WO2004097413A1 - Procede de determination de la concentration d'un analyte - Google Patents

Procede de determination de la concentration d'un analyte Download PDF

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Publication number
WO2004097413A1
WO2004097413A1 PCT/EP2004/004553 EP2004004553W WO2004097413A1 WO 2004097413 A1 WO2004097413 A1 WO 2004097413A1 EP 2004004553 W EP2004004553 W EP 2004004553W WO 2004097413 A1 WO2004097413 A1 WO 2004097413A1
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WO
WIPO (PCT)
Prior art keywords
signal
assay
sample
sensitivity
analyte
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Application number
PCT/EP2004/004553
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German (de)
English (en)
Inventor
Jae-Chul Pyun
Ji-Won Chung
Sang-Doo Kim
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Kist-Europe Forschungsgesellschaft Mbh
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Application filed by Kist-Europe Forschungsgesellschaft Mbh filed Critical Kist-Europe Forschungsgesellschaft Mbh
Publication of WO2004097413A1 publication Critical patent/WO2004097413A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • 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.
  • 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.
  • a signal is usually generated on a detection layer which converts the degree of occupation of the receptors into a detectable signal.
  • enzyme immunoassays are used that take advantage of the specific interaction between antigens and the associated antibody for highly specific and selective molecular recognition.
  • 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.
  • 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).
  • 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.
  • the present inventive method uses a Re 'Zeptor-ligand binding assay such that the assay is contacted with a sample in which
  • Analyte 'is as ligand 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.
  • 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.
  • 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.
  • the degeneration of the sensitivity of the sensor due to regeneration and the effort associated with regeneration are avoided.
  • 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).
  • 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.
  • Bovine serum albumin BSA
  • anti-BSA polyclonal antibodies 11-mercaptoundecanoic acid
  • EDAC N- (3-dimethylaminopropyl) -N '-ethylcarbodiimide
  • NHS N-hydrosuccinimide
  • 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 ⁇ l. This flow cell was connected to a peristaltic pump.
  • an anti-BSA layer was produced on the gold surface of the Spreeta R chip, in which was first incubated with 1 ⁇ M 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.
  • 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.
  • Sample was 500 ⁇ l, 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.
  • a standard sample with a known concentration was taken. of 16.33 ⁇ g / ml was added to the Spreeta R chips.
  • 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
  • the accumulated signal indicated in the penultimate column in FIG. 1 also includes the signal of the standard sample given first.
  • 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 ° / ( ⁇ g / 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%).
  • 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 ° ( ⁇ g / l) and the inaccuracy of the measurements to 1.93 ⁇ g / ml.
  • a slope (sensitivity) of 2.47 m ° / ( ⁇ g / 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 ° / ( ⁇ g / ml) and the inaccuracy of the measurement to 1.93 ⁇ g / ml were determined.
  • 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.
  • 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.
  • the sensor response has good linearity within each cycle, even without chemical regeneration of the immunoaffinity layer.
  • 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.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
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Abstract

L'invention concerne un procédé de détermination de la concentration d'un analyte, en tant que ligand dans un échantillon, par étude de la liaison ligand-récepteur, procédé dans lequel un nombre prédéterminé de récepteurs ou de points de liaison, au niveau desquels l'analyte peut se lier spécifiquement en tant que ligand, sont immobilisés ou présents sur un substrat, l'essai étant mis en contact avec l'échantillon, et aussi bien un signal global qu'un signal individuel produit par contact avec l'échantillon sont mesurés, la concentration de l'analyte étant déterminée à partir du signal individuel, en tenant compte du signal global.
PCT/EP2004/004553 2003-04-30 2004-04-29 Procede de determination de la concentration d'un analyte WO2004097413A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE2003119572 DE10319572A1 (de) 2003-04-30 2003-04-30 Verfahren zur Bestimmung der Konzentration eines Analyten
DE10319572.6 2003-04-30

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4331444A (en) * 1979-03-01 1982-05-25 Fuji Photo Film Co., Ltd. Competitive immunoassay using silver halide fogging agent
EP0345732A2 (fr) * 1988-06-09 1989-12-13 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Méthode pour déterminer un titre d'anticorps
JPH1059068A (ja) * 1996-08-23 1998-03-03 Yoshihisa Furuta 車両の死角確認装置
WO2002031478A2 (fr) * 2000-10-06 2002-04-18 Quantech Ltd. Procedes et appareils relatifs a des epreuves biologiques utilisant des partenaires de fixation d'analysat

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SE8902043L (sv) * 1988-11-10 1990-05-11 Pharmacia Ab Foerfarande foer karakterisering av makromolekyler
SE462454B (sv) * 1988-11-10 1990-06-25 Pharmacia Ab Maetyta foer anvaendning i biosensorer
DE19736641A1 (de) * 1997-08-22 1999-03-11 Michael G Dr Weller Verfahren und Vorrichtung zur parallelen Messung von mehreren Analyten in komplexen Mischungen

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4331444A (en) * 1979-03-01 1982-05-25 Fuji Photo Film Co., Ltd. Competitive immunoassay using silver halide fogging agent
EP0345732A2 (fr) * 1988-06-09 1989-12-13 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Méthode pour déterminer un titre d'anticorps
JPH1059068A (ja) * 1996-08-23 1998-03-03 Yoshihisa Furuta 車両の死角確認装置
WO2002031478A2 (fr) * 2000-10-06 2002-04-18 Quantech Ltd. Procedes et appareils relatifs a des epreuves biologiques utilisant des partenaires de fixation d'analysat

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MOUVET C ET AL: "Determination of simazine in water samples by waveguide surface plasmon resonance", ANALYTICA CHIMICA ACTA, vol. 338, no. 1-2, 1997, pages 109 - 117, XP002297037, ISSN: 0003-2670 *
PATENT ABSTRACTS OF JAPAN vol. 0132, no. 63 (P - 886) 19 June 1989 (1989-06-19) *

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