WO2006082318A1 - Immunochromatographic method for the quantitative measurement of analytes in a liquid sample - Google Patents

Abstract

The invention relates to a method for the quantitative measurement of at least one analyte in a liquid sample by means of immunochromatography, said method comprising a weighted measurement of the quantity of analyte in relation to a control. The method is particularly suitable for medical diagnosis in the hemostasis field, for example in order to exclude a risk of venous thromboembolism diagnosis.

Description

Method of quantitative measurement by immunochromatography, of analgests in a liquid sample

The present invention relates to a method for quantitatively measuring at least one analyte of interest in a liquid sample by immunochromatography, as well as the device used for this purpose.

Appeared in the late 1980s, the technology of immunochromatography has grown significantly. It now offers a wide range of applications, from the traditional fields of medical or veterinary diagnosis, to the environment or the food industry.

Immunochromatography is one of a group of diagnostic methods based on affinity binding reactions between members of specific binding pairs, grouped under the generic term of immunoassays.

Overall, according to the approach used, the immunoassays are divided into two broad categories: those involving competition for a specific ligand between the desired analyte and a labeled analyte and those called "sandwich" type, in which the binding of the analyte to a first specific ligand is revealed by a second labeled specific ligand attached to said analyte.

Immunoassays have progressively evolved towards devices that are more and more simple to use, allowing the development of routine diagnostic methods that are quick and at a moderate cost. This evolution has been particularly noticeable in the medical field, with the emergence of "point of care" or "home testing" diagnosis, in which a diagnosis is made directly near the patient's bed or at home, without it is necessary to use automated laboratory analysis techniques.

Immunochromatography is one of the technologies used for this type of rapid diagnosis.

It is a solid phase diagnostic method, using dry chemistry and lateral migration on an inert membrane. This approach uses a porous support in the form of a strip, in which are integrated in dry form the reagents necessary for carrying out the test. The membrane is treated so that a recognition reaction between specific binding partners (usually antigen / antibody) occurs at a given area and can be revealed at this level. Schematically, when the sample to be analyzed is deposited on the support, it migrates along the membrane by a capillary phenomenon. The desired analyte is captured by a specific ligand immobilized on a determined area of the membrane, and the reaction is revealed by a second labeled specific ligand, according to the principle of the sandwich method.

It is also possible to use a competition-type reaction in immunochromatography. In this case a labeled analyte analyte of the desired analyte for binding with the immobilized ligand is used. For this same type of test, it is also possible to use the labeled ligand: in this case, the desired analyte is in competition with the immobilized analyte.

Finally, it is also possible to use in immunochromatography a serological type reaction concerning the search for antibodies. In this case a dosage possibility is described. The specific antigen of the pathology is immobilized on the support. It captures the human antibodies specific for the antigen and pathology present in the sample. The presence of human antibodies is revealed after addition of a secondary antibody against human immunoglobulins. For these serology tests, it is even possible to make a typology of antibodies to determine the class of human antibodies detected (IgM, IgG or IGA) (The principle of an immunochromatography strip is shown schematically in Figure 1).

Numerous state-of-the-art documents describe immunochromatography tests and devices and particular adaptations of this technique to certain applications. For example, US5,591, 645, US5,120,643, EP299428 or EP250137, which are intended for immunochromatography tests in their general principle, comprising a test strip allowing liquid migration by capillarity and the capture of the desired analyte by a specific reagent immobilized at a given zone of the strip, EP291184 which describes a specific analysis apparatus composed of a hollow case containing a reaction support, EP383619 which relates to a specific membrane support , or EP267006, which describes a qualitative immunochromatography method for the simultaneous detection of the presence of several analytes in a sample.

However, most immunochromatographic unitary rapid tests described in the state of the art are qualitative "yes / no" tests based on naked-eye reading. However, the subjectivity of this type of reading is a source of variability and confusion. In addition, these tests can not be used in certain applications for which a decision threshold exists, as is the case for example with certain thrombosis exclusion markers, such as D-dimers, in the field of hemostasis.

It quickly became apparent that there was a need for approaches to quantify the results obtained in the simplest possible way.

One of the oldest approaches commonly used by those skilled in the art is that using a specific internal control called "Good Functioning Control" (TBF) (see for example EP 0 542 627, WO 96/10179, WO 94 / 07163 or AN ₁ CD., YOSHIKI T., LEE G. and OKADA Y., Cancer Letters, 2001, 162: 135-139 CHAN, CPY, SUM KW, CHEUNG KY, JFC GLATZ, SANDERSON JE, HEMPEL A., LEHMANN M., RENNEBERG I. and RENNEBERG R., J. Immunol Methods, 2003, 279: 91-100).

Schematically in this system, the sample to be analyzed is placed in the presence of a specific conjugate of the analyte sought during the deposit on the immunochromatography strip.

The conjugate binds to the desired analyte and the complex formed migrates along the membrane to the specific test capture zone of the analyte. The color that develops at this zone is proportional to the desired analyte concentration. Excess conjugate that is unrelated to the analyte is captured at a TBF zone downstream of the test catch area on the strip. This TBF zone comprises an immobilized reagent specific for the conjugate used. In practice, in this approach, the conjugate is generally a monoclonal antibody of animal origin and the reagent immobilized in this zone of TBF is an anti-species antibody directed against the animal species of the conjugate. Although relatively simple in principle, this system has some disadvantages. Thus, the same conjugate reagent serves to reveal the test and control. As a result, the TBF signal is highly dependent on the concentration of the analyte. For a high concentration, the signal can be greatly reduced or absent for extreme cases. Furthermore, in such a control system, there is no weighting of the sample and membrane related variations.

In other approaches, EP088636 describes a quantitative analysis method based on the use of several successive specific reaction zones of the same analyte spaced apart on the migration support.

WO 91/12258 discloses a method of quantitative immunochromatography, also using a multi-zone strip, in which, schematically, the desired analyte is sandwiched between two specific reagents, one of which is coupled to a marker and the other other than biotin. The product resulting from these interactions migrates along the strip and is captured at the detection zone by a capture agent consisting of avidin fixed on latex particles, the assembly being immobilized on the strip.

WO 97/09620 relates to a method for the quantitative or semi-quantitative detection of an analyte in a sample, consisting in using a calibration agent and based on the presence of several zones, at least one for the calibration, one for the measurement and one last for the control of the good functioning of the test.

WO 00/43786 describes an approach for quantifying the results obtained by immunochromatography with a visualization of the results associated with a coloration, in which values or domains of values at staining or signal staining intensities, and comparing the staining of the result with that of an internal calibration indicator. The approach used in this reference is in fact that of a semi-quantitative test, with definition of a reference staining intensity corresponding to a reference concentration of the desired analyte. It does not use a measuring and pre-calibration instrument, the only means to guarantee reliable results when it comes to quantitative tests.

WO 97/37222 and WO 02/077646, relate to quantitative immunochromatography methods implemented with a RAMP ™ device (Rapid Antigen Measurement Platform). These methods are relatively complex in their application. In particular, they involve a differentiation between the sample application zone and the zone brought into contact with the specific reagent of the desired analyte. Moreover, they use, as detection reagent, populations of particles capable of binding specifically to the desired analyte, which migrate therewith to a detection zone.

The present invention aims to provide a general method of simple quantitative immunochromatography, ensuring reliability of the results despite certain fluctuations due to factors such as the sample (viscosity, composition, etc.) or the membrane (micro heterogeneity of structure), thanks to a specific weighting system.

In particular, the invention provides means for collecting a signal related to the presence of the desired analyte and also a control signal, the latter being chosen to be independent of the main reaction involving the analyte and thus to ensure a constancy of the control signal and also the objectivity of the test result. Thus, the present invention provides a method and a device of this type in which a biological sample is deposited on one end of a strip of porous material and migrates by capillarity to the second end of the strip, the sample coming at the beginning of the strip. its migration in contact, on the one hand, with a test product (capture reagent) able to form a specific binding complex with the substance (analyte) to be assayed and, on the other hand, with a control product (reagent control). The complex formed by the substance with the test product is displaced, during migration of the sample, towards the second end of the strip which comprises a test zone in which a capture reagent of this complex has been deposited. The control product is also displaced by the migration of the sample towards the second end of the strip which comprises a control zone in which a reagent for capturing the control product has been deposited, the test zone being in the vicinity of the control area.

The test reagent and the control product comprise detection markers or markers of any type, which are detectable for example by optical means, magnetic means, electromagnetic means or other.

According to a particular aspect, the present invention thus relates to a method for quantitatively measuring at least one analyte of interest in a liquid sample by immunochromatography, said method comprising the steps of: a) providing an immunochromatography device comprising a solid support in the form of a strip, said support comprising a sample application zone, a migration zone, a specific capture zone for each of the desired analytes and a ratiometric measurement control zone (CMR), the said specific capture areas each comprising a capture reagent immobilized on the support, said capture reagent forming a specific binding pair with one of the desired analytes, and said CMR zone comprising a control capture reagent immobilized on the support, said reagent forming a specific binding pair with a control reagent carrying a detectable marker, b) contacting the sample with the control reagent carrying a detectable label and with a conjugate or mixture of conjugates, each conjugate consisting of a reagent forming a specific binding partner with one of the desired analytes and carrying a detectable detectable label in the same length the control reagent marker, c) deposit the sample in the presence of the control reagent and the conjugate or mixture of conjugates on the area of application of the support, d) maintain the device in sufficient conditions to allow the desired analyte (s) to form binding pairs with the specific conjugate (s) and to capillary ansport along the solid support of the desired analyte (s) and the control reagent to each of the specific capture zones of the desired analyte (s) and to the CMR zone; (e) maintain the device in sufficient conditions to further enable binding of the desired analyte (s) with the capture reagent (s) at the capture area (s), and binding of the control reagent with the control capture reagent at the CMR region, f) measuring the intensity of the test signal produced by the conjugate attached to the desired analyte at each of the capture zones and that of the CMR control signal produced by the control reagent at the CMR zone, wherein the amount (or rate) of each analyte of interest in the sample is directly proportional to the ratio of each test signal on the CMR control signal. The present invention also relates to an immunochromatography device for the quantitative analysis of one or more analytes in a sample according to the method of the invention, comprising a solid support in the form of a strip, said support comprising a zone of sample application, a migration area, a specific capture area for each of the desired analytes and a ratiometric measurement control (CMR) area, wherein the CMR area comprises a control capture reagent immobilized on the support said reagent being capable of forming a pair by specific binding with a control reagent carrying a detectable label.

According to the present invention, the control reagent that migrates along the immunochromatography membrane and is subsequently captured by the immobilized control capture reagent in the CMR zone does not have cross-reactivity with the desired analytes. It thus provides a signal whose intensity is independent of the concentration of the desired analyte in the sample. Moreover, the control capture reagent may be chosen so as to have no interference, in particular cross-reactivity in the case of antibodies, with the other reagents of the assay, in particular that for the capture of analytes.

The signal which develops at this level thus makes it possible to secure the observed results by a weighting of the influences of certain experimental conditions, such as the viscosity and the composition of the sample, the variations of the physicochemical properties of the membrane, etc. .

The signals obtained according to the method of the invention are advantageously measured by a signal reading apparatus, which makes it possible to provide quantified results and to eliminate any subjectivity. In the context of the method of the invention, the conjugate or mixture of conjugates specific for the desired analyte (s), as well as the CMR control reagent, may be added to the sample prior to deposition on the area of application of the support. In this embodiment, the sample / conjugate (s) / CMR reagent mixture is then deposited on the application zone in accordance with steps b) and c) of the method announced above.

According to a second preferred aspect of the invention, the conjugate or mixture of conjugates, as well as the CMR control reagent, are used in dry form. They are then contained in a reservoir at the sample application zone and the liquid sample is brought into contact with the conjugate or mixture of conjugates and with the CMR control reagent at the time of deposit of the sample. the sample on the area of application of the support. The deposition of the sample will result in the solubilization of the conjugates and the CMR control reagent, the binding of the conjugate (s) on their specific analyte (s) and the capillary migration along the support, the complexes. formed between each analyte and its specific conjugate, CMR reagent and, optionally, excess conjugates that will not react with the analytes.

In a particular embodiment of the invention, the deposition of the sample takes place on a previously saturated reservoir (for example in a saturation solution PBS - Régilait 1% - Sucrose 5% (at a rate of 0.5 ml to 1 ml / test) in the saturation tray).

Thus, according to this particular variant, the subject of the invention is: a method for quantitatively measuring at least one analyte of interest in a liquid sample by immunochromatography, said method comprising the steps of: a) providing an immunochromatography device comprising a solid support in the form of a strip, said support comprising a sample application zone, a migration zone, a specific capture zone for each of the desired analytes and a control zone ratiometric measuring device (CMR), said application zone comprising a reservoir containing a control reagent carrying a detectable label and a conjugate or mixture of conjugates, each conjugate consisting of a reagent capable of forming a specific binding partner with the one of the desired analytes bearing a detectable detectable label in the same wavelength as the control reagent label, and said CMR area comprising a control capture reagent immobilized on the support, said reagent forming a binding pair specific with the control reagent carrying a detectable marker, b) deposit the sample on the and (c) maintaining the device under conditions sufficient to allow the analyte (s) to form binding pairs with the specific conjugate (s) and to permit capillary transport along the solid support of the desired analyte (s) and from the control reagent to each of the specific capture zones of the desired analyte (s) and to the CMR zone; d) keeping the device under conditions sufficient to further enable the binding of the desired analyte (s) to the the capture reagents at the capture zone (s), and the binding of the control reagent with the control capture reagent at the CMR zone; e) measuring the intensity of the test signal produced by the fixed conjugate; the desired analyte at each capture zone and the CMR control signal produced by the control reagent at the CMR zone, wherein the amount of each analyte of interest in the sample is directly proportional to the ratio of each test signal on the CMR control signal.

According to a first embodiment, an assay is carried out in the following manner: an operator deposits the sample to be analyzed on the first end of the strip, possibly with a migration buffer, then triggers a stopwatch. The strip is then placed in a device for reading the results and the operator controls this reading when a predetermined duration has elapsed since the start of the stopwatch. The reading of the results is carried out by detecting the signals generated by the markers in the test and control zones of the strip, after which the ratio of the signals picked up is carried out and the quantity of substance present in the liquid sample is determined from of this report by means of a table or a calibration curve. The ratio of the signals detected in the test and control zones makes it possible to overcome the variations due to certain characteristics of the sample (its nature, its viscosity, its protein or ionic content, etc.) and the strip (such as only micro-variations of its structure and its components). The ratio of the signals detected in the test and control zones is essentially a function of the concentration of the substance to be assayed in the sample and is, at least as a first approximation, independent of the aforementioned characteristics of the sample and the strip. It has been found, however, that the accuracy of the assays thus carried out depends very much on the operator, who must in principle always follow the same procedure for each assay in order to guarantee the quality and reliability of the result, which is not always possible in convenient. In particular, if the operator triggers the stopwatch early or late after the deposit of the sample and possibly the migration buffer on the strip, the result of the assay may vary relatively important.

It is also possible that the operator makes an error, such as not triggering the stopwatch or not adding the migration buffer, which forces him to restart the dosing when he has noticed his error. that is to say in general the absence of result, which results in a relatively significant loss of time.

For these reasons, the invention describes, according to another embodiment, a method and a quantitative dosing device that improve the accuracy and reliability of the assay results.

According to this embodiment, the subject of the invention is a method and a quantitative dosing device of the aforementioned type, which make it possible to avoid operator manipulation errors and which ensure good repeatability and good reliability of the assays.

It proposes for this purpose a quantitative assay method by immunochromatography of a substance present in a liquid sample, according to the description of the present application and characterized in that it also consists in automatically detecting the passage of markers in a given area. of the strip at the beginning of the migration of the sample, to automatically trigger the countdown of a predetermined period of time at the detection of this passage of the markers and, at the end of this period of time, to automatically trigger the acquisition signals generated by the markers in the test and control areas of the strip.

In particular, the assay method in question consists in bringing the liquid sample into contact with a conjugate capable of reacting with the substance to be assayed to form a complex, and with a control product, on the other hand. conjugate and the control product comprising detection markers, to capillarily migrate the sample with the conjugate and the control product into a strip of porous material comprising a test zone which contains a reagent capable of reacting with the complex and a control zone which contains a reagent able to react with the control product, to detect the signals generated by the markers in the test zone and in the control zone, to report these signals and to deduce from this ratio the quantity of substance present in the sample, said method being further characterized in that it also comprises automatically detecting the passage of the markers in a given area of the strip at the beginning of the migration of the sample, to automatically trigger the countdown of a period of time predetermined at the detection of this passage of the markers and, at the end of this period of time, to automatically trigger the acquisiti signals generated by the markers in the test and control zones of the strip.

The described method comprising the automatic detection step can be carried out by integrating the different variants of the characteristics of the quantitative analyte measurement method or the device for the execution of this method.

According to this embodiment incorporating an automatic signal detection, the method according to the invention therefore provides for triggering the countdown of the time when the sample has already migrated for a certain distance in the strip, that is to say after a a preliminary phase during which disturbances related to the assay reaction may occur upon deposit of the sample or migration buffer on the strip or solubilization of the conjugate or formation of the complex. The triggering of the time counting after this preliminary phase guarantees the same migration time in the strip for all the samples before acquisition of the results, which would not be the case if the time count started at the beginning or end of the sample or migration buffer deposit or during solubilization of the conjugate or formation of the complex.

This significantly improves the quality of the results and the reliability of the assays.

The acquisition of the results consists in a preferred embodiment, measuring the areas of the signals generated by the markers in the test and control zones, to report these areas, and to deduce from this ratio and from a curve or calibration table the amount of the substance present in the sample.

According to another characteristic of the invention, the markers have a light absorption line at a given wavelength, and the method consists in illuminating the strip at this wavelength, in measuring the reflected or diffused light intensity. by the markers at this wavelength by means of at least one photodetector, and to process the output signals of this photodetector to obtain the areas of the signals generated by the markers.

Advantageously, this photodetector is associated with scanning means of the strip for sensing the signals generated by the markers and determining the areas of these signals as a function of a scanning distance.

The subject of the invention is also an immunochromatography device for implementing the method defined above, said device comprising a solid support in the form of a strip, said support comprising a zone for application of the sample, a migration area, a specific capture area for each of the desired analytes and a ratiometric measurement control area (CMR), wherein the application area comprises a reservoir containing a control reagent carrying a detectable label and a conjugate or mixture of conjugates in form each conjugate consisting of a reagent forming a specific binding partner with one of the desired analytes and bearing a detectable detectable label in the same wavelength as the control reagent label and the CMR zone comprising a reagent capture agent immobilized on the support, said reagent forming a specific binding pair with the control reagent carrying a detectable marker. Such a device is shown schematically in FIG.

According to one embodiment of the device of the invention, the membrane of the strip is made of porous inert material so as to allow a capillary flux of between 50 and 150 sec / 4 cm., In particular less than 100 sec / 4 cm. for example 90 sec / 4 cm. The pore size of the support forming the strip may be 10 μm.

According to a particular embodiment of the invention, the conjugate capable of specifically reacting to form a binding with an analyte is an antibody, for example a monoclonal antibody directed against a first antigenic site of the analyte. The conjugate-bound analyte capture reagent may also be an antibody, for example a monoclonal antibody directed against a second analyte site.

In a particular embodiment of the invention, the CMR control capture reagent (or compound) is also an antibody, for example a polyclonal antibody. The reagents forming the analyte binding conjugate and the control capture reagent may be sprayed onto the strip.

In the context of the method of the invention set out above, the use of a TBF becomes accessory. The function of this type of control in this case is limited to checking whether all the specific conjugate of the analyte sought has been complexed or if there remains after the reaction an excess of unused conjugate.

The supply of conjugates in a reservoir makes it possible firstly to store, on the other hand, a uniform transfer of the conjugates and the sample on the migration membrane. Indeed, after addition of the sample on the test device comprising the conjugate reservoir, there is solubilization of the tracer. Thus, the tracer is "released" or "salted out" of the reservoir on the migration membrane. Then, the rest of the reactions leading to the detection and / or assay will normally proceed under optimal conditions.

The types of materials used to serve as a conjugate reservoir are mostly nonwoven materials. There may be mentioned: the glass fibers or Glass Fiber (GF) which are the most commonly used, then the cellulose filters and the hydrophilic polyesters.

Conjugate reservoirs are usually pretreated; the treatment being a prior saturation with saturating agents including proteins and / or surfactants. After this saturation followed by rapid drying at 37 ^° C. (in general), the conjugate is deposited by immersion or by "printing". The use of a reservoir makes it possible to control the amount of salted conjugates, the rate of release and migration of the conjugates on the membrane.

The location of the CMR zone on the support does not matter to carry out the method of the invention, since it is distinct from that of each of the capture zones. The remoteness of the CMR zones on the one hand and capturing on the other hand can be in a range of 3 to 10 mm, advantageously around 5 + 0.5 mm. For reasons of simplification of use, the CMR zone is preferably located downstream of the other test capture zones.

The method of the invention may advantageously be implemented for the simultaneous quantification of several analytes, each analyte then being stopped in its migration on the support at a specific capture zone, and revealed by a specific conjugate.

The quantification of the concentrations of each analyte is advantageously carried out with a single CMR. In this case, it suffices to calculate the ratio of each of the test signals on the CMR signal.

As already indicated, the control reagent captured at the CMR zone and the desired analytes do not have cross-reactivity, which guarantees a CMR signal intensity independent of the concentration and nature of the analytes. research.

For example, in the case of an assay carried out on a sample of human origin, the CMR is chosen from molecules of animal origin, without cross-reactivity with the type of analytes sought. Thus, the control reagent may be selected from certain immunoglobulins of animal origin such as chicken immunoglobulins.

In this case, the capture reagent is an antibody directed against the animal species of said control reagent, namely an anti-chicken immunoglobulin antibody.

The binding pair formed by the CMR control reagent and its specific capture reagent is preferentially antigen / antibody type.

However, it may be constituted by other affinity partners, for example of the latex or liposome type or pairs such as: Avidin (streptavidin) / biotin, Protein A / Immunoglobulin (Ig), Protein G / Ig, Lectins / Concanavalin A (Con A), anti-Haptoglobin / Hemoglobin.

The marker used on the conjugates and on the CMR reagent is generally of two types:

- Direct, such as particulate markers (gold particles, latex, carbon, polycarbonate) and dyes, colored dextran, fluorescent tracers, etc .... These markers are detectable in itself, without further reaction ,

-indirect, such as enzymatic markers that require a revelation reaction whose product is detected and / or measured.

Preferably, the marker on the conjugate or mixture of conjugates is identical to that used on the CMR control reagent. Nevertheless, it is possible to use different markers, provided that they absorb in the same wavelength, so that a correlation can always be established during the measurement. Preferably, the marker used is of the particulate type, in this case gold particles, such as colloidal gold.

As has already been pointed out, the quantitative measurement of the concentration of the desired analyte is advantageously carried out using a strip reader which can measure, for example by reflectance or transmittance, the test and CMR signals in arbitrary units. . The final measurement referred to is the test signal / CMR ratio (also referred to as "ratio" in the examples below). Thus for each test performed corresponds a measured ratio.

As a first step, a standard curve is established from the multi-concentration reference analyte assay. This standard curve serves, for a given analyte, reference to determine the amount of an analyte of the sample, corresponding to the ratio test signal / CMR signal measured. The standard curve values also correspond to the signal ratios obtained with the reference analyte at a given concentration / single CMR signal. The CMR signal is chosen by those skilled in the art to establish the standard curve and corresponds to a value of the complex formed between the CMR-CMR capture reagent which gives a signal (called CMR signal) lying in the ascending part of the signal curve / complex, as opposed to a value that would be in the plateau part of the curve.

In a second step, the ratio measured for a performed assay is plotted on this standard curve. Thus, it is possible to translate the measured ratio into the desired analyte concentration.

Any assay of a sample is thus made with respect to a standard curve which is traced or memorized by the reader in a form of adequate presentation (curve, bar code ...). The device described is advantageously made such that the solid support constituted by the strip is contained in a housing. The housing intervenes in the flow of the sample at the reservoir and the membrane, preventing leakage of liquids. It can also be adapted to the reading system.

The detection agent generally used in immunochromatography tests is colloidal gold which has its absorption peak around 535 nm. In this case, for detection, the reader can be equipped with two light emitting diode (LED) sources emitting at 530 nm.

For this use, both types of light (transmitted and reflected) have different characteristics:

the reflectance is more or less independent of the environment and there is no influence of the material used except for the tape itself,

the transmittance depends very strongly on the material and more particularly on the material in which the test strip is inserted. This implies that depending on the nature and thickness of the material used, it is more or less possible to measure the transmittance.

Reflected and transmitted light measurements are proportional to the concentration of analyte captured by a specific reagent coupled to the colloidal gold particles (conjugate).

For the illustrations of the invention, it is the reflectance which has been preferentially retained. The invention also relates to a method of manufacturing the measuring device described and a device obtained by this method comprising the following steps:

coating (or immobilization) on a membrane permitting the flow of a liquid by capillarity, by means of a part of the assay reagent of the desired analyte in a sample, on the other hand of the capture reagent of control ;

- Drying, for example at a temperature between 35 and 40 ⁰ C, including 37 ° C, for example for 30 minutes, for example in a ventilated oven of the previously coated membrane.

In one embodiment of the invention, the manufacturing method of the measuring device does not include a saturation step or washing the membrane after coating.

In one embodiment of the invention, the manufacturing method of the measuring device is carried out by means of coating reagents which are capture antibodies, for example deposited reagents must be determined so as to produce coating lines well. defined.

The post-coating drying temperature is determined as a function of the molecules constituting the capture reagents of the analyte and the control capture reagents.

The markers (or tracers) of the test analyte capture and control capture reagents are used without the need to bind them to a matrix (such as latex particles). In other words, the capture reagents are labeled after a coupling process, for example by adsorption of the label on the capture reagent to form a conjugate which can then be purified, for example by centrifugation.

The invention proposes in particular a device for carrying out the method, comprising a strip support formed with at least one sample deposition orifice and a window for reading a result at the test and control zones of the sample. the strip, meeting the characteristics set forth in the present application and means for capturing the signals generated by the markers in the test and control zones, this device being characterized in that it comprises means for detecting the passage of the markers in a given zone of the strip at the beginning of the migration of the sample, time counting means, and means for controlling these time counting means from the detection of the passage of the markers in the given area of the strip and control means for capturing signals after counting a predetermined period of time.

Such a device can also be constituted by incorporating the variants described in the context of the present application.

Advantageously, the means for detecting the passage of the markers in this zone of the strip are constituted by the means for capturing the signals generated by the markers in the test and control zones.

These detection and capture means comprise means for illuminating the strip at a given wavelength and at least one photodetector associated with scanning means of the illuminated area of the strip. This device also comprises means for determining the area of the signals generated by the markers in the test and control zones, calculating the ratio of these areas and determining the quantity of substance sought in the sample from a table or calibration curve previously stored in memory.

The strip holder comprises a housing in which the strip is housed and immobilized and whose upper face has at least one opening for detecting the passage of the markers at the beginning of the migration of the sample and for capturing the signals generated by the markers in the test and control areas.

This opening is elongated in the direction of migration of the sample and one of its ends covers the aforementioned zone of passage of the markers at the beginning of the migration, while its other end covers the test and control zones.

As will be more specifically set out at the level of the examples, the method of the invention provides a medical diagnostic aid made under emergency conditions for the exclusion of certain pathologies, by making it possible to limit the use of certain invasive explorations or expensive analyzes.

This is for example the case, in the field of haemostasis, the diagnosis of venous thromboembolism which includes deep vein thromboses and pulmonary embolism.

It is estimated that in France, the only pulmonary embolism could affect nearly 100,000 patients per year, and be responsible for 10,000 to 20,000 deaths. It is therefore a serious pathology that can affect all categories of patients whether hospitalized or outpatient. Born Clinical examination may be limited to diagnosis, which involves the use of heavy (and sometimes invasive) imaging tests such as angioscan, angiography or scintigraphy, most often associated with Doppler ultrasound. lower. This diagnosis therefore requires expensive equipment and specialized personnel, not always available in health care facilities. In addition, performing several examinations may be necessary to exclude with certainty the diagnosis of pulmonary embolism or, conversely, to confirm it, thus justifying the introduction of an effective anti-coagulant treatment. It should be noted, however, that the diagnosis of pulmonary embolism is confirmed by exploration only in less than one-third of outpatients admitted to an emergency department. Hence the interest in the development of noninvasive biological tests to confirm or otherwise reverse the diagnosis of pulmonary embolism and more generally venous thromboembolism. Most of the tests concerned activation markers of coagulation and especially fibrinolysis, in particular D-dimers, fibrin-specific degradation products. The interest of dosing D-Dimer to exclude a diagnosis of pulmonary embolism has been clearly identified.

D-dimers appear as degradation products of the fibrin clot under the action of plasmin. Indeed, the thrombin generated during the activation of coagulation cleaves the fibrinogen molecule resulting, after release of two small peptides (FPA and FPB), the generation of fibrin monomers which then polymerize (soluble clot). Stabilization of the clot (insoluble clot) is dependent on factor XIII, whose activated form is a transglutaminase that allows the binding (cross-link) of fibrin molecules involved via their D domains. Fibrinolytic system will then lead, via the generation of plasmin, thrombus lysis constituted. The protease action of plasmin on fibrin results in the release of many cleavage fragments of variable size including D-Dimer which appear relatively late compared to the initial thrombotic process. It is nonetheless a set of biochemically heterogeneous degradation products. It should be noted that plasmin is also capable of cleaving fibrinogen, as well as other coagulation factors, during pathological activation of the fibrinolytic system. Products derived from the gradation of fibrinogen or fibrin are grouped under the generic term of PDF (Fibrin Degradation Products / Fibrinogen). D-dimers are therefore a reflection of plasmin lysis of polymerized fibrin molecules having undergone the action of factor XIII.

The plasma D-dimer level is considered a good reflection of the formation of a fibrin clot and its lysis. It increases in several clinical cases: deep vein thrombosis, pulmonary embolism, disseminated intravascular coagulation (DIC), surgery, cancers and cirrhosis, etc. The dosage of D-Dimer has become a very valuable tool used as an aid to the diagnosis of exclusion venous thromboembolism (VTE), either deep vein thrombosis (DVT) or pulmonary embolism (PE).

As a result, the development of a rapid quantitative assay that is sufficiently reliable and independent of user experience and environmental conditions is particularly useful in limiting the use of examinations such as angiography. pulmonary, phlebography or pulmonary scintigraphy.

The dosage of D-dimer in a blood sample according to the method of the invention is illustrated in the examples below. In an advantageous embodiment of the invention, illustrated in the examples, the assay method offers a performance characterized by a coefficient of variation of the measurement (CV) around the threshold of 500 ng / ml of D-Dimer, which is on the order of 10%, or even 6 to 8%, if the reading lines of the signal are separated by an additional 1 mm.

The blood sample quantitatively assayed according to the invention is, for example, a plasma sample. About 30 μl of plasma can be deposited on the strip to perform the test. The reading of the test leads to a D-Dimer level, resulting from the ratio obtained between the measurement of the signal of the test and that of the CMR control signal.

The invention also relates to a kit for the implementation of a method for the quantitative measurement by immunochromatography of at least one analyte of interest in a liquid sample, comprising:

an inert support on which have been immobilized in distinct zones, different types of so-called capture compounds allowing (i) on the one hand, with respect to each analyte capture compound, the capture of a determined analyte of the sample tested by a specific binding reaction between said capture compound and an analyte contained in the sample, when said analyte forms a specific binding partner with a conjugate consisting of a reagent carrying a detectable label, and (ii) on the other hand, with respect to the capture reagent of the control reagent, the capture of a control reagent carrying a detectable marker;

a conjugate or a mixture of different conjugates, each conjugate being constituted by a reagent capable of forming a specific binding with a specific analyte, sought in a biological sample, said conjugate further bearing a detectable label, said conjugate (s) being contained in a saturated conjugate reservoir;

a control reagent (called CMR: ratiometric measurement control) carrying a detectable marker absorbing in the same wavelength as the marker of the conjugate (s);

for each analyte a standard curve; specific for said analyte, based on the multi-concentration assay of the conjugate-associated reference analyte and the control reagent (CMR) assay at a given concentration, under the same conditions, each point of the curve corresponding to the ratio the signal produced by the conjugate associated with the analyte and captured by the analyte capture compound, and the signal produced by the control reagent captured by the control reagent capture compound;

one or more supports (backing) intended to contain the inert supports for carrying out the test;

- if necessary a housing that can contain the inert supports and their accessories to form a cartridge to be placed in the signal reader,

- where appropriate, guidelines for the use of the kit for measuring the amount of analyte or analytes.

In particular, the capture compounds are immobilized (coated) on an inert membrane in the form of a strip, for example, a nitrocellulose membrane.

The different parts of the device comprising the strips are illustrated in the examples and in FIG. In a particular embodiment of the method or kit of the invention, the capture compounds are capture antibodies, particularly monoclonal antibodies for capture of the analyte and polyclonal antibodies for CMR capture.

The analyte capture antibody is, for example, immobilized on the inert support at a concentration of 1.5 to 4 mg / ml, for example around 2 ± 0.1 mg / ml.

The control reagent capture antibody is, for example, immobilized on the inert support at a concentration of 0.5 to 2 mg / ml, for example around 0.05 mg / ml.

The invention will be better understood on reading the description which follows, given by way of example with reference to the appended drawings, in which:

FIG. 1 very schematically represents an immunochromatography strip;

FIG. 2 illustrates the general principle of the assay according to the invention;

- Figure 3 schematically illustrates the use of a result reading apparatus;

- Figure 4 shows schematically a method of assembly of an immunochromatography strip; FIG. 5 represents a standard calibration curve;

FIG. 6 represents the essential components of a metering device according to the invention;

FIG. 7 is a graph of variation of the intensity of a light signal captured by a photo detector as a function of time; FIG. 8 is a graph illustrating the acquisition of a signal generated by the markers in a test or control zone of the strip.

Figure 1 is a reminder of the principle of an immunochromatography strip. This strip includes respectively:

a deposit zone 1 on which the sample is deposited; the deposition zone is represented with a reservoir containing the conjugate in dry form;

a capture zone 2 comprising an immobilized capture antibody specific for the desired analyte;

a zone of TBF 3 comprising an immobilized capture antibody specific for the conjugate. The excess of unreacted conjugate with the sample analyte migrates along the membrane to the TBF ₁ zone where it is stopped by the capture antibody;

an absorbent 4 which serves both as a "pump" and "trash can" immunological reactions triggered by the passage of the analyte sample on the test strip. It has the function of increasing the absorption capacity of the entire device to finally "clean" the migration membrane on which the immunological reactions resulting in the assay occurred.

Figure 2 schematizes the general principle of the device according to the invention. In this scheme, we consider that we are looking for only one analyte. The membrane 5 comprises respectively: a deposition zone 1 on which the sample is deposited. In this example, the conjugate and the CMR control reagent are present in dry form in a reservoir 6 at the deposition zone. a capture zone 2 comprising an immobilized capture reagent specific for the desired analyte,

a CMR 7 zone comprising an immobilized control capture reagent specific for the labeled control reagent. This control reagent made soluble by the deposition of the sample on the deposition zone migrates by capillarity along the membrane to the CMR zone where it is specifically captured by the control capture reagent. At this CMR zone a signal is thus developed which is totally independent of the concentration of the analyte to be assayed.

an absorbent 4 which serves both as a "pump" and "trash can" immunological reactions triggered by the passage of the analyte sample on the test strip. It has the function of increasing the absorption capacity of the entire device to finally "clean" the migration membrane on which the immunological reactions resulting in the assay occurred. Excess conjugate that has not reacted with the sample analyte or with the CMR capture reagent migrates along the membrane and is aspirated by absorbent.

The operating principle of the results reader is summarized in Figure 3. According to the diagram, the reader 8 measures the reflected light 9 and transmitted 10 by an immunochromatography strip 5.

Two separate light sources 11 and 11 'are used to illuminate the strip 5.

Figure 4 is an assembly diagram of the immunochromatography strip, in cross-section.

The actual dimensions of the supports used in the composition of the strip (dimension 7cm + 5mm) are:

_. 12 = Backing (adhesive backing) (70mm) 13 = Coated nitrocellulose membrane (40mm) 14 = Conjugate tank (7mm) 15 Filter (16mm) 16 = Absorbent (18mm)

Positioning of recoveries:

Absorbent / nitrocellulose membrane = AB = 4mm

Conjugate tank / nitrocellulose membrane = CD = 1 mm Filter / tank conjugates = DE = 6mm

Figure 5 illustrates an example of a standard curve for calibration, giving the D-Dimer concentration as a function of the test / CMR ratio.

The device of FIG. 6 essentially comprises a plastic housing 20 in which is positioned and held a strip 22 of porous material, the contour of which is represented by a dashed line, the upper face of the housing 20 having an opening 24 for depositing a liquid sample and optionally a migration buffer at one end of the strip 22, and a reading window 26 of elongate shape which extends for example about half of the length of the strip 22 to near its opposite end to that on which the liquid sample is deposited.

The strip 22 is made of nitrocellulose material for example, in which the liquid sample deposited through the orifice 24 can migrate by capillary action.

As already described above, the strip 22 may comprise, between the orifice 24 and the first end 28 of the window 26, a zone 30 on which, on the one hand, a conjugate intended to form a complex with the substance to be dosed in the liquid sample and, on the other hand, a migration control product.

The portion of the strip appearing in the window 26, in the vicinity of the second end 32 thereof, comprises a test zone 34 and a control zone 36, materialized by transverse lines of the strip on which have been deposited a test capture reagent and a control capture reagent, respectively.

The reagent of the zone or test line 34 is intended to capture the complex formed by the conjugate and the substance to be assayed. The reagent of the control line 36 is intended to capture the control product deposited in the aforementioned zone 30 of the strip.

For example, in the case of D-Dimer assay, the conjugate deposited in zone 30 in dry form is a F (ab) ' ₂ anti-D-Dimer / F (ab)' ₂ 9C3 fragment of the designated antibody. 9C3 or 8D2 in the Liatest® kit

D-Dl from Diagnostica Stago (France), and the control product, also deposited in dry form, is an anti-biotin antibody or a chicken IgG antibody, the capture reagent of the test zone 34 being an anti-D-Dimer / 2.1.16 antibody and the capture reagent of the zone of control 36 being a gamma biotin or anti-IgG hen, the control product and the capture reagent of this product having no cross-reactivity with the substance to be assayed.

The conjugate and the control product deposited in the zone 30 contain detection or labeling markers which may be of any type, such as particulate markers (gold, latex, carbon, polycarbonate particles). .) or dyes, fluorescent tracers or enzymatic markers.

In a preferred embodiment of the invention, the markers used for the conjugate and the control product are colloidal gold particles which have a light absorption line at a wavelength of

530 nm so that these markers can be detected by illumination of the strip by a light source 38 emitting at the wavelength of 530 nm or at a very close wavelength and by forming an image of the illuminated area on a photodetector or a set of photodetectors 40 sensitive to this wavelength.

Preferably, the light source 38 and the photodetector 40 are on the same side of the strip above the window 28 of the housing 20, the measurement of the light intensity being made by reflection. Alternatively, the light source 38 and the photodetector 40 may be on either side of the strip, the measurement being then in transmission.

The photodetector 40 or the set of photodetectors is controlled by information processing means 42, which also controls the operation of the light source 48 and receives the signals. output of the detection means 40. When a single photodetector is used, or a photodetector array aligned with a test or control zone 34,36, these photodetection means are associated with scanning means which enable them to aim successively the different zones of the strip traversed by the liquid sample during its migration. For example, these scanning means may comprise means for moving the casing 20 in the direction indicated by the arrow 44 with respect to the detection means 40. It is also possible to use optical scanning means or else a matrix assembly of photodetectors. which then renders useless the use of a scanning means.

The assay method according to the invention is the following, in the case of assaying the D-Dimer in a sample of blood plasma:

A fixed volume of plasma sample is deposited in the orifice 24 of the support 20, then a fixed volume of a migration buffer is deposited in this orifice. The liquid deposited in the orifice 20 moves by capillarity in the strip 22 towards its opposite end and passes first through the zone 30 which comprises the conjugate for the test and the control product. The conjugate is solubilized by the sample and the migration buffer and reacts with the D-dimer to form a complex. This complex migrates in the strip 22 with the capillary control product and therefore moves towards the first end 28 of the window 26.

The casing 20 is during this time placed in a reader equipped with the light source 38, detection means 40, information processing means 42 and possibly scanning means associated with the detection means 40. In this reader, the first end 28 of the window 26 is illuminated by the light source 38 and observed by the detection means 40. When, at the beginning of the migration in the strip 22, the complex and the control product reach the first end 28 of the window 26, their markers are detected by the detection means 40.

The output signal of the detection means 40 corresponding to the detection of the markers is shown schematically in FIG. 7 which is a graph representing the variation of the light intensity picked up by the detection means 40 as a function of time. The light intensity I captured by the means 40 decreases as soon as the markers appear at the first end 28 of the window 26, passes through a minimum and then increases gradually to a plateau during which measurements are made on the test areas. 34 and control 36.

The falling edge of this signal at the moment when the markers appear at the first end 28 of the window is rather steep and can be used for the triggering of a time count, carried out by the information processing means 42, which, conventionally, are equipped with a clock generating a time base.

The measurement signal generated by the detection means 40 observing the test zone 34 or the control zone 36 is shown schematically in FIG. 8, where the curve C corresponds to the variation of the luminous intensity as a function of a distance of sweep d. This signal is transmitted to the information processing means 42 which calculate the area A of the signal, represented by the hatched portion in FIG.

The amount of D-Dimer present in the sample is determined from the ratio R of the area of the signal measured in the test zone 34 and the area of the signal measured in the control zone 36, and a table or a pre-established calibration curve directly giving the concentration of D-Dimer from the ratio R (Figure 5).

The detection of the markers at the first end 28 of the window 26 has a certain number of advantages:

the measurement is carried out in the test and control zones 34, 36 a certain time (for example 10 minutes) after detecting the appearance of the markers at the end 28 of the window 26, this time interval being the same for all the samples and not depending on the solubilization conditions of the conjugate in the zone 30 of the strip and the formation of the complex between this conjugate and the substance to be assayed,

an error of the operator, such as forgetting the deposition of the migration buffer in the orifice 24 of the housing 20, or the use of a strip 22 which has already been used, is detectable rapidly insofar as the signal I of FIG. 7 does not have the falling edge characteristic of the presence of the markers.

Example 1

Coating or immobilizing process on the nitrocellulose membrane.

Equipment

Anti-D-Dimer capture antibody to be immobilized: monoclonal 2.1.16 (Stago, France) at a concentration of 2 mg / ml.

CMR capture antibody to be immobilized: goat anti-chicken IgG antibody (Goat anti-Chicken antibody (Sigma, France) at a concentration of 1 mg / ml). Coating Membrane: Nitrocellulose SHF0900405 (Millipore, USA)

Coating buffer: 0.15 M phosphate buffer pH 7.4.

Dispensing equipment for antibody solutions: Linomat IV (Camag, Switzerland) or Bio Jet Quanti 3000 (Bio dot, USA).

Method

The coating (or immobilization) of the antibodies and capture proteins on an inert support is carried out according to the method which is briefly recalled. On the pre-cut nitrocellulose membrane in a format useful for the test (for example 40mm x 200mm), distributed (by spraying) using a Linomat or Bio Jet Quanti deposit apparatus, the capture antibodies at a rate of 1 μg per test for the capture antibody Test 2.1.16 and 0.25 μg per test for CMR anti-lg hen catch antibody produced by the goat.

After depositing the antibodies on the membrane leading to the absorption of antibodies on the membrane, dry immediately in a ventilated oven for 30 minutes at 37 ^° C. The membrane thus coated with the antibodies is kept in a sealed aluminum bag, room temperature (22-25 ^° C).

Example 2

Method of coupling antibodies to colloidal gold particles.

Equipment Anti-D-Dimer monoclonal antibody: Fragments F (ab) ₂ 9C3 (Stago, France).

CMR revealing antibody: IgG hen (Sigma, France)

Tetrachloroauric acid (Sigma, France)

Tri sodium citrate (Prolabo, France)

PEG 20,000 (Prolabo, France)

2 mM Borate buffer.

0.2 M K ₂ CO _{3 solution}

Conjugate tank: PT-R5 (MDI, India).

Method

Preparation of Colloidal Gold Particles

The colloidal gold particles of size 50 nm are prepared according to a slight modification of the Frens - Tinglu method. This method is briefly recalled.

Add 2 ml of 1% tetrachloroauric acid solution to 198 ml of ultra pure water in the clean flask.

Put a magnetic stirrer in the flask and heat to a boil.

Add 275 μl of the 10% Tri sodium citrate solution, still with magnetic stirring. Continue to heat.

After the appearance of the purple color, heat another 10 minutes.

Allow to cool to room temperature (22-25 ^° C). Keep the colloidal gold particles at 4 ° C and away from light in an amber glass bottle.

The particles thus prepared are characterized: measurement of the maximum wavelength and the OD at this wavelength, measurement of the pH.

Preparation of Conjugate Anti-D-Dimer Test - Colloidal Gold Particles

This preparation comprises several stages.

a) Preparation of the antibody for coupling

The anti-D-Dimer 9C3 antibody (Fragment F (ab) ' ₂ , Stago) is coupled to the gold particles according to the following protocol.

Dilute the antibody in 2 mM Borate pH 6.5 in order to have a final concentration of 1 mg / ml and then dialyze against this buffer for 3 hours at room temperature (22-25 ^° C.).

Centrifuge the antibody solution for 15 minutes at 5,000 rpm at 4 ^° C. After recovering the supernatant containing the antibody, check its concentration by spectrophotometric measurement at 280 nm, before coupling.

b) Preparation of colloidal gold particles for coupling

Adjust the OD _{530 nm} of the colloidal gold particles by the addition of MiIIi-Q water.

Adjust the pH of the solution of colloidal gold particles to 6.5 +/- 0.1 by adding 0.2 M K ₂ CO ₃ . c) Adsorption coupling

Mix the antibody solution at 0.2 mg / ml with the solution of colloidal gold particles at a ratio of 1/10 (volume Antibody / volume gold particles), ie, 1, 85 ml of antibody for 18.5 ml of colloidal gold particles.

Stir for two minutes at room temperature (22-25 ^° C.).

d) Stabilization after coupling and recovery of the conjugate

Add 1.2 ml of PEG ₂ o ooo 1% and vortex for 2 minutes room temperature (22-25 ⁰ C).

Add 2.4 ml of 1% BSA and vortex for 2 minutes at room temperature (22-25 ^° C.).

Centrifuge for 1 hour at 9000 rpm, at ^40C .

Remove the supernatant and recover the pellet in 20 mM Tris buffer pH 8.2, PEG 20,000, 1% BSA, 0.09% sodium azide.

Filter the conjugate recovered on 0.2 μm.

The conjugate is spectrally characterized.

Measure the λ _max . and the DO _max .

Preparation of Conjugate CMR IgG Poule - Colloidal Gold Particles.

The same approach is followed for the preparation of this CMR conjugate. The difference is mainly in the coupling concentration and the coupling pH: 0.1 mg / ml and pH 9.5. Example 3

Saturation of the conjugated reservoir and deposition of the conjugate

Immerse the PTR5 sheet (MDI, India) in the saturation solution (PBS, Milk 1% Sucrose 5%), with 1 ml of solution per test. Leave for 1 hour at ambient temperature, with stirring.

After removal of the saturation solution, dry the reservoir sheet for 3 hours at 37 ^° C. in a ventilated oven.

The mixture of the two conjugates (Test and CMR) is deposited on the saturated conjugate reservoir at a rate of 8 .mu.l per cm, ie 4 .mu.l per test, the width of the conjugate reservoir being 0.5 cm. The deposit is made by the Air Jet system (Bio dot).

After the deposition, the conjugate reservoir is dried for 1 hour at 37 ° C. in a ventilated oven.

Example 4

D-Pimer Assay Test Cartridge: Immunochromatography Tape.

The different constituent elements of the strip used are the following:

- absorbent (17CHR ₁ Whatman, USA)

- sample filter (GF 3596, Schleicher & Schϋell, Germany),

nitrocellulose membrane (SHF0900405, Millipore, USA) coated, reservoir (PT-R5, MDI, India) of deposited conjugate.

They are assembled on a rigid inert support called "backing" (010 "white polyester" GL-187, G & L, USA).

The assembly diagram is shown in FIG.

After assembly, strips 5 mm wide and 70 mm long are cut with a cutter (Bio dot, USA).

Each strip is inserted into a plastic case whose diagram is given as an illustration in Figure 6. After insertion of the strip, the plastic case is closed and packaged in an aluminum bag (Soplaril, France) with a desiccant ( BO54 / lndice 1, Airsec, France) then kept at 2-8 ° C.

Example 5

Determination of D-Dimer According to the Method of the Invention

Equipment

Migration buffer: PBS Casein 0.5% Triton X100 0.1%

Calibrant D-Dimer: D-Dimer prepared from human plasmas (Stago, France).

Tape reader: Readers: Rapi-kit (77 Elektronika, Hungary).

Method Operating Procedure

The following protocol is used: • Allow strips, buffers, samples to stabilize at room temperature (22-25 ^° C).

• Place 15 μl of D-Dimer or plasma sample calibrator on the sample well of the plastic housing.

• Add 150 μl of migration buffer.

• Leave for 10 minutes at room temperature

• Read the result with an immunochromatograph strip reader (77 Elektronika).

All tests are done in duplicate.

Obtaining and interpreting the results

Firstly, the standard curve is established from the assay of the calibrated D-dimers calibrator after reconstitution at different concentrations from 0 to 4000 ng / ml. For each assay, both the Test and CMR signals are measured by the reader and the Test / CMR ratio is calculated. By plotting this ratio as a function of the concentration by calibrating D-Dimer, the standard curve is constructed. It is valid for a given lot of test cartridge. The CMR signal is chosen by those skilled in the art in the ascending part of the signal / complex curve formed between the CMR capture reagent and the CMR located around 4000 ± 500.

In a second step, the ratio measured for each unknown D-dimer concentration plasma assay is plotted on this standard curve. Thus, it is possible to translate the measured ratio into the desired analyte concentration.

The steps for producing the calibration curve are as follows: a) reconstitute the calibrant approximately (4500 ng / ml) with ¹ H ₂ O-MiIIi-Q, b) allow the calibrator to stabilize for 30 minutes. The calibrant is stable for 4 hours at RT once it is reconstituted, c) dilute the calibrant in migration buffer so as to obtain the following D-Dimer levels:

250, 500, 1000, 2000 and 4000 ng / ml, d) the tests are performed in duplicate, e) deposit 15 μl of sample in the sample well (for point 0 of the calibration, 15 μl of buffer are deposited (f) add 150 μl of migration buffer (10mM PBS - 0.5% Casein - 0.1% Triton x100 - 0.095% Sodium Azide), g) wait 10 minutes at room temperature, h) read results on reader, i) report the results on the results sheets, j) plot the calibration curve [rate in D-Dimer = f (Ratio Test /

CMR)], k) assay plasma and control following the protocol from a to i.

Example of results for the standard curve

Example of plasma dosing

The assay results obtained with the method of the invention are compared with those obtained with a reference method which is based on I ¹ ELISA, Asserachrom DDimer (Stago, France). We can see that the results obtained with the method of the invention are quite close to those of the reference method.

It is therefore a method that allows a quantitative determination that can provide results equivalent to those of the reference method. It is the object of the invention, the CMR, which makes it possible to weight the variations inherent in the technique of immunochromatography (variations due to the supports and to the sample, etc.).

It is possible to estimate this weighting from the results obtained.

Weighting effect of CMR

Each dosage is done in duplicate. The effect of CMR weighting is estimated from the absolute difference between the two values obtained for each assay. For a perfect theoretical weighting system, the difference between the two values of the same assay must be equal to 0.

With these results, it is shown that the smallest deviations are obtained only with the system of the invention. On the other hand, in the absence of the CMR, thus using only the only signal Test, the assay is possible but with a greater uncertainty on its measurement. The weighting effect is further demonstrated with the repeatability tests. In this case, the same sample is dosed 21 times according to the method of the invention.

REPEATABILITY TEST

Plasma 1

With these dosages made in repeatability, the method of the invention makes it possible to weight the variations. Indeed, a gain of 4 to 6% of variation is observed, compared to the signal Test only.

Claims

A method of quantitatively measuring at least one analyte of interest in a liquid sample by immunochromatography, said method comprising the steps of:

a) providing an immunochromatography device comprising a solid support in the form of a strip, said support comprising a sample application zone, a migration zone, a specific capture zone for each of the desired analytes and a control zone ratiometric measuring unit (CMR), the at least one specific capture zone each comprising a capture reagent immobilized on the support, said capture reagent forming a specific binding pair with one of the desired analytes, and said CMR zone comprising a an immobilized control capture reagent on the support, said reagent forming a specific binding pair with a control reagent carrying a detectable marker, b) contacting the sample with the control reagent carrying a detectable label and with a conjugate or a mixture of conjugates, each conjugate consisting of a reagent forming a specific binding partner a with one of the desired analytes and bearing a detectable label absorbing in the same wavelength as the control reagent marker, c) depositing the sample placed in the presence of the control reagent and the conjugate or mixture of conjugates on the area of application of the support, d) maintaining the device under conditions sufficient to allow the desired analyte (s) to form binding pairs with the specific conjugate (s) and to permit capillary transport along the solid support of the desired analytes and control reagent to each of the specific capture zones of the desired analyte (s) and to the CMR zone, e) maintaining the device under conditions sufficient to further enable the binding of the desired analyte (s) to the capture reagent (s) at the capture area (s), and binding of the control reagent to the capture reagent; control at the CMR zone ₁ f) measure the intensity of the test signal produced by the conjugate bound to the desired analyte at each of the capture zones and that of the CMR control signal produced by the control reagent at the level of of the CMR zone, in which the amount of each analyte of interest in the sample is directly proportional to the ratio of each test signal on the CMR control signal.

2) Method for quantitative measurement of at least one analyte of interest in a liquid sample by immunochromatography, said method comprising the steps of:

a) providing an immunochromatography device comprising a solid support in the form of a strip, said support comprising a sample application zone, a migration zone, a specific capture zone for each of the desired analytes and a control zone ratiometric measuring device (CMR), said application zone comprising a reservoir containing a control reagent carrying a detectable label and a conjugate or mixture of conjugates, each conjugate consisting of a reagent forming a specific binding partner with one desired analytes bearing a detectable detectable label in the same wavelength as the control reagent label, and said CMR area comprising a control capture reagent immobilized on the support, said reagent forming a specific binding pair with the control reagent carrying a detectable marker, b) deposit the sample on the area of application support, c) maintaining the device under conditions sufficient to allow the desired analyte (s) to form binding pairs with the specific conjugate (s) and to permit capillary transport along the solid support of the desired analyte (s) and control reagent up to each of the specific capture zones of the desired analyte (s) and up to the CMR zone, d) keeping the device under conditions sufficient to further allow the binding of the desired analyte (s) with the capture reagent (s). at the capture area (s), and binding of the control reagent to the control catch reagent at the CMR zone, e) measuring the intensity of the test signal produced by the conjugate attached to the analyte at the level of each of the capture zones and that of the CMR control signal produced by the control reagent at the CMR zone, in which the quantity of each An analyte of interest in the sample is directly proportional to the ratio of each test signal on the CMR control signal.

3) Method according to claim 2, characterized in that the conjugate or mixture of conjugates, as well as the control reagent are used in dry form.

4) Method according to claim 1 or 2, characterized in that the control reagent captured at the CMR zone and the desired analytes do not have cross-reactivity.

5) Method according to claim 1 or 2, characterized in that in the case of an assay performed on a sample of human origin, the CMR is chosen from molecules of animal origin. 6) Method according to claim 5 characterized in that the control reagent is selected from immunoglobulins of animal origin and its capture reagent is an antibody directed against the animal species of said control reagent.

7) Method according to claim 1 or 2, characterized in that the binding pair formed by the CMR control reagent and its specific capture reagent is antigen / antibody type.

8) Method according to claim 1 or 2, characterized in that the marker used on the conjugate or mixture of conjugate and that used on the CMR control reagent absorb in the same wavelength.

9) Method according to claim 8, characterized in that the marker used on the conjugate or conjugate mixture and that used on the CMR control reagent are identical.

10) Method according to claim 8, characterized in that the marker used is of the particulate type.

11) Method according to claim 10, characterized in that the marker used is colloidal gold.

12) Method according to claim 1 or 2, characterized in that the reading of the test signals and CMR is performed using a strip reader.

13) Method according to claim 12, characterized in that the measurement is carried out by reflectance or transmittance. 14) Method according to claim 1 or 2, characterized in that the ratio test signal / CMR (ratio) is compared to a standard calibration curve established from the assay of the multi-concentration reference analyte and the RMC assay each point of the calibration curve corresponding to an analyte level directly proportional to the ratio of the analyte signal to the CMR signal.

15) Method according to any one of claims 1 to 14, characterized in that it also consists in automatically detecting the passage of the markers in a given zone (28) of the strip at the beginning of the migration of the sample, to automatically count down a predetermined period of time from the detection of this passage and, at the end of this period, automatically trigger the acquisition of the signals generated by the markers in the test and control zones of the strip.

16) Method for the quantitative determination by immunochromatography of a substance present in a liquid sample, consisting in bringing the liquid sample into contact with a conjugate capable of reacting with the substance to be assayed to form a complex and other together with a control product, the conjugate and the control product comprising detection markers, to capillarily migrate the sample with the conjugate and the control product into a strip (22) of porous material comprising a test zone (34) which contains a reagent capable of reacting with the aforementioned complex and a control zone (36) which contains a reagent adapted to react with the control product, to detect the signals generated by the markers in the test zone (34). ) and in the control zone (36), to report these signals and to deduce therefrom the quantity of analyte present in the sample, characterized in that it also consists in automatically detecting the passage of the markers in a given zone (28) of the strip at the beginning of the migration of the sample, to automatically count down a predetermined period of time from the detection of this passage and, at the end of this period, to automatically trigger the acquisition of the signals generated by the markers in the test zones (34 ) and control (36) of the strip.

17) Method according to any one of claims 1 to 16, characterized in that it comprises measuring the areas A signals generated by the markers in the test and control areas, to report these areas and to deduce from this report the quantity of the substance present in the sample.

18) Method according to claim 16 or 17, characterized in that the markers having an absorption line at a given wavelength, it consists in illuminating the strip (22) at this wavelength, measuring the light intensity reflected or scattered by the markers at this wavelength by means of a photodetector or a set of photodetectors (40), and processing the output signals of this or these photodetectors to obtain the areas A of signals generated by the markers.

19) Method according to claim 18, characterized in that it consists in using a matrix of photodetectors (40) for sensing the signals generated by the markers at different points of the strip (22).

20) Method according to claim 18, characterized in that it consists in using a photodetector (40) associated with scanning means of the strip (22) for receiving the signals generated by the markers as a function of a scanning distance and determine the areas A of these signals. 21) Method according to one of the preceding claims, characterized in that the period of time between the detection of the passage of markers in the given area (28) at the beginning of the migration of the sample and the acquisition of signals in the Test (34) and Control (36) zones are approximately 10 minutes.

22) Use of the method according to one of claims 1 to 21 for the determination of D-Dimer in a blood sample.

23) Use according to claim 22, characterized in that the assay is performed on a plasma sample.

24) Use of the method according to claim 22 or 23 in the diagnosis of exclusion of venous thromboembolism.

25) A kit for implementing a method for quantitatively measuring by immunochromatography at least one analyte of interest in a liquid sample, comprising: a. an inert support on which were immobilized in distinct zones, different types of so-called capture compounds allowing (i) on the one hand, with respect to each analyte capture compound, the capture of a specific analyte of the sample tested by a specific binding reaction between said capture compound and an analyte contained in the sample, when said analyte forms a specific binding partner with a reagent consisting of a reagent carrying a detectable label, and (ii) d on the other hand, with respect to the capture reagent of the control reagent, the capture of a control reagent carrying a detectable marker; b. a conjugate or a mixture of different conjugates, each conjugate consisting of a reagent capable of forming a specific binding with a determined analyte, searched in a biological sample, said conjugate further carrying a detectable label, said conjugate (s) being contained in a saturated conjugate reservoir; vs. a control reagent (called CMR: measurement control metric ratio) carrying a detectable marker absorbing in the same wavelength as the marker of the conjugate (s); d. for each analyte a standard curve, specific for said analyte, established from the multi-concentration assay of the conjugate-associated reference analyte and the assay of the control reagent (CMR) at a given concentration under the same conditions, each point the curve corresponding to the ratio of the signal produced by the conjugate associated with the analyte and captured by the analyte capture compound, and the signal produced by the control reagent captured by the control reagent capture compound; e. one or more supports (backing) intended to contain the inert supports for carrying out the test; f. if necessary, a housing that can contain the inert supports and their accessories to form a cartridge to be placed in the signal reader, g. where appropriate, guidelines for the use of the kit for measuring the amount of analyte or analytes.

26. Kit according to claim 25, wherein the inert carrier is a nitrocellulose membrane.

27. Kit according to claim 25 or 26, in which the capture compounds are capture antibodies, in particular antibodies. monoclonal antibodies, for the capture of analyte and polyclonal antibodies for CMR capture.

28. Kit according to claim 27, wherein the capture antibody is immobilized on the inert support at a concentration of 2 ± 0.1 mg / ml.

29) Kit according to one of claims 25 to 28, wherein the CMR capture antibody is a IgG bound to biotin at a concentration of 1 + 0.05 mg / ml.

30) Kit according to any one of claims 25 to 29, wherein the deposition zones of the different capture compounds are separated from each other by about 5 ± 0.5 mm.

31) Kit according to any one of claims 25 to 29, wherein the desired analyte is D-Dimer, the analyte capture compound is an anti-D-Dimer monoclonal antibody, and the standard calibration curve is established for varying D-Dimer levels from 0 to 4000 ng / ml and a CMR control value corresponding to the range 1200-1600 ng / ml D-dimer.

32) Device for carrying out the method according to one of the preceding claims, comprising a support (20) strip formed with at least one orifice (24) for depositing a sample and a window (26) for reading the result at level of the test zones (34) and control (36) of the strip, and means (40) for capturing the signals generated by the markers in the test and control zones, characterized in that it comprises means (40) for detecting the passage of the markers in a given area (28) of the strip (22) at the beginning of the migration of the sample, time counting means, and means (42) for controlling these means counting time from detection passing the markers in the given area (28) of the strip, and controlling the means (40) for capturing the signals generated by the markers in the test and control zones after counting a predetermined period of time.

33) Device according to claim 32, characterized in that the means for detecting the passage of the markers are constituted by the means (40) for capturing the signals generated by the markers in the test (34) and control (36) zones. .

34) Device according to claim 33, characterized in that the detection and capture means comprise means (38) for illuminating the markers at a given wavelength and at least one photodetector (40) associated with means for scanning the illuminated area of the strip.

35) Device according to one of claims 32 to 34, characterized in that it comprises means for determining the area A of the signals generated by the markers in the test (34) and control (36) zones and calculating the ratio of these areas.

36) Device according to one of claims 32 to 35, characterized in that the strip holder comprises a housing (20) in which the strip (22) is housed and immobilized and whose upper face has at least one opening (26). ) for detecting the passage of the markers at the beginning of the sample migration and for capturing the signals generated by the markers in the test (34) and control (36) zones.

37) Device according to claim 36, characterized in that said opening (26) of the housing is elongate in the direction of migration of the sample and one of its ends (28) covers the detection zone passing the markers at the beginning of the migration, its other end (32) covering the test areas (34) and control (36).