FR2788856A1 - Quantitative immunochromatographic assay device, useful e.g. for determining antibody levels, based on comparison of test color with that of internal standard - Google Patents

Abstract

Production of a test device (A) for quantification of an analyte by immunochromatography in which results are read as a color intensity (CI). Production of a test device (A) for quantification of an analyte by immunochromatography in which results are read as a color intensity (CI). A reference CI is first defined, corresponding to detection of a reference level of (I), and the amount of ligand (L) used to capture the detection reagent (DR), for monitoring proper functioning of the test, is adjusted to provide a CI equal to the reference CI. The color is read by eye or instrumentally. Independent claims are also included for the following: (1) a method for producing a test device as for (A) but where the concentration of a ligand (L') for a reference analyte (I') is adjusted so that the CI for detecting (I') is equal to reference CI; (2) a method for producing a test device as for (A) but where different colored molecules are used to provide a variation in color according to the concentration of (I) in the test sample; (3) (A) produced the methods above; and (4) analytical kits containing (A).

Description

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It is a question of protecting an original test device making it possible to quantify the results obtained by immunochromatographic methods, that is to say to assign values or ranges of values of the element sought to the results obtained by these methods. The immunochromatographic methods to which the present invention applies are characterized by the fact of comprising at least one internal operation control or operation indicator.

The test device, calling for a comparative reading of the result (s) obtained with one or more indicators of internal operation, makes it possible to establish a correspondence between intensities of coloring of signal or signals and values or ranges of result values .

This test device makes it possible in particular to assign the coloring intensity of a signal obtained to a value or range or range of quantitative or digital values, and therefore to specify the rate of an element in a sample.

This device can be used for any detection of an element liable to intervene in an affinity reaction with a counter-element.

This invention relates mainly to a test device for assigning value (s) to a result of a qualitative nature: the intensity of coloring of a signal.

Element is understood, in the context of this presentation, any substance, composition, particle capable of intervening in a ligand-refining system, in particular in an affine reaction with a counter-element.

The generality given to the word element therefore also extends to the expression counter-element. Without limitation, element and counter-element may consist of a mutually complementary, affine antigen and antibody, or vice versa. For example, this test device makes it possible to estimate the rate of a humoral response to one or more antigens and / or the level of an antigen present in a sample.

The present invention is applicable to research in biology and in the field of medical diagnosis, as well as in all fields of diagnostic and analytical sciences, such as veterinary, agricultural, agro-food, food and expertise sciences. , where it is necessary to evaluate or estimate the rate of an element, compound or

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 substance. It is also applicable in industry, and to any qualitative detection process used industrially.

The method of applying this test device uses the comparison of the intensity of coloring of the signal of the result with that (s) of one or more internal operating indicators which have been adjusted during previous tests.

The present invention relates to a test device the purpose of which is to allow internal calibration of unit tests intended for the detection of one or more elements in a sample. More specifically, the invention relates to a device and a method using the operating control (s) of said tests in order to obtain one or more referenced indicators of coloring or abacus intensity, linked to the operation of the same tests.

In particular, the purpose of this original test device is to allow the quantification of the results obtained with the immunochromatography tests by migration or membrane filtration, using a colored visual indicator, which may be colloidal gold, latex beads. , any particulate substance or not, or the product of an enzymological reaction, and giving a coloration for the indication of the result. The test device, using a precise methodology and the aforementioned techniques, makes it possible to establish a correspondence between the intensity of coloring of the signal obtained and ranges of quantitative values of the element or elements sought.

Analyzes using the specific affinity of reagents have shown their great utility in recent years, both in the field of biological analysis and for other applications. Several techniques thus use the specific affinity existing between two or more reagents: this is the 'sandwich' technique, and competition.

These methods use one or both members of couple (s) of specific affinity for the detection and determination of one or more element (s).

All of these techniques have the characteristic of using a reagent fixed on a support and a free development reagent. Depending on the nature of the test, the reagent attached to the support can be an antibody, an antigen or any

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 substance member of a couple of specific affinity. Likewise for the development reagent, which also has the particularity of not being attached to a support but of being composed at least in part of an indicator molecule or which may give rise to an indicator reaction. The most commonly used markers are dyes, radioactive substances, enzymes, gold colloids and latex beads.

Many forms of solid supports on which one of the members of an affinity couple is fixed, have been described. Currently, the most common supports for RIAs and ELISAs are polystyrene tubes, plates or beads. But paper filters, glass and various plastics have been used for a long time.

Radioimmunological (RIAs) and enzymatic (ELISAs) tests are known and used worldwide. More recently developed, immunochromatographic tests have taken and continue to take an increasingly important place in the field of diagnosis. These tests mainly use membranes of various natures and compositions as a solid support.

The development of non-radioactive and non-enzymatic markers has facilitated the use of immunological diagnostic procedures outside specialized laboratories, up to the doctor's office and even in patients' homes. These procedures should be quick and simple, providing diagnostic assistance almost instantly. The development of such tests has therefore focused, in addition to the need for appropriate sensitivity and specificity, on the improvement of three main characteristics: speed, stability (at room temperature), and ease of job and reading the results.

However, it is sometimes useful or necessary to have a quantitative result for the accuracy of a diagnosis or the follow-up of a therapy.

The only advance known in this direction is the appearance of reflectometry reading devices. But we then lose two interesting characteristics of these tests: simplicity, but above all the lack of instrumentation necessary for the test. In addition, the problem of choosing a reference or a standard curve allowing

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 to establish the correlation between the intensity of coloring of a result and one or values remains.

In Immunochemistry - Practical applications in pathology and biology (eds Polak J. M and Van Noorden S., 1986), the preparation and use of gold as a colored marker is described. The absorption of a reactive substance on gold colloids is described by Geoghegan W. and Ackerman G. in J. Histochem. Cytochem. (1977), 25, 1187. The use of gold colloids for the visualization of antigen-antibody reactions in analyzes using the deposition of immunological reagents on membranes is described by Moeremans M. et al. in J. Immunol.

 Meth. (1984). 74, 353.

The immunochromatographic analysis methods use an immunochromatography support through which the sample will diffuse, either laterally and we speak of migration, or transversely and we speak then of filtration. In the case of immunochromatography tests by migration, gold colloids and latex beads are generally used for the optical (colored) development of the result, as components of the development or coloring reagent. However, enzymes are also used, the reaction of which with a substrate directly or indirectly produces a coloration associated with an insoluble colored product.

According to the sandwich technique which uses two ligands of the molecule sought, one of the ligands is fixed and the other coupled with an indicator allowing the visualization of the result, by direct coloration when it is a colored substance or by the appearance of a color after an enzymological reaction when it is an enzyme. According to the competition technique, a single ligand is used as well as the desired substance or its analog; ligand is then fixed or free and coupled with the colored or enzymatic indicator, the desired substance or its analog being inversely fixed or coupled.

Immunochromatography has been the subject of numerous patent applications, including EP 0 306 772 and EP 0 262 328 from Abbott (registered trademark), EP 0 286 371 and EP 0 183 442 from Syntex (registered trademark), EP 0 276 152 from Synbiotics Corporation (registered trademark), EP 0 296 724 from Quidel (registered trademark) and WO 88/00322 from Unilever (registered trademark). We can

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5 10 15 20 25 30 35 also cited US Pat. Nos. 4,366,241 to Tom et al. , US No. 4,632,901 from VaIkirs et al., US No. 4,522,923 from Deutsch A. and Platt HA, WO 8606488 from Barnett B., EP 0 022 669 from Graham HA et al., US No. 4,094,647 , 4.235.601 and 4.361.537 from Deutsch ME and Mead LW, FR 2537724 from Friedenberg RM, EP 0206779 from Lipp V. and Buck RL, EP 0189925 from Deutsch A. et al., WO 84/02397 from Friedenberg, US n 3,893,808 to Campbell, and US No. 3,895,914 to Alberty et al.

Immunochromatography on a strip containing a particulate immunoreactive conjugate is described in Rosenstein patent EP 284 232 by Becton Dickinson (registered trademark).

The immunochromatograph (strip) in a plastic case with two windows: sample well and reading window, is described in patent EP 291,194 to Unilever (registered trademark).

Other patents relate to the revealing reagent used, such as the Leuvering patents from Akzona EP 7654 (US 4,313,734), EP 398,913 (WO 89/06801) from Nycomed (registered trademark), EP 0 250 137 and 0 258 963 from Ortho Diagnostic System (registered trademark), EP 0 158 746 and 0 293 947 from Janssen Pharmaceutica (registered trademark), and EP 0 310 872 from Hygeia Sciences, for the use of gold colloids, patents for the use of latex beads being also numerous (US N 4,434,150, N 31,712, N 4,478,914, N 4,141,965, N 4,210,622, N 4,331,649, N 4,582,810).

Mention may also be made of US patents N 4,803,154, N 4,446,232, N 4,752,572 for the enzymatic development and the use of a ligand coupled to an enzyme.

These patents relate to devices, methods and applications making it possible to detect an element, and therefore leading to obtaining a solely qualitative result, indicating the presence or absence of said element in a sample.

These immunochromatographic methods are mainly used in unit tests allowing rapid analysis of a single sample. These tests present the result of the analysis in the form of a spot or a visible or invisible line depending on the result after a time generally less than 20 minutes. The validity of the result is generally ensured by an internal operating control.

These tests are made up of 3 distinct parts:

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 1. A first reactive zone, called reservoir, composed of a membrane intended to receive a sample of fluid to be tested and containing half a pair of specific affinity of the ligand sought and coupled to a reagent allowing the demonstration of the presence of the ligand sought. This membrane is composed of a material which can act as a container (for example cellulose or cellulose derivatives, nylon, fibrous or microporous polymers of high density polyester type for example, fibers of plant, animal, mineral or synthetic origin, under in the form of powder, tablets or multiple layers) and having, with or without treatment, properties of low adsorption of biological molecules.

2. A second reactive zone, called the reaction support, composed of an activated membrane having properties of selective adsorption of molecules (for example by ionic interactions, by hydrophobic interactions, by affinity, by immunoaffinity) and migration properties and capillarity allowing the sample to be tested to move and thus to drive the element to be assayed through the reactive zones. This membrane may or may not be composed of two distinct and contiguous parts having or not the properties set out above.

This membrane can be composed of one or more materials having the properties set out above (for example cellulose or cellulose derivatives, nylon, fibrous or microporous polymers of high density polyester type for example, fibers of plant, animal or mineral origin. or synthetic, in the form of powder, tablets or multiple layers).

3. A third part, called absorbent, whose role is to cause and / or promote the flow through the two previous parts. This element can be composed of a material having hygroscopic properties (for example cellulose wadding or cellulose derivatives, nylon, fibrous or microporous polymers of high density polyester type for example, fibers of plant, animal, mineral or synthetic origin. , in the form of powder, tablets or multiple layers)

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 These three parts are placed contiguously and without discontinuity, often in a plastic module revealing two zones of the reactive membrane through a window:. the first zone constitutes the test zone showing the result of the analysis.

. the second constitutes the control zone proving that the test was carried out in good conditions: of operation.

In the case of series tests, it is generally provided one or more positive and negative samples making it possible to verify the correct functioning of the tests by simultaneously providing a calibration / calibration curve or at least a reference point. This makes it possible to quantify the results obtained.

This calibration method cannot be used for unit tests. It is indeed not conceivable to use a test for the establishment of a frame of reference for each analysis test, one of the interests of which is to be practiced in a unitary manner.

These unit tests therefore have the particularity of presenting an internal operating control. On the same test, we obtain the result of the analysis and the result of the function check. These results, of a qualitative nature and most often visual, are presented in the form of colored lines, dots or characters, for example red in color for tests using colloidal gold.

Generally these tests use the direct capture of the development reagent (complex composed of an affine substance and a colored indicator or an enzyme) for controlling the functioning of the test.

In order to assure the user of the correct functioning of the test, the signal is generally intended to have a maximum intensity, and therefore optimal visibility.

Some producers of such unit tests have tried unsuccessfully to provide standard ranges or color intensity charts in order to quantify the results obtained. The use of such graphs comes up against the difficulty on the one hand of its establishment under given conditions, but above all with the variability of the operating conditions and of the samples. Indeed, the immunological reactions due to their nature vary according to the operating conditions (temperature, deposit of the sample, etc.), but especially of the nature and quality of the sample

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 (pH, viscosity, presence of more or less interfering substances, inhibitors or accelerators of affinity reactions, etc.).

However, the reaction of the operational control being of the same nature as that of the analysis, it is therefore affected in an analogous manner by these variables.

This is the description of a test device allowing this operating control to be used for establishing an intensity indicator or an abacus element. By reference to the intensity of coloring of the signal of the operating indicator, one can then define three levels of intensity of coloring of the signal of the result corresponding to three domains of positive values of the analysis result: greater than, equivalent or close to , and less than a selected value or range of values.

The use of an internal indicator for calibration ensures the validity of the correspondence. Indeed, the internal indicator being the witness of good functioning of the reaction, it is influenced in an identical way to the result by the operating mode, the quality of the operating practice, the quality of the production batch, the nature of the sample and its characteristics, the reading conditions as well as the operator or reader vision characteristics.

In a first version, the test device comprises an operating indicator giving an intensity A of coloring when the analysis is carried out. This same intensity A observed for the coloring of the signal of the result (signal obtained in the test zone) indicates that the element sought is present in the sample at a rate of x ± y ng / ml, or also indicates that the element sought is present in the sample at a rate between # and p ng / ml. When the intensity of the colored signal of the result is greater, it can be affirmed that the element sought is present in the sample at a rate greater than p ng / ml.

When the intensity of the colored signal of the result is lower, it can be affirmed that the rate in the sample of the element sought is less than # ng / ml.

In a second version, the test device includes two operation indicators giving two different intensities, A and B, of coloring for each operation indicator during production.

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 of the analysis. Arbitrarily and for the understanding of this version, the intensity B is said to be greater than the intensity A.

If the coloring of the signal of the result has an intensity equal to or close to A, the level in the sample of the element sought is between x and # ng / ml.

When the colored signal of the result has an intensity close to B, the level of the element sought is between o and # ng / ml.

When the colored signal of the result has an intensity Less than A, the rate of the element is less than x ng / ml.

When the coloration of the result signal has an intensity between intensities A and B, the rate of the element sought is between ≈ and o.

When the colored signal of the result has an intensity greater than B, the element rate is greater than #.

We have thus defined 5 ranges of result values to which correspond the intensities of coloring of the signal of the result. This gives the interpretation table of the results presented in Figure 1 of Plate 1.

In derivative versions thereof, the number of operating lamps with different signal coloring intensities can be increased to the extent desirable or necessary and within the limits of the possible both in implementation and in the possibility of reading. differential.

In another version mainly applicable to tests using lateral immunochromatography and the result area of which is upstream of the operating indicator (s) relative to the direction of migration, the development reagent is not present in large excess, but in quantity just sufficient to give a low intensity of coloring of the operating indicator in the presence of very high levels of the element sought in the sample, that is to say with a maximum intensity of coloring of the signal of the result. This arrangement results in a competition system for the development reagent between the test and control zones.

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There will then be an inverse variation in the coloring intensities of the signals of the result zone or zones and of the signals of the operating indicator or indicators.

This version has the advantage of increasing the variation in the ratio of the intensities of coloring of the result (s) / control (s) signals, which leads to easier reading and interpretation of the results.

Indeed, in the presence of small quantities of sought element, the intensity of the signal of the result will be weak and that of the witness will be strong. It will then be all the more visible and obvious as the intensity of the result is lower than the control.

In the presence of intermediate quantities of the element sought, the two intensities will be identical.

In the presence of high quantities of the element, not only will the intensity of the result be high, but that of the operating indicator will be reduced accordingly.

This version not only has the effect of facilitating comparative reading and therefore interpretation, but also of reducing the extent of the range of values where the intensity of the result and control signals is identical.

In a last version which can moreover be coupled to one of the previous versions, an unmarked substance and for which the reagent for capturing the development reagent which is used for the functional indicator has an affinity, is incorporated into the device test. At the level of the operating indicator, there will then be a competition reaction between the development reagent and this non-development substance. This makes it possible to reduce and fix the coloring of said operating indicator at a desired intensity.

The recommended method for obtaining an operating indicator having a coloring intensity determined during the carrying out of the test or analysis is as follows: All the other parameters of the test are optimized and fixed according to the desired characteristics of the test. In particular, the parameters relating to the development reagent on the one hand, and those relating to the test zone will have been definitively established beforehand, it being understood that the development reagent is in large excess.

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In previous studies and possibly by analysis of available scientific data, the critical rate (s) of the element sought will be determined, corresponding to a threshold for interpretation of quantitative results.

The desired intensity is then determined by carrying out the test with a range of samples having various levels of the element sought, including the critical rate or rates. The critical rate (s) will then give a coloration to the signal of the result, the intensity of which will be called the reference intensity.

We will thus determine the correspondence between the reference intensity and a critical value or range of values and on the other hand we will make sure that this intensity is well situated in a range of intensity variation of the colored signal of the result as a function of the rate of the element sought in the sample, that is to say that we observe a lower intensity for a lower rate and a higher intensity for a higher rate.

The characteristics of the deposition of the operating control zone will then be determined by various tests by varying the concentration of the ligand capturing the development reagent, until the reference intensity is obtained. These tests will be conducted on the one hand with samples containing the element sought at the rate producing the reference intensity for coloring the signal of the result (test area), which allows direct comparison and on the other hand with samples negatives not containing the element sought and strongly positive samples to ensure that the functional check is operational in all cases.

When several operation indicators are desired, the same procedure will be carried out by adjusting the operation indicators one by one, and starting with the operation indicator which should have the lowest intensity of the colored signal, or the lowest reference intensity, and by preferably placing upstream of the other function indicator (s) in the case of a test using lateral immunochromatography.

A second method can be used to obtain a determined intensity of coloring of the operating indicator:

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 Instead of, or in addition to, the modification of the deposition characteristics of the ligand capturing the development reagent, a substance which is non-revealing and competitive for this ligand is incorporated into the device. The quantity of this substance necessary to obtain the desired intensity of coloring of the operating indicator is then determined.

Here is a mode of application of the invention to which the latter is not limited in any way.

Example 1: This example relates to a rapid lateral immunochromatography test for the detection of total IgE in human serum, according to the sandwich method.

The development reagent is an antibody specific for human IgE coupled with colloidal gold.

In the test area (of the analysis result), an antibody specific for human IgE is attached.

In the control zone, a functioning indicator, an antibody is affixed which has an affinity for the revealing antibody coupled to colloidal gold.

Concerning the level of total IgE in the serum, the following ranges of values can be distinguished: The values lower than 100 kUI / 1 are not pathological.

Values between 100 and 400 kUI / 1 may or may not be pathological.

The values between 400 and 1000 kUI / 1 are pathological, indicative either of atopy, of developing pathologies, or of parasitoses.

Values greater than 1000 kUI / 1 are pathological and indicate a certain severity, either of allergy, or of other pathologies.

The intensity of staining of the result for an IgE level of approximately 700 kUI / 1 was therefore chosen as the reference intensity. It has been confirmed that lower (lower than 400 kUI / 1) and higher (higher than 1000 kUI / 1) rates give intensities of coloring which can easily be distinguished from that chosen as a reference.

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 All the other parameters of the test having been previously defined, various deposits were made with various concentrations of the antibody having an affinity for the revealing antibody coupled to colloidal gold and which is fixed in the control zone. It was thus determined the deposit concentration which made it possible to obtain a coloring intensity of the operating indicator equivalent to that obtained for the IgE detection result signal at the rate of 700 kUI / 1 in a serum sample.

A test was thus obtained, the interpretation of the results of which is as follows: # when no signal is detected in the result zone or test zone, the total IgE level is less than 100 kUI / 1.

 # when the result signal has a coloring intensity lower than that of the operating indicator, the total IgE rate is between 100 and 400 kUI / 1, # when the result signal has an intensity close to that of the operation, the IgE rate is between 400 and 1000 kUI / 1, # when the result signal has a coloring intensity greater than that of the control, the IgE rate is greater than 1000 kUI / 1, This device has could thus be validated on a sample of all sera, with obtaining a satisfactory agreement between the interpretation of the results (reading) and the values of the IgE levels determined by a recognized quantitative method.

The test device of the present invention is applicable to any test using an affine reaction, comprising an internal operating control, and characterized by the presence of a reagent fixed on a support, which can be, without limitation, a membrane, and the use of a development reagent. This development reagent can comprise particulate components giving a coloration or reactive molecules, for example and without limitation, enzymes, the reaction of which allows a coloration to be obtained, or an absence of coloration, indicative of the nature of the result. .

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 The element sought by this test device may be of a biological nature, an antibody or an antigen, of a chemical or biochemical nature.

This test device and this method is applicable to the detection of any biological marker of one or more infectious pathologies, for example and in a non-exhaustive manner serology tests, and antigen detection tests.

This test device and this method are also applicable when the element sought is a single type of antibody or several types, classes or subclasses. A specific example to which the claims of this patent is not limited in any way is the serology Chlamydia, and for which this device and this method make it possible to determine or evaluate the level of anti-chlamydia antibodies present in serum or samples of other nature of patients.

This test device and this method are also applicable when the element sought is one or more antigens specific for one or more pathologies.

This test device and this method are also applicable in the context of therapeutic monitoring.

This test device can be used either with a visual reading, carried out by a human operator, or with a reading device coupled or not with a data processing device.

The median of the range of values corresponding to the intensity of reference coloration indicated by the coloration of the operating indicator is determined as a function of the desired and necessary characteristics of the test and of the values of the element sought. The same applies when several reference intensities are indicated by several operating indicators.

On the other hand, when the reading is carried out visually, the extent of the range of values corresponding to the intensity of reference coloring must be determined experimentally and is dependent on the nature of the test, of the reagents the components, in particular the reagent of revelation, and production parameters of the test concerned. This remark is of course also valid for each reference intensity when there are several.

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Reading can also be carried out by a machine or using a tool or instrument. This reading device then uses the intensity of coloring of the signal from the operating indicator as a reference for reading and interpreting the signal of the result of the detection of the element or elements sought. The use of such a reading instrument makes it possible to limit the correspondence of the reference intensity to a precise and unique value of the element sought. Similarly, when several reference intensities are indicated by several operation indicators, each intensity can be matched with a single numerical value of the element sought.

A much more precise interpretation of the results can then be obtained, by assigning a single numerical value to any intensity.

 This value can be calculated according to the following formula: = Vr x le / Ir Ve = Value of the element sought in the sample Vr = Value of the element sought giving the reference intensity Ir of coloring of the signal of the result.

Ir = Color intensity of reference = Color intensity of the operating indicator, read during the test. le = Intensity of coloring observed for the signal of the result.

Similarly, when several operating indicators indicate several intensities of reference coloring, it is possible to draw a standard curve, making it possible to establish the correspondence between any intensity of coloring of the result signal observed and the value or the rate of the element sought.

For example, in the case where two internal operating indicators give two intensities of coloring reference Irl and Ir2, corresponding to two values Vrl and Vr2 of the element sought producing each corresponding intensity for coloring the signal of the result, the following standard curve for assigning a value Ve to an observed intensity the coloring of the signal of the result. A standard curve model is described in Figure 2 of Plate 1, the legend of which is as follows: Vrl = Value of the element sought giving the reference intensity Irl for coloring the signal of the result.

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 Vr2 = Value of the element sought giving the reference intensity Ir2 of coloring the signal of the result.

Irl = Color intensity of reference N 1 = Color intensity of operation indicator N 1, read during the test.

Ir2 = Standard coloring intensity N 2 = Color intensity of the operating indicator N 2, read during the test. le = Intensity of coloring observed for the signal of the result.

Ve = Value of the element sought in the sample

Claims (10)

1) Test device for the quantification of the results relating to the detection of an element and obtained by an immunochromatographic technique in which the visualization of the results is associated with a coloration, characterized on the one hand by the fact that the internal control of operation of the tests concerned gives a defined intensity of coloring of the signal during the carrying out of the test, and further characterized by the fact that the intensity of coloring of the signal obtained with said internal control or internal function control is used to evaluate by direct comparison, visually or using a device, of the coloring intensity of the signal obtained during the detection of the element sought.  2) Test device according to claim 1, characterized in that the internal operating control has been calibrated and manufactured so as to give a determined intensity of coloring of the signal, less than the maximum observable intensity for coloring the signal of the result, and thus making it possible to define three areas of intensity of coloring of the signal of the result: close to, and greater than the intensity of coloring of the signal of the internal operation indicator.  3) Test device according to claim 1 or 2, characterized in that the intensity of coloring of the signal of the internal control of operation is equivalent to the intensity of coloring of the signal of the result obtained for a known value or range of value known to the element sought, this correspondence being indicated to the user of the device.  4) Test device according to any one of claims 1 to 3, characterized in that several internal operating controls are calibrated to give different signal intensity, and are present and used to define different areas of coloring intensity of signal and thus quantify the test result.  5) Test device according to any one of claims 1 to 4, characterized in that the correspondence between one or more reference intensities of coloring of the signal of the result and one or more values of the element sought is determined, indicated , and used for the interpretation of the results, the intensity or the reference intensities of <Desc / Clms Page number 18>  coloration being that (s) obtained for one or more internal operating indicator (s).  6) A test device according to any one of claims 1 to 5 wherein the colored reagent comprises a colored particulate substance which may be a complex of colloidal gold or metal salts or latex beads.  7) Test device according to any one of claims 1 to 5 wherein the development reagent comprises an enzyme whose reaction causes the appearance of a coloration, allowing the visualization of the result.  8) Test device according to any one of the preceding claims, characterized in that it comprises a reading device or instrument, which can be coupled to a data processing device.  9) Method making it possible to fix the coloring intensity of the signal of the internal operating indicator at a determined intensity, corresponding to the coloring intensity of the signal obtained for the detection of the element sought at a value or range of values of said element in a sample.  10) assembly or analysis kit comprising a test device according to any one of claims 1 to 8, containing a development reagent reacting with another reagent in order to indicate the correct functioning of the analysis and giving a signal coloring intensity used as an internal reference element.