WO2004097413A1

WO2004097413A1 - Method for determining the concentration of an analyte

Info

Publication number: WO2004097413A1
Application number: PCT/EP2004/004553
Authority: WO
Inventors: Jae-Chul Pyun; Ji-Won Chung; Sang-Doo Kim
Original assignee: Kist-Europe Forschungsgesellschaft Mbh
Priority date: 2003-04-30
Filing date: 2004-04-29
Publication date: 2004-11-11
Also published as: DE10319572A1

Abstract

The invention relates to a method for determining the concentration of an analyte used as a ligand in a sample by means of a ligand-receptor assay. According to said method, a predetermined number of receptors or binding points to which the analyte can specifically bind as a ligand are immobilized or provided on a substrate. The assay is contacted with the sample, both an overall signal and the individual signal generated through said contact with the sample are measured, and the concentration of the analyte is determined from the individual signal by taking into account the overall signal.

Description

Method for determining the concentration of a

analytes

The present invention relates to a method for determining the concentration of an analyte. Such methods are used in the entire field of analysis, especially medicine and environmental analysis.

Immunoaffinity assays which function according to the ligand-receptor principle are used in particular for such methods. The receptor represents a binding site to which an analyte as a ligand can bind in a highly specific manner. The different configurations of such ligand-receptor assays each generate a signal in different ways when some of the binding sites (receptors) are occupied by an analyte.

Such ligand receptor assays are particularly popular used in the field of biosensors as immunoaffinity assays. However, the present invention is not limited to immunoaffinity assays, but encompasses the entire range of ligand-receptor binding assays, both ligand and receptor being inorganic molecules, organic molecules or also biological molecules such as antigens, antibodies, proteins, haptens and the like should include.

In these assays, a signal is usually generated on a detection layer which converts the degree of occupation of the receptors into a detectable signal.

For example, in the field of biosensors, enzyme immunoassays are used that take advantage of the specific interaction between antigens and the associated antibody for highly specific and selective molecular recognition. For this purpose, for example, the antibody is placed on a metal surface

Transducers immobilized so that the transducer emits a specific signal when an antigen binds to the antibody.

Since the recognition layer binds a limited number of binding sites (receptors), the number of binding sites occupied by analytes increases when the same recognition layer is used repeatedly, since a receptor-analyte binding is usually not readily detachable due to the highly specific and selective interaction between receptor and analyte is. This means that the signal strength of the sensor becomes smaller and smaller with each further contact of the sensor with an analyte-containing sample and ultimately results in a saturation of the biosensor response. If all the binding sites are occupied by the analyte on the detection layer, the sensor generates a maximum accumulated sensor signal (total sensor signal).

For this reason, receptor ligand

Binding assays, in particular immunoaffinity sensors, are usually chemically regenerated between two uses by removing the analyte, in particular the antigen in antibody-antigen assays, from the immunoaffinity layer. The aim of such regeneration methods is to restore the same or at least a large part of the activity of the sensor through this treatment. The usual regeneration methods, such as washing with acidic or basic solutions, however, have the disadvantage that they partially or completely denature the antibodies immobilized on the immunoaffinity layer. This leads to a reduced number of binding sites when the immunoaffinity layer is reused and thus to a reduced sensitivity or activity.

The object of the present invention is to provide a method for determining the concentration of an analyte, in which the disadvantages mentioned above can be avoided.

This object is achieved by the method according to claim 1. Advantageous further developments of the method according to the invention are described in the dependent ones

Given claims.

The present inventive method uses a Re ^{'Zeptor-ligand} binding assay such that the assay is contacted with a sample in which

, is optionally an Analyte ^'is as ligand. It will now determines not only the signal generated by contacting the sample with the immunoaffinity assay, but also the overall signal that has been produced since the binding assay was used for the first time. The overall signal is the signal that results as the difference between the signal of the new and unused binding assay and the signal after contacting the sample. Alternatively, if the sensor is based on a purely differential principle, the sum of all signals that result from the individual measurements that have already been carried out overall with the binding assay can also be considered as the total signal. Overall signal is understood here in the description as well as in the claims to mean the accumulated overall signal.

Surprisingly, it has been found that it is possible in this way to reliably determine the sensitivity of the sensor from the overall signal, even if it has already been used several times without being regenerated in between. The present invention therefore makes it possible to dispense with regeneration of the binding assays or to carry out no regeneration and thus to avoid the disadvantages associated therewith. In particular, the degeneration of the sensitivity of the sensor due to regeneration and the effort associated with regeneration are avoided.

This is possible because the reduction in sensor sensitivity correlates with the occupation of the binding sites for the analyte. However, in the case of highly specific and selective binding assays, this in turn correlates with the overall signal as described above since the assay was first contacted with the Analytes.

The entire signal of the sensor can thus be between 0 and the total signal which is present when all of the receptors are fully occupied.

Binding parts results from analytes. It has now advantageously emerged that this area can be divided into a few or more sections within which the sensitivity of the sensor can be regarded as constant. The sensitivity can be regarded as constant if a certain degree of inaccuracy (standard deviation) or linearity is maintained within this range of the overall signal. The corresponding values can be determined from comparative tests on sensors, for example by means of a numerical linear regression. The limit values for a range are to assume the standard deviation to <0.1 or a linearity> 0.98, advantageously> 0.99. If a specific overall signal is then measured, the corresponding sensitivity range corresponding to this overall signal can be selected and the concentration of the analyte can be determined from this individual signal on the basis of this sensitivity (sensitivity).

In the case of new, still unused immunoassays, the method according to the invention makes it possible to reuse it until the total signal reaches the first limit value at which the area ends with the initial sensitivity of the binding assay and the next area begins with reduced sensitivity. It is therefore also possible, in a further variant of the present invention, for a binding assay up to a limit value of the overall signal to be used several times without regeneration without taking into account possible changes in sensitivity and still being able to determine a sufficient accuracy of the analyte concentration.

Examples of methods according to the invention are now described below.

1 shows the surface plasmon resonance when samples are used repeatedly in four different Spreeta ^R chips;

2 shows the averaged signal from four Spreettaa ^1R1 --CChhiippss with repeated application of samples; and

3 shows the surface plasmon resonance with repeated application of samples to a single Spreeta ^R chip.

For the following examples only chemicals of analytical purity were used. Bovine serum albumin (BSA), anti-BSA polyclonal antibodies, 11-mercaptoundecanoic acid, N- (3-dimethylaminopropyl) -N '-ethylcarbodiimide (EDAC), N-hydrosuccinimide (NHS) were obtained from Sigma-Aldrich Chemical Co. (Deisenhofen , Germany).

A Spreeta ^R chip (Texas Instruments, Dallas, USA) was used as the immunoaffinity layer, which was arranged in a flow-through cell with a capacity of 50 μl. This flow cell was connected to a peristaltic pump. To generate the immunoaffinity layer, an anti-BSA layer was produced on the gold surface of the Spreeta ^R chip, in which was first incubated with 1μM 11-mercaptoethanol for two hours at room temperature. The gold surface was then observed using an excess volume of ethanol and PBS (10 mg / ml). Bovine serum albumin (BSA) was now immobilized on this surface. For this, the surface was treated with 100 mM EDAC / 10 mM NHS for 10 min. incubated and then a BSA solution (10 mg BSA / ml PBS) for one hour at room temperature on the gold surface. In order to block remaining reactive centers on the gold surface, this was then again with 0.1 M ethanolamine solution for 10 min. treated.

The measurements were then carried out using an anti-BSA antibody as analyte, which specifically and selectively binds to the BSA immobilized on the gold surface of the Spree- ^R chip.

The sample was placed in the flow cell using an autosampler (MIDAS, Spark Holland BV, Emmen, Holland) and a peristaltic pump (Amersham-Pharmacia Biotech AB, Uppsala, Sweden). Both the peristaltic pump and the autosampler were controlled using a microprocessor. The surface plasmon resonance signal (SPR signal) generated by the Spreeta ^R chip was transferred online to a PC using a 12-bit converter. The entire assembly was tempered in an incubator at a temperature of 37 ° C.

Before the measurements, the Spreeta ^R chip was calibrated by first introducing air and then deionized water with a refractive index of 1.33 into the flow cell was given. The pumping rate was set at 1.0 ml / min. The baseline fluctuation was set to less than 5 m ° / h. PBS was circulated in the flow cell between the individual sample injections into the flow cell. A 0.1% Tween 20 solution was used in each case as a washing step. The volume of each injected. Sample was 500 μl, with the Spreeta ^R chip in each case for 10 min. was incubated with each sample. After such an incubation, 1 min. with Tween 20 and then twice for 1 min each. washed the surface of the Spreeta ^R chip with PBS. The individual SPR signal was determined as the difference signal before the Spreeta ^R chip contacted the sample and after the Spreeta ^R chip contacted the sample.

FIG. 1 now shows the SPR signals when samples are repeatedly used with four different Spreeta ^R chips, which were prepared as described above. Before the first sample measurement, a standard sample with a known concentration was taken. of 16.33 μg / ml was added to the Spreeta ^R chips. As can be seen in Figure 1, the signal for this standard sample was 37.22 m ° with a standard deviation of 0.56 m ° (1.5%). This means that the BSA

Layers on all four Spreeta ^R chips react with high reproducibility to the Antd-BSA antibodies. In other words, the BSA layer was reproducibly produced on the four Spreeta ^R chips. The Spreeta ^R chips were then contacted with a total of five different concentrations of anti-BSA antibodies and the associated individual signals were recorded. This is indicated in the last column in Figure 1. The concentrations of samples (1), (2), (3), (4) and (5) were 8.2 μg / ml, 9.8 μg / ml, 16.3 μg / ml, 24.5 μg / ml or 49.0 μg / ml. The accumulated signal indicated in the penultimate column in FIG. 1 also includes the signal of the standard sample given first.

Although the AntABSA antibodies were placed on the chips one after the other without regeneration of the chips, it can be seen that the signal curves of the Spreeta ^R chips have a high linearity and a sensitivity (sensitivity) of 2.36 m ° / (μg / ml) with a standard deviation of 0.10 (4.2%). The accumulated signals from the second to last column are a measure of the coverage of the immunoaffinity layer with anti-BSA antibodies.

The individual signals were summed up for each individual chip in order to determine the accumulated signal (total signal) before each new sample determination. The total signal over all five samples and the standard sample was determined to be 271.7 m ° to a standard deviation of 12.8 m ° (4.6%). In this area of the accumulated signal, the sensitivity of the sensor was changed only slightly. This means that the SPR chip is used several times within a predetermined degree of occupation of the receptors on the immunoaffinity layer without regeneration between the individual measurements. The linearity of the individual sensitivities was determined here to be 0.99. The sensitivity was determined from the slope of a graph, which was evaluated with linear regression, to 2.45 m ° (μg / l) and the inaccuracy of the measurements to 1.93 μg / ml. In measurements with an unused and new Spreeta ^R chip, a slope (sensitivity) of 2.47 m ° / (μg / ml) with a linearity of 0.99 was determined from the corresponding curve. This shows that the sensitivity of the sensor does not change significantly within an accumulated total signal, which can be up to 200 m °, and therefore the immunoaffinity layer can be used several times within this overall signal range.

FIG. 2 shows the application of the average signal size of the four Spreeta ^R chips shown in FIG. 1 when the samples are used repeatedly. As can be seen in FIG. 1, the order of the sample application was varied. FIG. 2 now shows the individual measured values with the associated standard deviations and a straight line according to a linear regression analysis. From this straight line the sensitivity to 2.45 ° / (μg / ml) and the inaccuracy of the measurement to 1.93 μg / ml were determined.

Since the immunoaffinity layer has only a limited number of binding sites, the sensor signal becomes smaller and smaller with increasing number of measured samples at the same sample concentration. The sensor response will therefore saturate when the chip is used repeatedly, so that a maximum, accumulated overall signal is finally achieved.

In order to investigate the influence of further uses of the Spreeta ^E chip, samples were repeatedly applied to a single SPR chip. Cycles of five measurements of samples were carried out. These five cycles are shown in FIG. 3 with the respectively determined average sensitivities, inaccuracies, linearity and the accumulated signal obtained for the respective cycle at the end of the respective cycle. The total accumulated signal for each cycle results from the sum of the accumulated signal of the respective cycle plus all accumulated signals of the previous cycles.

As can be seen from FIG. 3, the sensor sensitivity gradually decreases over the cycles, this decrease in the accumulated signal having a linearity of R = 0.987 with a gradient of -33.06 m ° / cycle and a y-axis intersection of 200 , 68 m °. This means that up to 6.1 cycles can be carried out in succession until the maximum accumulated total sensor signal is reached.

It can be seen that the linearity and the inaccuracy within the individual cycles are within a tolerance level, so that the sensitivity can be regarded as constant within each individual cycle and thus the individual es- Solutions of the individual cycle can be evaluated with the sensitivity specific to the cycle.

This makes it possible to use the accumulated total signal that results from a single measurement to place the individual measurement in a specific cycle and thus evaluate it, even if the chip was used many times before without regeneration.

The division of the cycles can take place on the basis of a tolerance level for the linearity constant, for example> 0.98, advantageously> 0.99. Depending on the desired accuracy of the evaluation of the measurement, however, other tolerance levels, for example 0.80, 0.90 or 0.95, can also be used.

In FIG. 3, the same concentrations as in FIG. 1 were used as samples (1) to (5).

As shown in FIG. 2, the sensor response has good linearity within each cycle, even without chemical regeneration of the immunoaffinity layer. This means that measurements as in FIG. 3 can also be used as a standard curve (calibration curve) in order to evaluate signals in the case of structurally identical receptor-ligand assays via the accumulated signal and a comparison with such a calibration curve. For example, if the accumulated signal of an identical sensor is 200 m ° and the individual signal of an applied sample is 15 m °, the concentration of the analyte in the sample can be determined, in which is referred to in Figure 3. In this case, this means that apparently a measurement within the range for the accumulated total signal in cycle 2 (between 171.8 m ° and 308.77 m °) was carried out and consequently a sensitivity of 1.55 m ⁰ / (μg / ml) can be used for evaluation.

Claims

claims

1. Method for determining the concentration of a

Analytes as ligands in a sample by means of a ligand-receptor binding assay in which a predetermined number of receptors or binding sites to which the analyte can specifically bind as ligand are immobilized or present on a substrate, characterized in that the Assay is contacted with the sample and both an overall signal and that by the

Contact with the sample generated individual signal is measured, and the concentration of the analyte is determined from the individual signal taking into account the total signal.

2. The method according to the preceding claim, characterized in that from the overall signal using a calibration curve or calibration values determines the sensitivity of the assay corresponding to this overall signal and from the individual signal and the sensitivity of the sample thus determined determines the concentration of the analyte in the sample.

3. The method according to the preceding claim, characterized in that the sensitivity of the assay is assumed to be constant within predetermined limit values of the overall signal.

4. The method according to any one of the two preceding claims, characterized in that the sensitivity of the assay is assumed to be constant within predetermined limit values of the overall signal if the sensitivity determined over this range of total signal has a certain degree of accuracy and / or linearity.

5. The method according to the preceding claim, characterized in that the sensitivity of the assay is assumed to be constant within predetermined limit values of the overall signal if the linearity is> 0.98, advantageously> 0.99.

6. The method according to any one of the preceding claims, characterized in that the range between a total signal of 0 and a maximum total signal with one or more areas before divided with constant predetermined sensitivity.

7. The method according to any one of the preceding claims, characterized in that the assay is contacted several times in succession with the same and / or different samples for successive measurements.

8. The method according to any one of the preceding claims, characterized in that the assay is contacted several times in succession for successive measurements with the same and / or different samples without him chemically, physically or in any other way before a measurement and / or between the individual measurements to regenerate.

9. The method according to any one of the preceding claims, characterized in that the assay is contacted several times in succession with the same and / or different samples for successive measurements until a first limit level for the overall signal is reached or exceeded.

10. The method according to any one of the preceding claims, characterized in that the individual signal from or as a difference from the measurement signal after contacting the assay with the sample and the measurement signal before contacting the assay with the sample.

11. The method according to any one of the preceding claims, characterized in that antibodies, antigens, proteins, haptens or other biological, organic and / or inorganic receptor molecules are used as receptors.