HK1106025B - Method for increasing the dynamic measuring range of test elements, especially immunological test elements, that are based on specific bonding reactions - Google Patents

Description

Method for increasing the dynamic measurement range of a test unit, in particular an immunoassay test unit, based on specific binding reactions

The invention relates to a method for increasing the dynamic measurement range of a test element, in particular an immunoassay test element, in particular an optically detectable immunochromatographic test strip, based on a specific binding reaction.

An immunoassay strip is a widespread aid for the rapid determination of drugs, progestogens, infectious diseases or so-called "heart disease markers" (cardiac markerns) such as troponin T. In this context, it has been widely used not only for qualitative tests which are read purely visually and provide only a "yes-no" answer, but also for quantitative tests which are analyzed with the aid of a reading device.

For example, according to WO97/06439, EP0291194, US5,591,645, US4,861,711, US5,141,850, US6,506,612, US5,458,852, US5,073,484, the use for the rapid detection of immunodetectable substances has long been known for a large number of different parameters. Here, the immunodetection reagents (essentially labeled and unlabelled antibodies or antigens) are mostly provided in dry form on a carrier, wherein the carrier allows transport of sample liquid (in particular body fluids, such as blood, serum, plasma, urine, saliva, etc.) on or in the carrier. For this purpose, the support is preferably capillary-active, such as a membrane or a synthetic material support with capillary channels (such as in US5,458,852). Often referred to in the art as an immuno or immuno-chromatographic test strip or device. This concept or expression "carrier-bound immunoassay" or "carrier-bound immunoassay unit" is generally used as synonyms and is also interchangeable hereinafter.

Such immunoassay devices are typically analyzed purely visually in simple systems and especially in purely qualitative assays (where the expression "analyte present or absent" is of interest only). This principle has gained wide acceptance in the market place, particularly in the field of rapid progestogen testing.

The (quasi-) quantitative immuno-rapid tests are mostly analyzed by means of corresponding measuring devices which cooperate with the individual test strips. Different measurement principles are used depending on the kind of reagent label used by the test device for detecting the analyte. Optical detection methods, in particular the measurement of reflectance and fluorescence, are widely widespread and simple to use.

Many systems according to the prior art are responsible for: the analyte detection region (also referred to below simply as "detection region") and the control region are narrowly defined in space and are arranged on the test device in a clearly separated manner from one another. For this purpose, it has first of all been found to be advantageous to arrange the respective binding reagents on the test device in a linear or dashed line. For the analysis of the analyte detection and control regions, position-resolving optical systems, such as camera chips or 2-or 3-dimensional photodiode arrays, are usually provided in the measuring devices for analytical test devices. The signals of the optical system are then converted into concentration values by appropriate analysis software and displayed.

In the case of prior art immunoassay devices, it is not possible to quantitatively detect any concentration of analyte in a sample. Downward, i.e. in terms of the lower limit of detection, the measurement range is limited in terms of the labels (labels) used, for example, due to the affinity and selectivity of the binding partners (mostly antibodies) used and the sensitivity of the detection optics. Upward, i.e. in terms of dynamic measurement range, saturation effects limit the measurement range. Thus, in the case of analytes that may be present in very high concentrations in a sample, it is often not possible to provide an appropriate amount of binding partner in the test device. In particular in analyte detection and control zones, in which the binding partners are arranged on the test device in a spatially narrowly limited manner, it is not possible to arrange any number of binding partners. This is particularly problematic in cases where a low detection limit for the analyte is required (so that one strives to place binding partners as intensively as possible in the detection zone, i.e. in a compact space, and thus only a relatively small number of binding partners may be provided on the test device due to only limited availability of binding sites), but the amount of analyte in the sample may fluctuate dramatically, i.e. very low and very high analyte concentrations may occur. In the case of high analyte concentrations, saturation of the detection zone with the corresponding detection reagent is achieved, so that a saturation behavior of the analyte-detection signal relationship is obtained: above a certain analyte concentration the detection signal does not rise any more; the analysis curve becomes flat and no longer can be analyzed meaningfully.

And becomes serious: in sandwich immunoassays, firstly, in the case of very high analyte concentrations, it is possible to observe not only a gradual curve which reproduces the relationship between analyte and detection signal, but also a decrease in signal with increasing analyte concentration. Referred to herein as the so-called "High-Dose Hook-Effect" (High-Dose Hook-Effect) ": in the case of very high analyte concentrations, a strong decrease in the signal intensity, which first increases with increasing analyte concentration, is observed in the sandwich immunoassay. This is explained by the following aspects: the amount of antibody provided in the test is no longer sufficient to form a sandwich complex (i.e. a complex with two antibodies per antigen) with the analyte molecule (antigen) in any case. The complexes consisting of the analyte and one antibody each are formed more, however, such complexes are no longer detectable by themselves. Which may lead to false negative measurements or to low measurements, which should naturally be avoided.

Quantitative immunoassay strips, in particular, which determine the signal by reflectance measurements, sometimes exhibit significant disadvantages relative to conventional analytical systems, which are mostly employed in large laboratories. In particular, the accuracy and dynamic measurement range are mostly poor in test strips. This limits the field of application of highly sensitive sandwich assays, in particular, for therapy monitoring for which an as large a measurement range as possible is desired.

Furthermore, on the one hand a low detection limit is required for certain parameters (such as myosin or D-dimer), and on the other hand very high concentrations of the analyte may be present in the sample material, sometimes well above the judgment limit, i.e. "normal pathological". In this case it is desirable to provide a test device which has as large a measurement range as possible in order to obtain reliable measurement values without prior dilution of the sample. This is particularly advantageous for applying such a test device in the process control of corresponding disease images.

The prior art is not an idea of solving the above-described problems. However, up to now there has been no convincing proposal in all aspects. The conversion of this concept has not been satisfactorily achieved at present, particularly in the field of immunochromatographic test devices.

US6,248,597 describes a heterogeneous agglutination immunoassay based on light scattering, wherein the dynamic measurement range is extended by using a mixture of particles with different scattering properties. Binding partners with a high affinity for the analyte are immobilized on the particles which cause large light scattering. In contrast, binding partners with a low affinity for the analyte are immobilized on particles that cause little light scattering.

A similar method is known from US5,585,241. In order to increase the dynamic measurement range, it is proposed here in conjunction with flow cytometry immunoassays: two different sized particles are loaded with two antibodies of different affinities with respect to the same antigen (small particles are loaded with high affinity antibodies; large particles are loaded with low affinity antibodies) and another detectable labeled antibody is used in order to detect the antigen by forming a sandwich complex. The proposed system works here with two different standard curves (one for each particle category) and allows quantitative analyte determination by means of developed software.

To avoid the hook effect at high analyte concentrations ("high dose hook effect"), US4,743,542 discloses a method in which a sample is simply provided with a quantity of the same but unlabelled antibody in addition to a detectable labelled antibody against the target antigen. Whereby the two antibodies compete for the analyte molecules and supersaturation typical for the hook effect (if this is the case) only occurs at higher antigen concentrations. This extends the dynamic measurement range to higher concentrations, but at the cost of sensitivity. For this purpose, low-affinity antibodies are proposed with the same effect.

US4,595,661 describes a heterogeneous sandwich immunoassay in which the hook effect is avoided by using two soluble antibodies in addition to an immobilized capture antibody, wherein the two soluble antibodies have different affinities and properties for the antigen. The antibodies with lower affinity here only contribute significantly to the measurement signal at high antigen concentrations, and thus avoid the hook effect being detectable.

It is known from US5,073,484: the immunodetectable analyte is quantitatively detected by means of a plurality of discrete, adjacent binding zones in a flowing carrier. The more analyte present in the sample, the more zones specific binding and detection reactions occur. The number of regions that have a color after contacting the sample is herein related to the amount of analyte in the sample. To improve the accuracy and to extend the measurement range, US5,073,484 suggests increasing the number of binding zones. The disadvantage here is that the automatic analysis of the binding zones requires a relatively complex optical system which can detect and analyze a plurality of zones if necessary in order to thus allow quantitative analyte determination. In this regard, the test device must be relatively long due to the relatively large number of discrete bonding areas that are spatially separated from one another. In order to ensure reliable diffusion of the sample through the test device, it is therefore necessary to work with relatively large sample volumes, which is likewise disadvantageous, in particular for sample collection reasons, in particular if a whole blood sample is to be used.

WO00/31538 describes immunochromatographic test strips in which one or more control zones are disposed on an inhalable Matrix (Matrix) in addition to the analyte detection zone. The binding partners of the analyte provided with a detectable label are bound to the matrix not only in the analyte detection zone but also in the control zone. In this case, a precisely defined amount of labeled binding partner is bound in the control zone, wherein this amount is independent of the amount of analyte in the sample. It is preferred to bind different amounts of labeled binding partners in the control zone so that a quasi-internal comparison scale is contained on the test strip. In the analysis of the analyte detection area, the control area is taken into account for calibration. In order to increase the dynamic measurement range, in particular with regard to the nonlinear concentration-measurement signal relationship, WO

00/31538 suggest additional control areas on the test strip.

It is known from "upper converting Phosphor reports in immunochromogenic Assays" (Analytical Biochemistry288, 176-187(2001)) J.Hampl et al: in immunochromatographic test strips for detecting analytes using fluorescent labels, measurement signals are analyzed in consideration of a control region ("control line") containing an immobilized form of a species-specific antibody in addition to the detection region ("target line") originally containing an immobilized form of an analyte-specific antibody. From OraSere Technologies, Inc., Bethlehem, PA, U.S. A., inWWW.orasure.comA similar application is described above. Thus, first of all, an evaluation of the "target line" and of the "control line" is carried out in order to eliminate a change in the measurement signal, wherein the change depends on the liquid in the optical measurement range of the test stripThe actual amount of (a). This also indirectly increases the sensitivity of the detection method (assay) (thus enabling an expansion of the dynamic measurement range to lower concentrations). In contrast, the dynamic measurement range is expanded to higher concentrations.

The dynamic measurement range of the immunochromatographic test device can be extended in fact by diluting the sample material accordingly before the analysis. The measurement range expansion achieved in this way is, however, only unsatisfactory, since an additional implementation step is required for this purpose, which potentially leads to errors in the analysis. In addition, in particular in the case where the analyte may be present in a similar sample in a possibly very high concentration and in a possibly very low concentration, controlled sample dilution is advisable only if the analyte is present in the sample in a high concentration, while in the opposite case it is not advisable, since here the lower detection limit may not otherwise be exceeded by the dilution and the analyte is erroneously not detected in the sample.

It has not hitherto been possible to easily and reliably extend the dynamic measurement range to higher analyte concentrations without compromising the lower detection limit for analyte detection.

The invention is based on the object of eliminating the disadvantages of the prior art. The object of the present invention is, in particular, to extend the dynamic measurement range of test elements, in particular immunoassay test elements, based on specific binding reactions to higher analyte concentrations, wherein this should be achieved in particular in such a way that no impairment in the lower detection limit must be tolerated.

This task is solved by the subject of the invention.

The subject of the invention is a method according to claim 1. Preferred embodiments of the invention are the subject of the dependent claims.

The invention makes it possible to shift the dynamic measurement range of test elements based on specific binding reactions, in particular immunoassay test elements, to higher analyte concentrations without having to tolerate damage in terms of detection limits. To this end, it is proposed according to the invention that at least two zones are provided in or on the test element which contain reagents which, due to different affinities for the analyte (for example in the case of antibodies with different affinities for the analyte) or due to different principles of interaction with the analyte or with other reagents involved in the detection of the analyte (for example antibodies directed against the analyte in one zone and analytes or analyte analogs in another zone), produce detectable signals of different intensities. The "different principles of interaction" can be, for example, different test principles, for example sandwich complex formation in one region and competition test guidance in another region. For the analysis of the analyte concentration-signal intensity relationship, the signals in at least two zones are taken into account and are used by suitable methods (calibration) for determining the analyte.

For better understanding and distinction, the two regions which are important in the method of the invention and which are located in or on the test element shall be referred to below as analyte detection region (shortly: detection region) and control region. The name should also be retained if it is not usual according to common language practice, for example if binding partners of different affinities are arranged in the region and thus a signal can only be observed from a threshold concentration of analyte.

The invention also encompasses methods wherein more than 1 probe region and/or more than 1 control region on a test unit are analyzed. Such as the method may also be used for analysing test elements comprising a probe line with a binding partner having a high affinity, a probe line with a binding partner having a low affinity and a control zone (such as comprising an immobilised analyte analogue).

The solution of the invention relates in particular to extending the measurement range of an immunoassay device in a sandwich assay by an additional quantitative analysis of the control line. This is typically only a functional control used by the user and is not considered for quantifying the analyte. However, as the analyte content increases, more and more antibody-labeled conjugate (Konjugat) is captured on the signal line or saturated with analyte, so that less and less antibody-labeled conjugate (such as antibody gold conjugate) is bound on the control line. The signal intensity of the control line therefore decreases with increasing analyte concentration. By measuring the signal intensities simultaneously on the control line and on the signal line (e.g. by reflectance or fluorescence measurements) and calculating both signal intensities using a suitable algorithm, the dynamic measurement range and thus the slope of the calibration curve (and thus the accuracy at higher concentrations) can be significantly improved.

Similarly this applies to test units based on specific binding reactions other than immunological binding reactions. The corresponding specific binding reactions are well known to the skilled person. Examples are the following binding pairs:

antibodies with haptens, antigens or other antibodies (such as species-specific antibody-interactions), where corresponding fragments of the species are sometimes sufficient;

biotin with avidin or streptavidin;

hormones and hormone receptors;

sugars and lectins;

nucleic acids and complementary nucleic acids; and so on.

For better understanding and clarity, the following discussion will focus on immunological binding pairs, i.e., binding pairs: antibodies with haptens or antigens or antibodies, however this should not be a limitation of the preferred, but not exclusive, embodiment of the invention.

The method according to the invention is used in particular for determining the concentration of an analyte in a sample by means of an immunoassay unit. The test element here has not only an analyte detection region but also a control region. The sample is contacted with the test element and with an analyte-specific reagent which, by interacting with the specific reagent, produces a detectable signal in the analyte detection zone, provided that the analyte is present in the sample. The measurement signal is dependent on the amount of analyte in the sample. A portion of the specific reagent that does not interact with the analyte or the reagent in the detection zone produces a detectable signal in the control zone. What is important here is that: the signal detectable in the control zone is also dependent on the amount of analyte in the sample. The signals in the analyte detection region and the control region are measured and correlated with each other, such as being calculated from each other. The result of the calculation is compared to a standard curve and finally the analyte concentration is determined.

According to the present invention, suitable analytes are analytes that can be detected based on a specific binding pair relationship. Especially preferred for immunodetection are antibodies, antigens, haptens (including fragments thereof, respectively). Particularly preferred are the immunodetectable analytes hCG, BNP, (NT-) proBNP, troponin I, troponin T, myosin, D-dimer, CRP, HIV, HCV, CD40, CK-MB, TSH, etc.

According to the invention, all sample materials which are flowable or transportable in a flowable form are suitable for the sample from which the analyte can be determined. Particularly suitable are body fluids such as blood and fractions derived therefrom (serum, plasma), saliva, urine, cerebrospinal fluid, semen, interstitial fluid, sweat and the like. Sample materials which do not flow but can be converted into a flowing state by dissolution or suspension in a solvent, in particular in an aqueous solvent, are also suitable.

Immunoassay test units that can be used according to the present invention are well known to the skilled person. The detection of analytes by means of such test elements is based on specific interactions between the analyte and the binding pair. Such interactions include binding pairs: antigen/antibody, antibody/antibody, hapten/antibody, antigen fragment/antibody, antibody fragment/antibody, and the like. As already mentioned, the test elements mostly contain flowable materials (e.g. paper, fleece, membranes, capillary channels), which are optionally fixed to a non-movable carrier. The test elements typically each have one or more sample application zones, an aspiration zone, a chromatography zone, a detection zone, a reaction zone, and a control zone. It is only important for the present invention to have at least one (analyte) detection zone and at least one control zone.

Spatially narrowly limited regions separated from the control region in or on the material capable of flow are typically used as analyte detection regions, wherein species (Spezies) as analyte measurement are combined during the use of the test element according to the purpose, so that they can be detected visually, optically or otherwise. Typically, a detectable binding partner for the analyte (e.g., a correspondingly labeled anti-analyte antibody) is bound in the analyte detection region by a specific interaction. To this end, there is a corresponding immobilized binding partner in the analyte detection region, such as an antibody to the antigen (so that a detectable sandwich complex can be formed from the immobilized antibody, analyte and detectably labeled antibody) or a species of other binding pair, such as immobilized (poly) (chain) avidin (so that a previously formed sandwich complex can be formed from the biotinylated antibody, analyte and detectably labeled antibody). The construction, function and other variations of such detection zones are well known to the skilled person.

The region which is narrowly spatially limited on or in the flowable substance, is separated from the analyte detection region and is mostly located downstream of the analyte detection region is typically used as a control region, wherein species are bound independently of the presence of the analyte in the sample during the use of the test element according to the purpose, so that they can be detected visually, optically or otherwise. The control area is typically used for functional control of the test unit. The signals in the control zone demonstrate: the sample has flowed through the flowable carrier in sequence and ideally the corresponding binding reagents are able to function. Typically, a detectable binding partner for the analyte (such as a correspondingly labeled anti-analyte antibody) is bound in the control zone by a specific interaction. For this purpose, corresponding, immobilized binding partners, such as antibodies against the labeled anti-analyte antibody (so that a detectable complex can be formed by the immobilized antibody and the detectably labeled antibody) or immobilized analyte analogs (so that a complex can be formed by the analyte and the detectably labeled antibody) are present in the control zone. The construction, function and other variations of such control zones are well known to the skilled person.

A specific reagent (also synonymously referred to as "specific binding partner") contained in the test element or to be added to the test element or sample undergoes a selective (binding) reaction with the analyte or immobilized binding partner on the carrier. The reagent allows the amount of analyte present in the sample to be inferred, either directly or indirectly.

Preferred binding partners are antibodies (AK; monoclonal antibody or AB in English), especially polyclonal antibodies (PAK; polyclonal antibody or PAB in English) or monoclonal antibodies (MAK; monoclonal antibody or MAB in English), as well as antigens and haptens and fragments thereof, as long as the preferred binding partner is active for the purpose of detection of a particular analyte.

Preferably, a part of the binding partners is provided on the test device in such a way that the binding partners can be separated from the test device by the sample liquid, for example by impregnation with a suitable carrier substance, such as a fleece, a membrane or the like, or by application and drying in a corresponding (capillary) pore structure.

However, it is also possible to add at least one of the binding partners in the form of a dissolved reagent for rapid testing, for example by incorporating a reagent solution into the sample or by applying a reagent solution independent of the sample to the testing device. According to the present invention, although less preferred, it is also possible to use all specific binding partners in one solution or in a plurality of solutions for rapid testing. Only one further binding partner is then present on the test device in the detection zone, wherein this further binding partner can capture the respectively labeled specific binding partner and thereby cause the analyte to bind indirectly to the solid phase of the rapid test. Similarly, a binding partner is present in the control region that can capture the corresponding labeled specific binding partner without requiring direct participation in the analyte.

In principle, a detectable signal is generated both in the detection zone and in the control zone by one of the binding partners immobilized there. Here, although not preferred, the signals in the two zones may be based on different principles. And preferably: the signals in both the analyte detection region and the control region are based on the same principle. Detectable signals are, for example, optically or visually detectable color changes, luminescence signals, in particular fluorescence signals, radioactive rays and the like. The detectable signal is thereby generated by the respective labeled species (binding partner) which, as previously described, is bound in the analyte detection zone or control zone. According to the invention, labels as binding partners can furthermore also be considered: individual labels (such as with colored latex, polymer labels, or semiconductor nanocrystals (so-called quantum dots) or metal (sol) labels (gold, selenium, etc.)) and non-individual labels (enzymes, radioisotopes, fluorescent labels) and the like.

Depending on the label used, other detection methods are naturally also required and possible (e.g.fluorescence measurements, radioactivity measurements, determination of enzyme activity, etc.). By means of these detection methods, the signals generated in the analyte detection region and the control region can be measured, in particular, with correspondingly designed measuring instruments. As is known to the skilled person from the prior art. However, important according to the invention are: not only the signal in the analyte detection region but also the signal in the control region is detected by the measuring device. Suitable measuring devices and methods for evaluating test elements are known to the skilled worker in the prior art. The system "Cardiac Reader" from Roche diagnostics Gmb H, Mannheim is a typical example of a measuring device. Here, the immunoassay strip is illuminated by means of one or more light sources (for example LEDs) and the gray-scale values of the detection region (signal line) and the control region are determined by means of position-resolved reflectance measurements. Corresponding measuring and analysis methods are known, for example, from US5,717,778.

According to the invention, not only the signals in the control zone but also the signals in the detection zone are detected and mutually calculated by suitable mathematical methods. This takes place, for example, in a central computing unit of the measuring device. According to the invention, it is important that both signals are dependent on the analyte concentration at least in a certain concentration range of the analyte. By suitable mathematical methods, which are further elaborated graphically in connection with the examples below, the dynamic measurement range of the measurement unit can be extended (compared to the unique analysis of the signal in the detection region).

The analyte concentration is typically determined from the measured signals of the detection and control regions by comparing the measured values to corresponding calibration curves obtained by measuring standard solutions having known analyte amounts. The calibration curve is preferably established using the signal of the probe line and the signal of the control line.

One possible implementation of the method of the invention provides for: reagents that produce detectable signals in the analyte detection and control regions have different affinities or reactivities for the analyte. Preferably, antibodies of lower affinity or less reactivity to the analyte in the control region participate in signal formation than in the analyte detection region.

An alternative possible implementation of the method of the invention provides for: the reagents that produce detectable signals in the analyte detection and control regions produce detectable signals of different intensities based on different principles of interaction with the analyte or with other reagents involved in analyte detection. In particular, an antibody directed to the analyte may be bound in the analyte detection region and the analyte or analyte analogue may be bound in the control region. It is also possible that the binding partners immobilized in the control region are bound to other epitopes of the antibody or to heterologous building blocks added synthetically to the antibody, or that antibodies specific to the type of antibody to be immobilized are immobilized in the control region. All these variants are known to the skilled worker from the prior art.

The measurement range can be extended to very high concentrations in the presence of a so-called "high dose hook effect". In this case, the signal line intensity drops again in the case of very high analyte concentrations, since the amount of antibody provided is no longer sufficient to form a sandwich complex in any case. More complexes are formed consisting of the analyte and one antibody (conjugate) each. The intensity or concentration dependence of the control line in this concentration range may be too low to be analyzed. And the decrease in the intensity of the signal line can be analyzed according to the concentration. There are then three analysis ranges for this case:

1. signal line strength increase according to concentration

2. Control line strength drop according to concentration

3. Signal line strength drop according to concentration

The automatic distinction between the three ranges mentioned above can be achieved, for example, by the following algorithm:

A) if the reflection at the signal line is greater than X1% and the reflection at the control line is less than Y1%, then only the signal line is analyzed and a standard curve of analyte concentration from A1 to A2mg/ml is used.

B) If the reflection at the signal line is less than X2% and the reflection at the control line is greater than Y2% and less than Y3%, then only the control line is analyzed and a standard curve of analyte concentration from A3 to A4mg/ml is used.

C) If the reflection at the signal line is greater than X3% and the reflection at the control line is greater than Y4%, then only the signal line is analyzed and a standard curve of analyte concentration from A5 to A6mg/ml is used.

The invention is explained in more detail with the aid of the following examples and the figures. Although in this example only an immunoassay test device is shown, which test device operates with a gold-labelled binding partner and is measured in reflectance photometry, the invention is not limited thereto. In addition to the immunological interactions that are indeed based on antigen (or hapten) antibody binding pairs, other binding pairs are also possible, in particular hormone-receptors, sugar-lectins, nucleic acid-complementary nucleic acids, biotin- (chain) avidin and the like. Besides gold labels, other separate labels are possible, such as using colored latex, polymer labels, or semiconductor nanocrystals (so-called quantum dots) or metal (sol) labels, as well as non-separate labels (enzymes, radioisotopes, fluorescent labels) and the like. Depending on the label used, other detection methods are naturally also required and possible (e.g.fluorescence measurements, radioactivity measurements, determination of enzyme activity, etc.). These variants are known to the skilled worker in many possible embodiments.

A preferred embodiment of a test device in the form of an immunochromatographic test strip usable in accordance with the present invention is schematically shown in FIG. 1.

The relative reflectance values (R in%) when analyzing the probe line (NS) and the control line (KS) are shown in fig. 2 as a function of the concentration of troponin T in the sample (C in ng/ml).

The calibration curve of the troponin T test strip is shown in fig. 3, wherein it is derived by applying the algorithms a) (prior art) and b) (present invention) detailed in example 2 to the measurement values of fig. 2.

In fig. 4 is shown a calibration curve of a troponin T test strip, wherein the calibration curve is derived by applying the algorithm c) (invention) detailed in example 2 to the measurement values of fig. 2.

The relative reflectance values (R in%) when analyzing the probe line (NS) and the control line (KS) are shown in FIG. 5 as a function of the NT-proBNP concentration (C in ng/ml) in the sample.

The calibration curve of the NT-proBNP test strip is shown in fig. 6, wherein the calibration curve is derived by applying the algorithms a) (prior art) and b) (present invention) elaborated in example 3 to the measurement values of fig. 5.

The relative reflection values (R in%) for the analysis of the probe line (NS) and the control line (KS) are shown in FIG. 7 in three partial graphs (each covering a different concentration range) as a function of the concentration of D dimer in the sample (c in ng/ml).

The numbers in the figures represent:

1 sample application zone

2 red blood cell compartment

3 detection zone

4 suction zone

5 support Material

6 sample application matrix ("Biotin wool" and "gold wool")

7 erythrocyte separation matrix

8 probing matrix

9 first linear fixing zone (detection line; analyte detection zone)

10 second linear fixing zone (control line; control zone)

11 inhalation matrix

Examples of the present invention

1) Production of a test device for determining an antigen from Whole blood (see FIG. 1)

The test device (fig. 1) comprises a carrier material (5) on which a sample application zone (1), a red blood cell separation zone (2), a detection zone (3) and an intake zone (4) are applied. In the sample application zone (1) a sample application matrix (6) is arranged, which partially overlaps the red blood cell separation matrix (7). The red blood cell separation matrix (7) is somewhat overlapped on one side with a detection matrix (8) (detection region), on which streptavidin in the form of a wire (9) is fixed as a detection wire and an antigen or antigen analogue, for example as a synthetic or recombinant antigenic peptide, in the form of a wire (10) is fixed as a control wire. The suction zone (11) overlaps somewhat with the detection substrate (8). All reagents required for the formation of complexes with the analyte to be detected are arranged in the sample application matrix (6). In this case, the sample application zone consists of 2 hairs placed one above the other, of which the first one ("gold hairs") is wetted with gold-labelled antibody against the analyte and the second one ("biotin hairs") contains biotinylated antibody against the analyte. The analyte is here an antigen present in the blood, in particular troponin T, NT-proBNP or D dimer.

A polyester film (petz) with a thickness of 350 μm was used as carrier layer 5. Polyester hairs (Roche Diagnostics) 360 μm thick were used as "gold hairs" or "biotin hairs" of the sample application matrix 6. A1.8 mm thick glass fiber wool (Roche Diagnostics) was used as the red blood cell separation matrix 7. A nitrocellulose membrane (Sartorius) 140 μm thick was used as the detection substrate 8. A1.8 mm thick glass fiber fleece (Roche Diagnostics) was used as the inhalation matrix 11. As shown in fig. 1, the individual components (6, 7, 8, 11) are bonded to the carrier layer 5 with a slight overlap by means of a hot-melt adhesive.

The impregnation formulation of "gold and biotin wool" of the cited examples is:

proBNP test strip：

"biotin fleece": 100mM Hepes pH7.4, 0.1%

Biotinylated antibodies to analytes

"gold hairs": 100mM Hepes pH7.4

Antibodies to analytes as gold conjugates

Troponin T test strip：

"biotin fleece": 100mM MES pH5.6

Biotinylated antibodies to analytes

"gold hairs": 100mM succinic acid, pH5.6, 0.1% Tween,

antibodies to analytes as gold conjugates

D dimer test strip：

"biotin fleece": 100mM Hepes pH7.4, 0.1%

Biotinylated antibodies to analytes

"gold hairs": 100mM Hepes pH7.4

Antibodies as gold conjugates against analytes

2) Analysis of test strips for the determination of troponin T (FIGS. 2 to 4)

The troponin T test strips according to example 1 were loaded with whole blood samples to which recombinantly produced troponin T was added in various amounts. The strips were analyzed not only according to the two methods according to the invention (variants b) and c), see below), but also for comparison purposes according to the usual method (variant a)). For the detection line (NS) (9) and the control line (KS) (10), the reflection is determined here with a conventional measuring device (Cardiac Reader, Roche Diagnostics GmbH) built on a CCD camera and the signal is calculated according to the following algorithm:

a)|Rem NS(0)-Rem NS(c)|

b)|Rem KS(0)-RemKS(c)|+|RemNS(0)-RemNS(c)|

c) i Rem KS (0) -Rem NS (c) i Rem NS (0) -Rem NS (c) i where the algorithmic alternative a) represents only the usual analysis of a probe line according to the prior art. According to the invention, both the probe line signal and the control line signal are taken into account in b) and c) for the analysis.

In the formula:

rem NS (0) denotes the reflection in% of the probe line at an analyte concentration of 0

Rem NS (c) denotes the reflection in% of the probe line at an analyte concentration of c

Rem KS (0) represents the reflectance in% of the control line at an analyte concentration of 0

Rem KS (c) represents the reflectance in% of the control line at an analyte concentration of c

The relative reflectance values (R in%) when analyzing the probe line (NS) and the control line (KS) are shown in fig. 2 as a function of the concentration of troponin T in the sample (c in ng/ml). Figure 2 shows that the control line signal decreases (reflectance increases) at the same time that the probe line signal increases (reflectance decreases) as the analyte concentration increases.

The calibration curve of the troponin T test strip is shown in fig. 3, wherein the calibration curve is derived by applying the above-described algorithms a) (prior art) and b) (present invention) to the measurement values of fig. 2. In fig. 4 is shown a calibration curve of a troponin T test strip, wherein the calibration curve is derived by applying the algorithm c) (invention) set out in detail above to the measurement values of fig. 2. Concentration determinations of only about to 10ng/ml can be carried out with the sole analysis of the probe line (algorithm a)) during the test, whereas concentrations above 20ng/ml can also be determined by the analysis of KS and NS according to algorithms b) and c).

3) Analysis of test strips for determination of NT-proBNP (FIGS. 5 to 6)

NT-proBNP test strips according to example 1 were loaded with whole blood samples to which synthetic NT-proBNP was added in varying amounts. The strips were not only analyzed according to the method according to the invention (variant b), see below), but also for comparison purposes according to the conventional method (variant a)). For the detection line (NS) (9) and the control line (KS) (10), the reflection is determined here with a conventional measuring device (Cardiac Reader, Roche Diagnostics GmbH) built on a CCD camera and the signal is calculated according to the following algorithm:

a)1-Rem NS(c)

b)Rem KS(c)：Rem NS(c)

wherein the algorithmic alternative a) merely represents a typical analysis of the probe line according to the prior art. According to the invention, not only the probe line signal but also the control line signal are taken into account in b) for the analysis.

The abbreviations in the formula have the same meanings as in example 2.

The relative reflectance values (R in%) when analyzing the probe line (NS) and the control line (KS) are shown in FIG. 5 as a function of the concentration of NT-proBNP (c in ng/ml) in the sample. Figure 5 shows that the control line signal decreases (reflectance increases) at the same time that the probe line signal increases (reflectance decreases) as the analyte concentration increases.

In fig. 6 a calibration curve of a NT-proBNP test strip is shown, wherein the calibration curve is generated by applying the above-described algorithms a) (prior art) and b) (present invention) to the measurement values of fig. 5. Only a concentration determination of about to 6ng/ml can be carried out at the time of the test with a unique analysis of the probe line (algorithm a)), whereas concentrations above 14ng/ml can also be determined by analysis of KS and NS according to algorithm b).

4) Analysis of test strips for determination of D dimer (FIG. 7)

The D-dimer test strip according to example 1 (biotinylated antibody directed to the analyte immobilized at the probe line; control line consisting of immobilized fibrin fragments comprising D-dimer building blocks; free gold conjugate antibody can be bound thereto) was loaded with a whole blood sample to which fibrin fragments comprising D-dimer were added in varying amounts. The strips were not only analyzed according to the method according to the invention (variant b), see below), but also for comparison purposes according to the conventional method (variant a)). The conventional measuring device (Cardiac Reader, Roche) designed on a CCD camera is used for the detection line (NS) (9) and the control line (KS) (10)

Diagnostics GmbH) determines the reflection and calculates the signal according to the following algorithm:

a)Rem NS(c)

b)1) if the reflection at the signal line is greater than 30% and the reflection at the control line is less than 40%, then only the signal line (Rem NS (c)) is analyzed and a standard curve with an analyte concentration of 0 to 3 μ g/ml is used.

2) If the reflection at the signal line is less than 50% and the reflection at the control line is greater than 40% and less than 70%, then only the control line (Rem KS (c)) is analyzed and a standard curve with an analyte concentration of 3 to 20 μ g/ml is used.

3) If the reflection at the signal line is greater than 30% and the reflection at the control line is greater than 70%, then only the signal line (Rem NS (c)) is analyzed and a standard curve with an analyte concentration of 20 μ g/ml to 1000 μ g/ml is used.

Wherein the algorithmic alternative a) merely represents a typical analysis of the probe line according to the prior art. According to the invention, not only the probe line signal but also the control line signal is taken into account in b) for the analysis.

The abbreviations in the formula have the same meanings as in example 2.

The relative reflectance values (R in%) when analyzing the probe line (NS) and the control line (KS) are shown in FIG. 7 as a function of the concentration of D dimer in the sample (c in ng/ml).

D-dimer concentrations up to about 3. mu.g/ml can be analyzed as usual by a decrease in reflection (increase in intensity) of the signal line. Up to about 20. mu.g/ml, the control line can be analyzed for increased reflection (decreased intensity). At analyte concentrations above 20. mu.g/ml, the concentration dependence of the control line signal is too small. From about 20. mu.g/ml up to > 1000. mu.g/ml, the increase in reflection (decrease in intensity) of the signal line can be used for analysis. Since the lower limit of the measurement range is < 0.5. mu.g/ml, >1000 dynamic factors can be obtained.

Claims (8)

1. Method for determining the concentration of an analyte in a sample by means of a test element based on a specific binding reaction, which test element has not only an analyte detection region but also a control region, wherein:

a) contacting the sample with the test element and with an analyte-specific reagent;

b) the analyte, so long as present in the sample, produces a signal detectable in the analyte detection region by interaction with an analyte-specific reagent, wherein the signal is dependent on the amount of analyte in the sample;

c) a portion of the analyte-specific reagent that does not interact with the analyte or the analyte-specific reagent in the analyte detection region produces a detectable signal in the control region, wherein the signal is also dependent on the amount of analyte in the sample;

d) signals in the analyte detection region and the control region are measured;

e) both signals are calculated according to a suitable algorithm;

f) the results thus produced are compared with a standard curve; and is

g) The analyte concentration is determined.

2. Method according to claim 1, characterized in that in step e) the two signals are compensated for each other.

3. Method according to claim 1 or 2, characterized in that not only the signal of the probe line but also the signal of the control line is used to establish the calibration curve.

4. A method as claimed in claim 1, 2 or 3 wherein the analyte-specific reagents that produce detectable signals in the analyte detection and control regions have different affinities or reactivities for the analyte.

5. The method of claim 4, wherein antibodies of lower affinity or less reactivity to the analyte at the control region are involved in signal formation than in the analyte detection region.

6. A method as claimed in claim 1, 2 or 3, wherein the analyte-specific reagent which produces a detectable signal in the analyte detection zone and the control zone produces detectable signals of different intensities according to different principles of interaction with the analyte or with other analyte-specific reagents involved in analyte detection.

7. The method of claim 6, wherein an antibody directed to the analyte is bound in the analyte detection region and the analyte or analyte analog is bound in the control region.

8. The method of claim 1, wherein the test element is based on an immunological binding reaction.