in the composition of a calibration system applied to the assay of multiple analytes in the same biological sample, said method implementing different categories of particles, each category of particles being sensitized by association with a specific ligand of one of the analytes to be assayed, said method comprising the steps which consist in: a) determining, for each category of sensitized particles and for each of n quantities ligand data associated with said particles, the response curve as a function of the concentration of homologous compound over a range of concentrations corresponding to the known measurement range of the analyte to be assayed; b) select, for each category of sensitized particles, the curve corresponding to the smallest quantity of ligand which gives a significant response signal and which is compatible with the use of a sample dilution and a labeling reagent common to all the analytes simultaneously dosed; c) evaluating, for each category of sensitized particles, from the curve chosen in step b), the average signal corresponding to the signal associated with a characteristic point of each of said curves, thus obtaining as many average signals as categories of particles; d) adjust, if necessary, the quantities of ligand associated with each category of particles so that the set of average signals evaluated in step c) is included in a ratio of 1 to 5, and e) mix, in an appropriate solvent, the different categories of sensitized particles which meet the criterion of step d).

The method which is the subject of the present invention therefore applies to assay methods using different categories of sensitized particles. More particularly, such methods consist mainly of immunoassays of the type:

- direct assay, by an immunometric or "sandwich" method, for example, of an antibody which has reacted with the specific antigen fixed beforehand on the particles,

direct assay, by an immunometric or "sandwich" method, for example, of an antigen which has reacted with the specific antibody fixed beforehand on the particles, or

- indirect assay, by a method called "by competition", of an antigen entering into competition with a similar labeled antigen, for its reaction with the antibody previously fixed on the particles, or vice versa.

These different dosages will be described in more detail below.

The reaction developed by bringing the different categories of particles, each sensitized by a ligand, into contact with the analytes to be assayed is measured by flow cytometry according to different modes of particle identification. Mention may be made, for example, of particles of different colors and of uniform size (identified according to their specific color, with quantification of the analyte-ligand reactions by addition of an external compound with excitation and emission lengths different from those of particles), particles of different sizes and identical color (identified according to their size, with quantification of the analyte-ligand reactions by measuring the size of the resulting aggregates) or particles of different sizes and of identical color (classified according to their size , with quantification of the analyte-ligand reactions by adding an external compound with excitation and emission wavelengths different from those of the particles). In practice, one or the other of these methods is selected as a function of the more or less large number of parameters to be tested.

These particles generally consist of polystyrene. However, they can also be made of any polymer which gives them specific physicochemical properties (functionality, density or even solubility) such as styrene-maleic acid, styrene-methacrylic acid, styrene-acrylamide or poly (methyl methacrylate). ).

By "ligand" is meant an antigen, an antibody, mono- or polyclonal, or any molecule capable of binding to the particles and which is capable of interacting, in any way whatsoever, directly or indirectly, with analyte to be assayed.

The term "antigen" means any substance from which the production of antibodies can be generated. Among these substances, mention may be made of proteins, haptens, allergens, peptides, medicaments or even drugs.

By “homologous compound”, it is necessary to understand an antibody, an antigen or any molecule capable of binding to a given ligand and whose interaction site with said ligand is similar to the interaction site between 1 analyte to be assayed and said ligand. The homologous compound can be the analyte itself.

The first step of the process therefore consists in determining for each category of sensitized particles, as described above, the correlation which exists, for a given quantity of ligand fixed to the particles, between the quantity of homologous compound and the magnitude of the signal emitted, representative of ligand / analyte interactions. In practice, for each category of particles, several tests are carried out by fixing a given quantity, different for each test, of the same ligand to said particles, then bringing the particles thus sensitized into contact with different quantities of homologous compound, identical for all the tests, and by measuring the signal resulting from their interaction.

More particularly, the quantities of ligand used vary in steps from 2 to 4. The different quantities of homologous compound are selected so as to be included in the measurement range of the analyte which it is sought to assay. Thus, if one seeks to assay an analyte whose concentration can vary between 0 and 100 biological units, one will choose, for example, six given quantities of homologous compound corresponding to 0, 20, 40, 60, 80 and 100 biological units . In practice, a population of samples characterized and certified on clinical-biological criteria is used, that is to say the presence or absence of a given analyte is validated by a clinical diagnosis and / or by the estimation of the analyte by one or more other assay method (s).

The result of this operation results in obtaining a dose / response curve between the quantity of homologous compound and the signals obtained for each of the n given quantities of ligand. The concept of expression of the concentration in biological units is based on the assignment, for example, of a range 0-150 to the measurement range of each of the analytes to be assayed. It is understood that these measurement ranges differ, in absolute terms, from one analyte to another and that, consequently, the same concentration of different analytes expressed in biological units, for example 95 biological units (for short 95 UB) does not correspond to the same concentration, in absolute terms, in these same analytes. The concentration of homologous compounds is expressed, in the same way, in biological units over an identical range for all the analytes.

At this level, it should be noted that the sensitization of the particles can be carried out in various ways, namely by covalence, by means of an intermediate molecular layer biologically and / or chemically reactive, or by the use of a system. of affinity interaction.

The sensitized particles, insofar as they contain a reactive functionality, can be sensitized by covalence. Thus, numerous immobilization protocols have been described as involving, without limitation, the following functional groups between the particles and the ligand:

- COOH / R-NH ₂ or R-OH,

- CO-NH ₂ / NH ₂ -NH ₂ ,

- C (= 0) -0- CH ₃ / NH ₂ -NH ₂ ,

- NH ₂ CHO / RCOOH, or

R-NH ₂ or NH ₂ - H,

It should be noted that, when using most of the couples mentioned above, it is necessary to activate certain functions beforehand, such as for example the COOH functions, so that they can react, for example, with the groups. functional ligand, such as aliphatic or aromatic amino groups. According to another preferred embodiment, the activation can be carried out by water-soluble carbodiimides, and be carried out in a single stage or, according to a particularly preferred embodiment, in two stages, with the carbodiimide alone or in the presence of N-hydroxysuccinimide. It is followed by the elimination of the excess activator before fixing the ligand.

According to yet another embodiment, a homo- or hetero-bifunctional spacer arm, having reactive chemical functions at each end, respectively identical or different, can be attached to the previously activated particles. These arms of variable length and functionality can be activated and covalently coupled, firstly, to the particles by one end, and secondly, to the compound to be fixed, by the other end.

Chemically or biologically active intermediates can also be used for sensitization. They create an intermediate molecular layer on the surface of the microparticles consisting of a protein (such as albumin), a mixture of proteins or a polymer (such as polylysine). This process is particularly used in the case of small particles which can then be coupled via bonding agents on the functions of this intermediate layer.

Another sensitization pathway, this time non-covalent, consists in using, without limitation, affinity interaction systems: - the biotin-avidin system, using particles coupled to avidin which are brought into contact with compounds having been previously biotinylated, - protein A or protein G, having a receptor for the Fc fragment of the immunoglobulins, allowing the immobilization of an antibody and the outward orientation of its fragments Fab, responsible for antigenic recognition, or - indirect immobilization via an antibody specific for the compound to be fixed. This list is in no way limiting and it is obvious that any similar sensitization method known to a person skilled in the art can also be used.

For each ligand to be fixed, the selection of one of the above systems is carried out initially during the initial development phase. The operating conditions are then fixed and subsequently applied systematically.

In a preferred embodiment, the sensitization medium consists of buffered water having a pH between 5 and 9 and an ionic strength between 0.01 and 0.1. In order to keep the medium constant, the ligand can be suspended in the same medium before carrying out said sensitization.

The advantage of such a system lies in its simplicity of use, its low cost and its reaction independence compared to the other simultaneous reactions. This is made possible by the fact that the reaction produced by each category of sensitized particles is standardized and reproducible, that the signal provided by this reaction is representative of all the ligand-analyte reactions developing simultaneously and that all of these reactions is controlled at the level of the sensitization process of each category of particles so that they all produce a similar signal in the presence of samples of the same degree of positivity, that is to say comprising substantially the same amount in the assayed analyte as expressed on its particular range of biological units.

Once the dose / response curves have been obtained, it is determined, for each category of sensitized particles, as described in step b), the curve corresponding to the smallest quantity of ligand giving, graphically, a significant read signal compatible with the use of a sample dilution as well as a common labeling reagent for all the analytes simultaneously detected. "Significant response signal" means a signal which is sufficiently high to ensure reading accuracy over the entire measurement range.

In practice, the curve selected must be substantially linear and have a sufficiently high measurement amplitude.

Each type of particle is therefore sensitized by one of the techniques described above using the quantity of ligand determined above.

The next step c) then consists in determining the mean signal (SM) associated with a concentration in biological units, identical for all the analytes.

To do this, a point characteristic of the dose / response curve used in step b) is selected and the signal corresponding graphically to said point is taken as the mean signal SM.

The characteristic point, otherwise called positive point or point of high positivity, is chosen arbitrarily in the upper quarter of the linear part of the curve. The average signals SM obtained for the different categories of sensitized particles are then compared with one another and, if necessary, readjusted so that they are all included in a ratio of 1 to 5. In practice, for the categories of particles having a SM not included in said report, the reactivity is then readjusted by selecting in the dose / response curve another quantity of ligand and recommencing the operation of sensitizing said particles with this new quantity of ligand until the resulting SM enters the ratio of 1 to 5 above. The fact that the SM of the different sensitized particles constituting the immunological reagent are all included in a ratio of 1 to 5 is decisive for ensure a relatively equivalent immunological reactivity for each type of sensitized particles used and therefore to obtain, in a range of measurements compatible with biological reality, values sufficiently close to allow the use of a single calibration system common to all the analytes.

The immunological reagent is then constituted by mixing, in a solvent, the different types of sensitized particles thus obtained.

Mention may be made, as possible solvents: mixtures of proteins and amino acids or else a mixture of proteins inert with respect to the analytes to be assayed and allowing particle stabilization. In any event, the solvents used are selected so as to allow the conservation of all the sensitized particles, and their choice is therefore dictated by the nature of the most restrictive ligands. Therefore, said solvent can be defined so that it is suitable and common to all of the ligands.

In order to make such a process for the preparation of the immunological reagent industrializable, involving the individual reactivity of different particles coupled to specific ligands, the experimental sensitization conditions, namely the pH, the ionic strength, the volume, the temperature or even the duration of the coupling, are previously fixed and standardized for all the particles. Only the quantity of ligand to be adjusted then varies, as described above.

In practice, the solvent used is buffered at a pH of between 7 and 9 and has an ionic strength of between 0.01 and 0.1.

According to a second aspect of the present invention, the latter relates to an immunological reagent intended for the determination of multiple analytes in biological samples, said reagent comprising a solvent and, mixed within said solvent, different categories of particles, each of which is sensitized by association with a given quantity of a specific ligand of one of the analytes to be assayed, with, for each of the categories of particles, and for a given concentration of compound homolog of the ligand, as expressed in biological units, the said given quantity of ligand resulting in a signal called "average signal", which is included in a ratio of 1 to 5 with the average signals obtained for the other categories of particles.

According to a third aspect, the present invention relates to a kit for assaying multiple analytes in biological samples using different categories of particles, each category of particles being sensitized by a specific ligand for one of the analytes to be assayed, which comprises : i) an immunological reagent resulting from the process as described above, ii) at least one calibration standard consisting of a single homologous compound reacting with one of the categories of particles entering into the composition of the immunological reagent, iii) a correlation table between the concentration, expressed in biological units, of the homologous compound constituting the calibration standard and that of each of the compounds homologous to the other ligands attached to the other categories of particles entering into the composition of the immunological reagent, and iv) a labeling reagent. More particularly, said calibration standard comprises a homologous compound reacting directly or indirectly with the ligand attached to the sensitized particle.

Indirect fixation can be done, for example, via a protein used to saturate the binding sites of the particle not occupied by the ligand. This protein must be chosen so as not to interfere with the specific system and be present on a single particle category. In this case, the quantification medium must contain a specific labeling reagent for the analytes to be assayed (consisting of one or more compounds) and a specific labeling reagent for the inert protein.

In a first embodiment, said homologous compound is of the same origin as the analyte to be assayed, for example, of human origin.

In a second embodiment, said homologous compound and the analyte to be assayed are of different origins.

While, for example, the analyte to be assayed is of human origin, such as autoantibodies present in human blood samples, the homologous compound may be, for example, of animal origin as long as it is capable of reacting specifically with the ligand. The quantification medium must, in this case, contain both a specific labeling reagent for samples of human origin and a specific labeling reagent for the standard of animal origin.

The preparation of the calibration standard involves prior optimization of its concentration so as to produce an SM comprised in a ratio of 1 to 5 compared to the SM obtained for each category of particles sensitized during the preparation of the immunological reagent.

As for the "correspondence table", this consists of a reference system, which can be in the form of a table, a graph, etc., which gives the different concentrations, expressed in biological units, of the homologous compound constituting the calibration standard and that of each of the compounds homologous to the other ligands attached to the other categories of particles entering into the composition of the immunological reagent. The correspondence table is produced empirically, prior to the preparation of the kits, in the following manner.

Several samples are reacted, comprising different and known amounts of analytes to be assayed with the immunological reagent which will be part of the kit. The resulting signals are measured and, for each analyte, the linear regression line is drawn linking the signals emitted to the known quantities, expressed in biological units, of the assayed analytes, according to an equation of type y = ax, with y = signal and x = concentration in biological units.

The signal obtained during the reaction between the calibration standard and the immunological reagent is likewise measured and the concentration, expressed in biological units, corresponding to this same signal value, is noted on each curve previously obtained. .

It is all of these analyte concentrations, expressed in biological units, which constitute the correspondence table. The concentration of the standard can then be checked from the correspondence table and adjusted if necessary.

In a variant, it may be desired to carry out a calibration range, that is to say from several calibration standards each consisting of the same homologous compound, but according to different concentrations.

In this case, the principle of realization of the correspondence table remains the same, except that we trace for each analyte, from the different signals obtained, the regression line linking the signals sent to the known quantities, in units biological, analytes to be measured according to a log equation of type y = a (log x), with y = signal and x = concentration in biological units.

This equation is then applied to the signals corresponding to the different known concentrations of calibration standard so as to deduce the correspondence between each of said concentrations and the concentration of each analyte, expressed in biological units.

As regards the "labeling reagent", this consists of an immunological compound capable of quantifying the reaction between the analytes and the immunological reagent, said immunological compound being coupled to a marker which is preferably a fluorochrome.

By "immunological compound" is meant any compound, natural or synthetic, capable of complexing specifically with a complementary compound, such as antibodies and antigens.

According to a preferred embodiment, said labeling reagent reacts with the complex of homologous compound or analyte to be assayed / ligand, in which case the assay is direct, or with the uncomplexed ligand, in which case the assay is indirect.

In addition, the labeling reagent can be prepared from a single compound, reacting with, for example, an antigen specificity common to all of the homologous compounds, or result from the mixture of different compounds reacting specifically with different homologous compounds.

According to a last aspect of the present invention, it relates to a method for assaying analytes present in a biological sample and, more particularly, a method for implementing the kit as described above which consists in measuring the signals resulting from the interaction between the immunological reagent and, on the one hand, the biological sample, on the other hand, the calibration standard, and determining and applying, to the various resulting signals, a correction factor so as to obtaining the titration, expressed in biological units, of each analyte in the sample, said correction factor being the ratio between the signal obtained for the calibration standard and the concentrations from the correspondence table. The method of implementing the kit according to the present invention therefore rests on three steps, namely a first step of determining the different signals emitted by flow cytometry, a second step of calculation to determine the different correction factors from the signal emitted by the calibration standard and a third calculation step for the titration of each analyte in the sample from the correction factor previously obtained and the signals emitted by the different samples tested.

More particularly, the method which is the subject of the present invention comprises the following steps: a) incubating, on the one hand, the biological sample and, on the other hand, the calibration standard, with a predetermined quantity of immunological reagent; b) add the labeling reagent; c) measuring, by flow cytometry, the signals emitted, on the one hand, by the calibration standard, on the other hand by the sample; d) determining, for each category of sensitized particles, a correction factor which corresponds to the ratio between the concentration in biological units of each analyte to be assayed as given by the correspondence table and the signal corresponding to the calibration standard and measured at step c); e) multiply by said correction factor, calculated for each analyte, the signal emitted by each category of particles and measured in step c) to deduce therefrom the concentration in biological units of each analyte in the test sample.

In a first variant, the method which is the subject of the present invention can be applied to direct dosages. In such assays, the labeling reagent is specific to the analytes which it is desired to assay and, therefore, the amount of analytes included in a sample is directly proportional to the signal emitted by said labeling reagent. A first application to the simultaneous and direct quantification of antibodies of different antigen specificities is envisaged with said analytes to be assayed which consist of antibodies, said ligands in antigens and said labeling reagent consists of one or more second antibodies labeled with a fluorochrome reacting specifically with the antibodies that one seeks to assay.

In this case, the immunological reagent consists of different categories of particles each sensitized by a specific antigen. The antibodies which one seeks to assay will complex with the said antigen (s) attached to the particles. The labeling reagent, which consists of one or more second antibodies labeled with a fluorochrome and specific for the antibodies that one seeks to assay, will bind to said antibodies previously complexed with the antigens forming the particle awareness layer. Thus, the signal emitted by the fluorochrome associated with each category of particles is proportional to the quantity of antibodies present in the sample.

By “second antibodies”, it is necessary to understand any substance capable of complexing with the antibodies which it is sought to assay, that is to say capable of reacting with an epitope of said antibodies different from that used to ensure their fixation to the ligand.

A second application to the simultaneous and direct quantification of different antigens is envisaged with said analytes to be assayed which consist of antigens, said ligands in antibodies and said labeling reagent in a mixture of second antibodies labeled with a fluorochrome reacting specifically with antigens that we are trying to dose.

The principle here is identical to that described above.

In a second variant, the method which is the subject of the present invention can be applied to so-called dosages indirect. Such assays are no longer based solely on the complementarity between two immunological species, but on the competition between two close immunological species to complex with a third immunological species.

Thus, according to a first possibility, it is envisaged the simultaneous and indirect quantification of different antigens with said analytes to be assayed which consist of antigens, said ligands in antibodies and said labeling reagent in a mixture of antigens labeled with an incoming fluorochrome in competition with the analytes to be assayed to complex with the ligands.

In this case, the immunological reagent consists of different categories of particles each sensitized by a specific antibody. The antigens that one seeks to assay will then enter into competition with the said antigen (s), labeled with a fluorochrome, constituting the labeling reagent. Thus, the increase in the concentration of antigen which it is sought to assay results in the increase in the concentration of antigen complexes which it is sought to assay / fixed antibody, to the detriment of the formation of labeled antigen / fixed antibody complexes. , with a corresponding decrease in signal. As a result, the signal emitted by the fluorochrome associated with each category of particles is inversely proportional to the quantity of antigens present in the sample.

A second application to the simultaneous and indirect quantification of different antibodies is envisaged with said analytes to be assayed which consist of antibodies, said ligands in antigens and said labeling reagent in a mixture of antibodies labeled with a fluorochrome competing with the analytes to be assayed to complex with the ligands.

The present invention will be better understood on reading the following examples. MATERIAL AND METHOD

The system of sensitized particles used in the examples below belongs to the Luminex system.

The particles used are of uniform size

(5.5 μm) and are distinguished from each other by a specific coloration (100 colors, from red to orange).

The detection system for these particles is a flow cytometer interfacing with a computer system for processing the signal emitted by different lasers: a first laser, classifying each category of particles on the basis of its unique fluorescence intensity, allows identify which compound is being analyzed. In parallel, a green laser excites an external fluorescent compound used to quantify the reaction specifically associated with each category of colored particles.

According to other embodiments, and according to the knowledge of a person skilled in the art, lasers other than a green laser can also be used.

Example 1: Simultaneous determination of antinuclear antibodies (ANA) directed against the following antigens: SSA, SSB, Sm. Sm / RNP, Scl70. Pretty. dsDNA, centromere.

a) Preparation of the immunological reagent. Eight categories of colored polystyrene particles functionalized by COOH groups are selected. Prior to sensitization, the particles are activated by a process which consists of:

- the separation of the eight categories of particles in eight different tubes,

- the addition of 1 ml of a solution of carbodiimide dosed between 10 and 40 mg / ml, freshly prepared, to 1 ml of each type of particle (i.e. approximately 10 particles),

- incubation for 20 to 60 minutes at room temperature, and - three washings of the particles with distilled water by centrifuging at 10000xg for 2 minutes to remove the excess carbodiimide.

In parallel, the quantities of each ligand, or antigen, to be attached to the different categories of particles were determined according to the invention, as described above.

The particles thus activated are then sensitized by different antigens, depending on the category of particles, by suspension of each solution of activated particles in a volume of 1 ml containing 50 to 500 μg of different antigens.

The following system is thus obtained:

The following steps consist of:

- incubation for 4 to 6 hours at room temperature, - washing of the particles with distilled water,

- Suspending in a pH 8 buffer containing a mixture of amino substances in order to saturate the free sites and reduce the particles to a concentration of the order of 10 to 5.10 particles per ml. The immunological reagent then consists of a mixture of an aliquot of 500 μl of each category of colored and sensitized particles, and it is stored at + 4 ° C until use.

Figures 1 and 2 give, by way of example, three dose / response curves obtained for two types of ligands (respectively SSA and SSB) expressing the fluorescence signal emitted (Y axis) as a function of the concentration of homologous compound in biological units (X axis). These curves make it possible to select the smallest quantity of ligand corresponding to a significant response signal, this over a measurement range from 0 to 150 biological units approximately.

For each system, five samples were selected according to their title in biological units, determined by ELISA (ENA-LISA kit, marketed by the applicant). The concentration of homologous compound, the test portion and the dilution are common.

With regard to the SSA system (Figure 1), the three curves shown A, B and C correspond respectively to concentrations of SSA antigens of 300, 150 and 75 μg / ml of particles. The curve used for the SSA system is curve B, corresponding to a concentration of 150 μg of SSA antigen per ml of particles. The higher concentration of 300 μg (curve A) results in saturation of the antigen-antibody reaction for the high values. The lower concentration of 75 μg

(curve C) does not make it possible to obtain a response of sufficient amplitude, hence a potential imprecision of the results.

With regard to the SSB system, the three curves represented A ', B' and C correspond respectively to SSB antigen concentrations of 100, 50 and 25 μg / ml of particles. The curve used for the SSB system is curve B ′, corresponding to 50 μg of SSB antigen per ml of particles. The higher concentration of 100 μg (curve A ') resulting in an almost identical curve is not retained. The lower concentration of 25 μg (curve C) does not make it possible to obtain a response of sufficient amplitude, in particular at low values.

b) Calibration.

Only one calibration standard is achieved. In this example, we choose the category of particles 1 attached to the SSA antigen. The calibration standard, that is to say the homologous compound, is here a solution, diluted in phosphate buffer pH 7.4, of human antibodies purified and directed against the SSA antigen.

The correspondence table for this calibration standard is as follows:

This table was obtained as indicated above.

c) Measurement of the signal emitted by the calibration standard For particles 1, sensitized to the SSA antigen, reacting with the calibration standard, an average signal (SM) is obtained by flow cytometry, tested at least in duplicate , corresponding to 80, for example.

d) Determination of correction factors.

In order to determine the correction factors to be assigned to each specificity, the above concentrations, expressed in biological units in the correspondence table, are divided by the value of the signal measured for the calibration standard, i.e. say the value obtained with the particles 1 sensitized with the SSA antigen (80).

The following correction factors are obtained:

These correction factors will be used when assaying analytes in biological samples. Thus, if we dose, using the immunological reagent which previously gave a signal of 80 with the calibration standard, the analyte SSB in a sample, and we obtain a signal corresponding to 120, we will be able to determine the titer of the SSB analyte by multiplying the value of this signal by the correction factor associated with the SSB analyte (here 0.75). The SSB titer in the sample will therefore, in this example, be 0.75 x 120 = 90 BU.

EXAMPLE 2 Simultaneous determination of anti-cytoplasm antibodies of neutrophils (ANCA) directed against the following antigens: myeloperoxidase (MPO) and proteinase 3 (PR3).

a) Preparation of the immunological reagent.

In this example, only two different colored particles are sensitized separately with the two antigens MPO and PR3, in an identical manner to example 1.

The immunological reagent is then constituted by a mixture of an aliquot of 500 μl of each category of colored particles. It is stored at + 4 ° C until use.

b) Calibration. For calibration, according to the very principle of the invention, a single calibration standard is used. In this example, the category of particles fixed to the MPO antigen is chosen. The calibration standard, that is to say the homologous compound, is here a solution, diluted in phosphate buffer pH 7.4, of human antibodies purified and directed against the MPO antigen. The protocol is the same as that used in Example 1. The correspondence table for this calibration standard is as follows:

c) Measurement of the signal emitted by the calibration standard The same protocol as in Example 1 is used to determine said signal. The average signal (SM) obtained for the calibration standard (tested in duplicate) reacting with MPO particles corresponds, for example, to 150.

d) Determination of the correction factors In order to determine the correction factors to be assigned to each specificity, the above concentrations, expressed in biological units, are divided by the value of the signal of the calibration standard, ie say the particles 1 sensitized to the MPO antigen (S = 150).

The following correction factors are obtained:

In a similar manner to example 1, if the dose, using the immunological reagent having previously given a signal of 150 with the calibration standard, the analyte PR3 in a sample, and that a signal is obtained corresponding to 110, the titer of the PR3 analyte can be determined by multiplying the value of this signal by the correction factor associated with the PR3 analyte (here 1.66). The title of PR3 in the sample will therefore, in this example, be equal to 1.66 x 110 = 183 BU. Example 3: evaluation of the reagents prepared in examples 1 and 2 and comparison with reference methods.

In order to evaluate the immunological reagents prepared in Examples 1 and 2 and compare their results with those of reference methods of the ELISA type, the specificity and the sensitivity of said reagents were studied.

a) Determination of specificity.

The specificity was assessed from 50 serum samples from blood donors and 34 samples selected for their potential biological interference (hypergammaglobulinelia, monoclonal gammopathies, other autoantibodies, plasma samples ...). These samples constitute group 1.

b) Determination of sensitivity.

The sensitivity was evaluated from serum samples, selected for each multiparametric assay, clinically characterized and resulting from the routine analysis of an immunology laboratory (Hôpital Tenon, Paris, France). These samples constitute group 2, that is 57 and 35 samples respectively for the detection of ANA and ANCA.

c) Protocol for evaluating the specificity and sensitivity of the samples:

This protocol consists in taking 50 μl of immunological reagent prepared in example 1 and mixing it, on the one hand, with 100 μl of calibration standard (deposited in duplicate) and, on the other hand, with 100 μl d 'samples prediluted to 1/200 in phosphate buffer (PBS type, pH 7.4). The deposits and mixtures are made in a 96-well microplate having at their base a 1.2 μm filter membrane. One well is reserved for mixing the immunological reagent with the buffer, alone, in order to constitute the “reagent blank”, making it possible to evaluate the signal associated with each category of particles which will then be systematically subtracted from the signal obtained for the other wells. The following steps consist of:

- incubate for 30 minutes at room temperature,

- wash twice with filtration through the filtering membrane of the microplate, - resuspend the medium in 100 μl of labeling reagent, consisting of a goat antibody reacting specifically with human immunoglobulins G and conjugated with phycoerythrine. Its concentration is adapted to all the "ligand-homologous compound" systems to be detected simultaneously,

- incubate for 30 minutes, and

- analyze by flow cytometry.

In doing so, at least 200 particles from each category are analyzed for 15 to 25 seconds depending on the number of categories present simultaneously. During this time, the computer system classifies each of the particles according to its color and then determines the average fluorescence emitted by the conjugate for each specificity of antibodies.

d) Comparative methods used.

The results obtained with the reagents prepared in examples 1 and 2 were compared with those resulting from the use of commercial assay kits (Biomedical Diagnostics, Marne-La-Vallée, France): ELISA kits (DNA-LISA, ENA-LISA, MPO-LISA and PR3-LISA) and Hep2000 substrate for the determination by indirect immunofluorescence of the centromere. RESULTS

Assessment of specificity (group 1 samples).

ANA detection: all the samples in group 1 were found negative by the multi-parameter test using the reagents prepared in Example 1 and by the protocol using an ELISA kit.

ANCA detection: all the samples in group 1 were found negative by the multiparametric test using the reagents prepared in example 2; only one result was discordant using an ELISA kit: it was a false positive for the determination of the PR3 antigen specificity developed by a sample from a patient with hypergammaglobulinemia IgG.

Sensitivity assessment (group 2 samples)

Detection of ANA: it was carried out on 57 clinically characterized samples for ^'detecting ANA from the group 2. A match of 98.7% was obtained of 399 tests carried out simultaneously with the multi-test using the reagents prepared in 'example 1 and with an ELISA kit. Figure 3 attached shows the comparative results obtained, expressed in biological units (BU), for the multi-parameter test (Y axis) compared to the individual ELISA test (X axis). The correlation coefficients are between 0.92 and 0.97 for the ANA specificities. For the centromere, total agreement was obtained with the indirect immunofluorescence (10 positive samples and 47 samples negative). The indirect immunofluorescence method consists of a semi-quantitative method using as substrate human transfected Hep-2 tumor cells. The autoantibodies attached to the substrate are revealed using a human anti-immunoglobulin conjugate labeled with fluorescein isothiocyanate. The presence of anti-centromere antibodies results in a speckled fluorescence on the nucleus at the interphase. The reading is carried out under a fluorescence microscope, determining the last dilution of the sample where this fluorescence remains visible.

ANCA detection: it was carried out on 35 samples clinically characterized for the detection of ANCA and from group 2. A concordance of 97.1% was obtained on 70 tests carried out simultaneously with the multi-parameter test using the reagents prepared in the example 2 and with an ELISA kit. Figure 4 appended presents the comparative results obtained, expressed in biological units (BU), for the multiparametric test

(Y axis) compared to the individual ELISA test (axis of

X). The correlation coefficients are between 0.87 and 0.85 for the ANCA specificities. These lower coefficients than those of ANA detection are due to the strong amplification of positive values in the case of multiparametric dosing.

Claims

1. A method of preparing an immunological reagent used in the composition of a calibration system applied to the assay of multiple analytes in the same biological sample, said method using different categories of particles, each category of particles being sensitized by association with a specific ligand of one of the analytes to be assayed, characterized in that it comprises the steps which consist in: a) determining, for each category of sensitized particles and for each of n given quantities of ligand associated with said particles , the response curve as a function of the concentration of homologous compound over a range of concentrations corresponding to the known measurement range of the analyte to be assayed; b) select, for each category of sensitized particles, the curve corresponding to the smallest quantity of ligand which gives a significant response signal and which is compatible with the use of a sample dilution and a labeling reagent common to all the analytes simultaneously dosed; c) evaluating, for each category of sensitized particles, from the curve chosen in step b), the average signal corresponding to the signal associated with a characteristic point of each of said curves, thus obtaining as many average signals as categories of particles; d) adjust, if necessary, the quantities of ligand associated with each category of particles so that the set of average signals evaluated in step c) is included in a ratio of 1 to 5, and e) mix, in an appropriate solvent, the different categories of sensitized particles which meet the criteria of step d).

2. Method according to claim 1, characterized in that, in step a), the quantities of ligand used vary in steps from 2 to 4.

3. Method according to claim 1 or 2, characterized in that the sensitization of said particles by said ligands is carried out covalently, by means of a biologically and / or chemically reactive intermediate molecular layer, or also by the use of an affinity interaction system.

4. Method according to any one of the preceding claims, characterized in that the concentration of homologous compound is expressed in biological units over a range which is identical for all the analytes.

5. Method according to any one of the preceding claims, characterized in that the solvent used in step e) is suitable and common to all of the ligands.

6. Method according to any one of the preceding claims, characterized in that said ligands consist of antigens and / or antibodies.

7. Immunological reagent intended for the assay of multiple analytes in biological samples, said reagent comprising a solvent and, mixed within said solvent, different categories of particles, each of which is sensitized by association with a given quantity of a specific ligand of l 'one of the analytes to be assayed, characterized in that, for each of the categories of particles, and for a given concentration of homologous compound of the ligand, as expressed in biological units, said given quantity of ligand results in a signal called "signal medium ", which is included in a ratio of 1 to 5 with the average signals obtained for the other categories of particles.

8. Kit for assaying multiple analytes in biological samples using different categories of particles, each category of particles being sensitized by a specific ligand of one of the analytes to be assayed, characterized in that it comprises: i) an immunological reagent from the method according to any one of claims 1 to 6, ii) at least one calibration standard consisting of a single homologous compound reacting with one of the categories of particles entering into the composition of the immunological reagent, iii) a correspondence table between the concentration, expressed in biological units, of the compound homologous constituting the calibration standard and that of each of the compounds homologous to the other ligands attached to the other categories of particles entering into the composition of the immunological reagent, and iv) a labeling reagent.

9. Kit according to claim 8, characterized in that said calibration standard comprises a homologous compound reacting directly or indirectly with the ligand attached to the sensitized particle.

10. Kit according to claim 8 or 9, characterized in that said homologous compound is of the same origin as the analyte to be assayed.

11. Kit according to claim 8 or 9, characterized in that said homologous compound and the analyte to be assayed are of different origins.

12. Kit according to any one of claims 8 to 11, characterized in that said labeling reagent consists of an immunological compound capable of quantifying the reaction between the analytes and the immunological reagent, said immunological compound being coupled to a marker which is , preferably a fluorochrome.

13. Kit according to claim 12, characterized in that said labeling reagent reacts with the complex homologous compound or analyte to be assayed / ligand, in which case the assay is direct, or with the uncomplexed ligand, in which case the assay is indirect.

14. Method of implementing the kit according to any one of claims 8 to 13, characterized in that it consists in measuring the signals resulting from the interaction between the immunological reagent and, on the one hand, the sample organic, on the other hand, the standard calibration, and to determine and apply, to the various resulting signals, a correction factor so as to obtain the titration, expressed in biological units, of each analyte in the sample, said correction factor being the ratio between the signal obtained for the calibration standard and the concentrations from the correspondence table.

15. The method of claim 14, characterized in that it comprises the steps which consist in: a) incubating, on the one hand, the biological sample and, on the other hand, the calibration standard, with a quantity predetermined immunological reagent; b) add the labeling reagent; c) measuring, by flow cytometry, the signals emitted, on the one hand, by the calibration standard, on the other hand by the sample; d) determining, for each category of sensitized particles, a correction factor which corresponds to the ratio between the concentration in biological units of each analyte to be assayed as given by the correspondence table and the signal measured in step c) for the calibration standard; e) multiply by said correction factor, calculated for each analyte, the signal emitted by each category of particles and measured in step c) to deduce therefrom the concentration in biological units of each analyte in the test sample.

16. The method of claim 15, applied to the simultaneous and direct quantification of antibodies of different antigenic specificities, characterized in that said analytes to be determined consist of antibodies, said ligands consist of antigens and in that said labeling reagent consists of one or more second antibodies labeled with a fluorochrome reacting specifically with the antibodies that one seeks to assay.

17. Method according to one of claims 15 or 16, applied to the simultaneous and direct quantification of different antigens, characterized in that said analytes to be determined consist of antigens, said ligands consist of antibodies and in that said labeling reagent consists of a mixture of second antibodies labeled with a fluorochrome reacting specifically with the antigens which are try to dose.

18. The method of claim 15 or 16, applied to the simultaneous and indirect quantification of different antigens, characterized in that said analytes to be determined consist of antigens, said ligands consist of antibodies and in that said labeling reagent consists of a mixture of antigens labeled with a fluorochrome competing with the analytes to be assayed to complex with the ligands.

19. The method of claim 15 or 16, applied to the simultaneous and indirect quantification of different antibodies, characterized in that said analytes to be determined consist of antibodies, said ligands consist of antigens and in that said labeling reagent consists of a mixture of antibodies labeled with a fluorochrome competing with the analytes to be assayed to complex with the ligands.