EP1787123A2 - Homogeneous method for determining an analyte in the presence of an excess of cross-reacting substances - Google Patents
Homogeneous method for determining an analyte in the presence of an excess of cross-reacting substancesInfo
- Publication number
- EP1787123A2 EP1787123A2 EP05782650A EP05782650A EP1787123A2 EP 1787123 A2 EP1787123 A2 EP 1787123A2 EP 05782650 A EP05782650 A EP 05782650A EP 05782650 A EP05782650 A EP 05782650A EP 1787123 A2 EP1787123 A2 EP 1787123A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- analyte
- signal
- prothrombin
- binding partner
- sample
- Prior art date
- Legal status (The legal status 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 status listed.)
- Withdrawn
Links
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
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
-
- 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
- G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
-
- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- 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
- G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
- G01N2333/974—Thrombin
Definitions
- the invention relates to a homogeneous method for the determination of an analyte in a sample, which additionally contains an excess of a substance which cross-reacts with one of the analyte-specific binding partners.
- binding tests are available, which allow specific detection and exact quantification by the use of analyte-specific binding partner. Examples of binding tests are immunoassays or methods in which nucleic acids are hybridized.
- homogeneous processes being characterized in that no washing and separation steps are required. Accordingly, homogeneous methods are particularly preferred for use in automatic analyzers, since the sequence of a test is reduced to a minimum of method steps, which firstly reduces the number of possible error sources and secondly the test duration.
- a particular category of binding assays are the so-called sandwich assays, characterized in that two analyte-specific binding partners form a "binding partner / analyte / binding partner" complex with the analyte, at least one of the binding partners being associated with a component of a signal-forming system, Heterogeneous sandwich assays are designed in such a way that, prior to the induction or measurement of the signal, a Separation of the free, unbound binding partner or unbound analyte is necessary, such. In ELISA testing.
- Homogeneous sandwich assays allow the determination of the measurement signal generated by the "binding partner / analyte / binding partner" complex, even in the presence of unbound, free binding partner or analyte molecules Components and the sample containing is measured after or even during the binding reaction without further separation and / or washing step and determines the corresponding measurement signal
- Examples of homogeneous sandwich assays eg., Boguslaski & Li (1982) Applied Biochemistry and Biotechnology 7: 401-414] are many turbidimetric or nephelometric methods, wherein the specific for the detection of analyte used binding partners may be linked to latex particles, such as the EMIT ® - Test; CEDIA ® -Teste.
- a particular embodiment of a homogeneous sandwich assay is based on the use of a signal-generating system in which the analyte-mediated pairing allows interaction between the two signal-forming components which then causes a measurable signal.
- assays are fluorescence polarization immunoassays or Luminescent Oxygen Channeling Immunoassay, LOCI TM for short [EP-B1-0 515 194; Ullman et al. (1994) Proc. Natl. Acad. Be. 91: 5426-5430; Ullman et al. (1996) Clinical Chemistry 42: 1518-1526].
- numerous analytes see also EP-B1-0 515 194, pages 7-14
- analyte concentrations in a range from 10 "4 to 10 -16 M can be measured.
- a prerequisite required for using this sandwich method is the availability of two analyte-specific binding partners. In order for pairing and thus the signal-inducing interaction to take place, it must be ensured that both binding partners can bind together to an analyte molecule. In the case of LOCI TM immunoassays, for example, this is accomplished by the use of two analyte-specific antibodies that bind to two different epitopes of the analyte. While one of the antibodies is associated with a photosensitizer, the other antibody is with one ChemilumineszenzENS associated. Only by the spatial proximity of the two signaling components of the energy transfer from the photosensitizer on the chemiluminescent compound is made possible, so that the amount of luminescence caused by the pairing correlated with the amount of analyte.
- Monoreactive in the context of the present invention means that the binding partner has no or negligible affinity for other substances in the sample and binds to the analyte with high specificity.
- An analyte-specific, polyreactive binding partner binds the analyte but also to at least one further substance which is contained in the sample.
- Prothrombin is the proenzyme of thrombin, the central enzyme in the blood clotting cascade.
- the prothrombin protein is modular and consists of an N-terminal Fi +2 -AnteiI and a C-terminal thrombin portion.
- Prothrombin is cleaved by the proteolytic activity of factor Xa, so that a thrombin molecule (30 kD) is formed per prothrombin molecule (70 kD) with the release of a prothrombin fragment F 1 + 2 (35 kD).
- the autocatalytic cleavage of prothrombin by thrombin can further lead to the cleavage of the fragments F 1 and F 2 .
- thrombin does not occur in the blood in free form, but is bound to inhibitors and fibrin immediately after its formation, the extent of thrombin formation must be detected indirectly, e.g. B. by the determination of the activation marker F 1 + 2 and / or F 2 .
- One possibility for the detection or exclusion of increased thrombin formation in vivo is therefore the determination of the F 2 / F 1 + 2 concentration in the plasma.
- the prothrombin concentration in the plasma of healthy individuals varies between 1 and 2 ⁇ mol / l, while the F 1 + 2 concentration is usually between 60 and 200 pmol / l and values of up to 9.5 nmol in acute venous thromboembolism / l can reach. This means that, compared to the F 1 + 2 concentration, there is a 1,000 to 10,000 fold molar excess of prothrombin in plasma samples.
- the immunological detection of F 2 / F 1 + 2 in the presence of prothrombin is complicated by the fact that the released prothrombin fragments F 2 / F 1 + 2 to intact, uncleaved prothrombin only a single F 2 / F 1 + 2 -specific, antigenic Epitope and namely the resulting after the cleavage of thrombin carboxy terminus of the F 2 / F 1 + 2 peptide (neoantigen). Only antibodies directed against the carboxy-terminal region of the F 2 / F 1 + 2 peptide are able to specifically bind to the prothrombin fragments F 2 / F 1 + 2 without at the same time having a specificity for intact prothrombin.
- the F 2 / Fi +2 neoantigen-specific antibodies are coupled to a solid phase and incubated with the sample, so that the F 2 / Fi + 2 peptides can bind to the immobilized antibodies.
- unbound proteins in particular prothrombin
- prothrombin-binding antibody is applied by addition of an antibody solution.
- this second antibody is associated with a signal-forming component that allows quantification of the F 2 / Fi + 2 concentration.
- the present invention has for its object to provide a sensitive homogeneous method that allows the determination of analytes for which only one monoreactive binding partner is available and which are to be determined in a sample which additionally contains an excess of at least one substance with a second analyte-specific but polyreactive binding partner cross-reacts.
- the object is achieved in that a sample, which presumably contains the analyte, an analyte-specific, monoreactive binding partner and an analyte-specific, polyrezine binding partner is added, the polyreactive binding partner in terms of the sum of all substances that a reaction enter with him, in 1.5 to 5-fold molar excess, preferably in 2 to 3-fold molar excess, is used.
- the sum of all substances which undergoes a reaction with the analyte-specific, polyreactive binding partner is composed of the usually expected amount of analyte (mol / l) and the total amount of cross-reacting substances (mol / l), which are usually present in the respective sample material are included, together.
- the analyte specific binding partner associated with each a signal-forming component which are brought by the formation of a binding partner / analyte / binding partner complex in spatial proximity and consequently in a detectable, quantifiable interaction.
- binding partner a member of a binding pair, the members of a specific binding pair being two molecules each having at least one structure complementary to a structure of the other molecule, the two molecules being linked by a complementary binding
- molecule also includes molecular complexes such as enzymes consisting of apo- and coenzyme, proteins consisting of several subunits, lipoproteins consisting of protein and lipids, etc.
- Specific binding partners may be naturally occurring but also eg be produced by chemical synthesis, microbiological techniques and / or genetic engineering methods, such as antibodies, receptors, enzymes or polynucleotides.
- the reaction of a monoreactive or polyreactive binding partner with the analyte is a specific binding, e.g. B. mutual recognition of complementary surface structures or an interaction of polar or non-polar groups may be based.
- specific binding reactions are antibody / antigen, antibody / hapten, receptor / ligand, enzyme / substrate, DNA / DNA, DNA / RNA; Operator / repressor, nuclease / nucleotide, biotin / avidin, lectin / polysaccharide, hormone / hormone receptor and other interactions.
- the polyreactive binding partner in the sense of the present invention reacts with at least one further substance which is contained in the sample in addition to the analyte.
- This reaction may also be based on specific binding, e.g. B. if the substance has identical surface structures as the analyte or if the polyreactive binding partner recognizes and binds the analyte and the other cross-reacting substance due to a common feature or a common property.
- polyreactive binding partners having specificity for more than one analyte are e.g. B. antibodies the are directed against such epitopes of a proenzyme which are retained on the proteolytic spaite products of the precursor molecule.
- the polyreactive binding partner binds specifically to the analyte and enters into a nonspecific interaction with one or more further substances. Such nonspecific reactions are usually characterized in that they can occur independently of complementary surface structures.
- the analyte-specific binding partners are monoclonal or polyclonal antibodies.
- the term "antibody” means an immunoglobulin, for example an immunoglobulin of the class or subclass IgA, IgD, IgE, IgGi, IgG 2a , IgG 2 b, IgG 3 , IgG 4 , IgM
- the method according to the invention for the determination of an analyte is further based on the use of analyte-specific binding partners, which are each associated with one component of a signal-forming system, wherein at least one component is a detectable label.
- a label is any molecule that can itself produce a signal or induce the production of a signal such.
- a fluorescent substance a radioactive substance, an enzyme or a chemiluminescent substance.
- the signal may be detected or measured by enzyme activity, luminescence, light absorption, light scattering, radiated electromagnetic or radioactive radiation, or a chemical reaction.
- a “signal-generating system” comprises components which, when in close proximity to one another, can undergo a detectable interaction with one another, for example in the form of energy donors and energy receivers such as, for example, photosensitizer and chemiluminescent substances (EP-A2-0 515 194), photosensitizer and fluorophores (WO 95/06877), radioactive (or 125 and fluorophores [Udenfriend et al., (1985) Proc. Natl. Acad., 82: 8672-8676] , Fluorophores and Fluorophores [Mathis (1993) Clin. Chem. 39: 1953-1959] or fluorophores and fluorescence quenchers (US 3,996,345).
- photosensitizer and chemiluminescent substances EP-A2-0 515 194
- photosensitizer and fluorophores WO 95/06877
- radioactive or 125 and fluorophores [Udenfriend et al., (1985) Proc. Nat
- the direct transfer of energy between the components e.g. As by light or electron radiation and short-lived reactive chemical molecules, mitein reminder.
- processes in which the activity of one component is inhibited or enhanced by one or more others for example, the inhibition or enhancement of enzyme activity, or the inhibition, enhancement, or alteration (eg, wavelength shift, polarization) of the affected one Component emitted electromagnetic radiation.
- the interaction between the components also includes enzyme cascades.
- the components are enzymes, at least one of which provides the substrate for another such that a maximum or minimum reaction rate of the coupled substrate reaction results.
- An effective interaction between the components usually takes place when they are spatially adjacent, so z. B. within a distance range of a few microns, in particular within a distance range of less than 600 nm, preferably below 400 nm, most preferably of less than 200 nm.
- the monoreactive binding partner is associated with a chemiluminescent compound and the polyreactive binding partner is associated with a photosensitizer or vice versa.
- chemiluminescent compounds and of substances which are suitable as photosensitizers are described in EP-B1-0 515 194 (chemiluminescent compounds see page 22, line 42 to page 34, line 15, photosensitizers see page 17, lines 26-50) ,
- the term "associated” is to be understood broadly and includes, for example, a covalent and a non-covalent bond, a direct and an indirect bond, the adsorption to a surface and the inclusion in a depression or a cavity etc.
- Antibodies or binding partners can be bound to the solid phase or bound to the label via a chemical bond
- noncovalent binding include surface adsorption, inclusion in cavities or the binding of two specific binding partners, in addition to direct binding to the solid phase or label
- the antibodies or binding partners are also bound to the solid phase or the label indirectly via specific interaction with other specific binding partners (see also EP-A2-0 411 945) .
- a biotinylated antibody can be bound via label-bound avidin to the Be bound label or it ka a fluorescein antibody conjugate is bound to the solid phase via solid phase-bound anti-fluorescein antibodies; or the antibody can be bound to the solid phase or label via immunoglobulin-binding proteins.
- the analyte-specific binding partners are associated with suspendible particles coupled to a component of a signal-forming system, preferably a chemiluminescent compound and a photosensitizer.
- suspendible particles comprise microparticles, preferably latex particles, which themselves may be associated with, for example, dyes, sensitizers, fluorescers, chemiluminescers, isotopes or other detectable labein.
- suspendable particles are particles which have an approximate diameter of at least 20 nm and not more than 20 ⁇ m, usually between 40 nm and 10 ⁇ m, preferably between 0.1 and 10 ⁇ m, particularly preferably between 0, 1 and 5 microns, most preferably between 0.15 and 2 microns.
- the microparticles may be regular or irregular in shape. They can represent spheres, spheroids, spheres with more or less large cavities or pores.
- the microparticles can be made of organic, of inorganic material or of a mixture or Combination of both exist. They may consist of a porous or non-porous, a swellable or non-swellable material. In principle, the microparticles may have any density, but preferred are particles having a density close to the density of the water, such as from about 0.7 to about 1.5 g / ml.
- the preferred microparticles are suspendible in aqueous solutions and as long as possible suspension stable. They may be transparent, partially transparent or opaque.
- microparticles may consist of several layers, for example the so-called "core-and-shell" particles having a core and one or more enveloping layers
- microparticles includes, for example, dye crystals, metal sols, silica particles, glass particles, magnetic particles, polymer particles, oil drops, Lipid particles, dextran, and protein aggregates
- Preferred microparticles are particles which can be suspended in aqueous solutions and consist of water-insoluble polymer material, in particular substituted polyethylenes.
- latex particles for example, of polystyrene, acrylic acid polymers, methacrylic acid polymers, acrylonitrile polymers, acrylonitrile-butadiene-styrene, polyvinyl acetate acrylate,
- Latex particles having reactive groups on their surface such as carboxyl, amino or aldehyde groups, which have a covalent bond, e.g. allow specific binding partners to the latex particles.
- the preparation of latex particles is described for example in EP-B1-0 080 614, EP-B1-0 227 054 and EP-B1-0 246 446.
- the present inventive method is further characterized in that the sample is diluted in a volume ratio of 1: 1 to 1: 250 and the analyte-specific, polyreactive binding partner in relation to the sum of all substances to which it can bind, in 1, 5 up to 5-fold molar excess is used.
- the samples such. As blood or plasma samples, in a volume ratio of 1: 2 to 1: 100, more preferably diluted in a ratio of 1: 4 to 1: 32.
- sample is understood as meaning the material which presumably contains the analyte to be detected.
- sample includes, for example, biological fluids or tissues, in particular of humans and animals, such as blood, plasma, serum, sputum, exudate, bronchoalveolar lavage. Lymphatic fluid, synovial fluid, seminal fluid, vaginal mucus, feces, urine, cerebrospinal fluid, hair, skin, tissue samples or incisions, cell culture samples, plant fluids or tissues, forensic samples, water and wastewater samples, food, pharmaceuticals, etc.
- samples must be pretreated Such pretreatment of samples may involve separation and / or lysis of cells, precipitation, hydrolysis or denaturation of sample components such as proteins, for example, to provide the analyte for the detection procedure or to remove interfering sample components.
- the centrifugation of sample n treating the sample with organic solvents such. As alcohols, especially methanol, treating the sample with detergents.
- the method according to the invention is characterized in that the signal is measured after a period of joint incubation of both components of the signal-forming system in the reaction mixture of less than 300 seconds, preferably less than 200 seconds and more preferably less than 160 seconds.
- the limitation of the incubation time to less than 300 seconds allows the measurement of a signal of high specificity and sufficient strength, which allows a precise quantification of the analyte.
- This measure solves the dilemma of two opposing effects.
- an increase in the incubation time leads to an increase in the specific signal accompanied, whereby a reliable measurement of the signal is made possible.
- a decrease in the signal strength also occurs, so that the sum of the measured signal no longer correlates with the amount of analyte.
- the signal-degrading effect may be due to the excess substance blocking the component of the signal-forming system associated with the polyreactive binding partner through excessive binding to the polyreactive binding partner, thus requiring the interaction with the second component of the signal Systems hindered.
- analytes can be determined, such.
- B. proteolytic cleavage products of coagulation factors Factor IX, Factor X and Factor Xl, preferably the cleavage products of factor II ( prothrombin) F 2 and Fi +2 , proteolytic cleavage products of fibrinogen such.
- fibrinopeptide A or B, or Fibrinogendegradations occur, such as.
- the sensitivity of the method according to the invention allows the determination of analyte concentration up to about 10 "12 mol / l in the presence of, for example, a 10,000 times molar excess of a cross-reactive with the polyreactive binding partner substance.
- the following examples relate to the determination according to the invention of the prothrombin fragment Fi +2 in human plasma samples, wherein the F 1 + 2 -specific binding partners are associated with suspendable polystyrene particles (beads), to which in turn a stimulable photosensitizer or a chemiluminescent compound is coupled.
- the photosensitizer-coupled polystyrene particles are also referred to as sensibeads
- the chemiluminescent compound-coupled polystyrene particles are referred to as chemibeads.
- the beads are dextran-coated polystyrene particles, with the sensitibeads labeled with phthalocyanine and the chemibeads with europium chelate.
- the preparation of the beads was carried out according to Ullman et al. (1996) Clin. Chem. 42 (9): 1518-1526 and the references cited therein or according to WO 01/67105.
- streptavidin-coated Sensibeads were used which allow via a streptavidin / biotin bond the association of the biotinylated binding partner to the Sensibeads.
- the photosensitizer is excitable by light of a wavelength of 680 nm and then produces singlet oxygen, which in turn is capable of exciting the chemiluminescent compound, producing a chemiluminescent signal which can be measured and correlated with the amount of Fi + 2 in the sample.
- a monoclonal antibody As a polyreactive binding partner that binds both Fi +2 and intact prothrombin, a monoclonal antibody (anti-prothrombin MAb) produced by the hybridoma cell line 92-195 / 097 used in the DSMZ - Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Mascheroder Weg 1 b, 38124 Braunschweig, Germany, under the accession number DSM ACC2607.
- a monoreactive binding partner with specificity for the carboxy-terminal neoantigen of F 1 + 2 a monoclonal antibody was also used (anti-Fi + 2 -MAK). The preparation of such an antibody is known from the prior art.
- Example 1 Preparation of reagents necessary for the homogeneous assay
- the anti-prothrombin antibody was first buffered in a 10 mM NaHCO 3 solution for the biotinylation and the IgG content of the solution was determined photometrically.
- Biotin-PEO 4 -NHS EZ-Link TM NHS-PEO 4 - Biotin, Pierce, Ill., USA
- DMSO DMSO DMSO
- the biotinylated anti-prothrombin antibody was purified on Sephadex® G25 (Amersham Biosciences Europe GmbH, Germany) and adjusted in a suitable buffer to a concentration of 2 mg / ml.
- An anti-F ⁇ + 2 antibody solution was purified on Sephadex® G25, taken up in 50 mM MES buffer (pH 6.0), quantified photometrically and adjusted to an IgG concentration of 20 mg / ml. 50 mg of the europium chelate-coupled chemibeads were washed by multiple sedimentation and resuspension in MES buffer (pH 6.0) and finally adjusted to a Chemibead concentration of 100 mg / ml.
- 500 ⁇ l of the anti-F 1 + 2 antibody solution were mixed with 500 ⁇ l Chemibead solution, 2 ⁇ l 4% Tween® 20 solution and 24 ⁇ l NaBH 3 CN solution (25 mg NaBH 3 CN / ml) and Incubated at 37 0 C for 60 hours. Subsequently, 50 .mu.l 0.5 M CMO solution was added and incubated for a further 2 hours at 37 ° C. Finally, the reaction mixture for the separation of unbound reactants and for the homogenization of the particles was treated with ultrasound.
- the Chemibeads were sedimented by centrifugation, resuspended in MES buffer (pH 6.0) and sonicated. This washing step and subsequent sonication were performed with MES buffer and twice with CB buffer (50 mM HEPES, 0.3 M NaCl, 1 mM EDTA, 1 mg / ml Dextran T-500, 12.5 ⁇ g / ml dextran Sulfate, 16 mg / ml BSA, 2 mg / ml bovine ⁇ -globulin, 0.1% [w / v] Triton X 405, 0.15% [v / v] Proclin 300, 0.1 mg / ml neomycin sulfate , pH 7.2). After the last wash, the anti-Fi + 2 antibody-coated Chemibeads were adjusted to a concentration of 50 mg / ml with CB buffer and subjected to a final sonication.
- Example 2 LOCI TM to quantify the Fi + 2 content of human plasma samples
- the reagents were used in the following concentrations: 75 ⁇ g / ml anti-F 1 + 2 chemibeads in CB buffer (pH 7.2), according to Example 1a) prepared biotinylated anti-prothrombin antibody in BA buffer (50mM HEPES, 0.3M NaCl, 1mM EDTA, 1mg / ml Dextran T-500, 0.1% [w / v] Triton X 405, 0.15% [v / v] Proclin 300, 0 , 1 mg / ml streptavidin-coated Sensibeads in SB buffer (50 mM HEPES, 0.3 M NaCl, 1 mM EDTA, 1 mg / ml dextran, 1 mg / ml neomycin sulfate, pH 7.2) diluted 1: 66 T-500, 16 mg / ml BSA, 2 mg / ml bovine saccharide
- a standard curve was prepared by adjusting purified F- ⁇ +2 -antigen with Ag buffer to a concentration of 1000 pmol / l. From this stock solution, a geometric dilution series (factor 2) was prepared. The standard samples were tested as described in Example 2, ie they were prediluted as well as the plasma samples before the test in a ratio of 1:20. The plotting of the logarithm of the signal level achieved for the individual standards against the logarithm of the F- I + 2 concentrations contained in the standards gives the calibration curve shown in FIG.
- an optimum can be selected which is less than 300 seconds, which ensures, on the one hand, a quantifiable signal level in the range of high analyte concentrations and, on the other hand, high sensitivity in the range of low analyte concentrations.
- Example 5 Increase of the assay sensitivity by increasing the amount of biotinylated anti-prothrombin antibody in the reaction mixture
- Standard human plasma was adjusted to defined concentrations by addition of purified F 1 + 2 antigen and tested with three different dilution steps of the biotinylated anti-prothrombin antibody prepared according to Example 1 (see Example 2).
- the test series with the highest anti-prothrombin antibody concentration (MAK 1:10) shows lower signals in the range of high analyte concentrations compared to the mean MAK concentration (MAK 1: 100). This effect can be explained by the blockage of sensibeads by the biotinylated anti-prothrombin MAb. At the lowest MAK concentration (MAK 1: 1000), the lowest signal levels were achieved.
- the determined data are summarized in FIG.
- the dilution of the plasma samples reduces the influence of prothrombin on the assay.
- An approximately 10,000-fold molar excess of prothrombin over F 1 + 2 blocks most of the biotinylated anti-prothrombin antibody by prothrombin molecules.
- the prothrombin is present at a normal concentration of 100 mg / ml in an approximately ⁇ fachmal excess to the antibody. Dilution of the plasma samples shifts the molar ratio of biotinylated anti-prothrombin antibody to prothrombin in favor of the antibody.
- the ratio of prothrombin to antibody in the reaction mixture is 2: 3.
- the antibody is thus present in excess of the prothrombin.
- the determined curves reflect the above-described influence of the prothrombin on the assay.
- the signals rise first, since the anti-prothrombin MAK is no longer blocked to the same extent as in undiluted samples of prothrombin molecules. Only at higher dilutions do the signal levels begin to decrease due to the decreasing Fi + 2 concentrations. In the range of low dilutions, the assay is therefore dominated by the excess prothrombin. With increasing dilution one arrives in a range of the dilution fastness, in which the signal strength correlates with the F 1 + 2 -concentration.
- FIG. 1 A first figure.
- Plasma samples used in the assay were also performed on the standard Fi + 2 samples.
- the concentrations given here thus do not correspond to the actual F 1 + 2 content, but are additionally due to the
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Application Number | Priority Date | Filing Date | Title |
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DE200410042124 DE102004042124B4 (en) | 2004-08-30 | 2004-08-30 | Homogeneous method for the determination of an analyte in the presence of an excess of cross-reacting substances |
PCT/EP2005/009284 WO2006024476A2 (en) | 2004-08-30 | 2005-08-29 | Homogeneous method for determining an analyte in the presence of an excess of cross-reacting substances |
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EP2177624A1 (en) * | 2008-10-02 | 2010-04-21 | Siemens Healthcare Diagnostics Products GmbH | Blood coagulation assays |
CN111781345A (en) * | 2020-06-30 | 2020-10-16 | 上海透景生命科技股份有限公司 | Chemiluminescent marker labeled antigen stabilizer and application thereof |
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DE3633497A1 (en) * | 1986-10-02 | 1988-04-14 | Hoechst Ag | IMMUNOMETRIC DETERMINATION PROCEDURE |
US6541275B1 (en) * | 1988-02-03 | 2003-04-01 | Dade Behring Inc. | Immunoassay for F1.2 prothrombin fragment |
US5512659A (en) * | 1989-08-04 | 1996-04-30 | Syntex (U.S.A.) Inc. | Compositions useful in heterogeneous immunoassays |
IE903930A1 (en) * | 1989-11-06 | 1991-05-08 | Akzo Nv | Immunoassays for and monoclonal antibodies to prothrombin¹activation peptides and their degradation products |
DE69233674T2 (en) * | 1991-05-22 | 2007-10-18 | Dade Behring Marburg Gmbh | Homogeneous test system for the determination of an analyte |
WO1995006877A1 (en) * | 1993-09-03 | 1995-03-09 | Behringwerke Ag | Fluorescent oxygen channeling immunoassays |
DE19521388C2 (en) * | 1995-06-13 | 1997-05-22 | Immuno Gmbh | Technique for the determination of a specifically bindable substance |
US7179660B1 (en) * | 2000-03-06 | 2007-02-20 | Dade Behring Marburg Gmbh | Carriers coated with polysaccharides, their preparation and use |
ES2370232T3 (en) * | 2000-05-04 | 2011-12-13 | Siemens Healthcare Diagnostics Products Gmbh | PROCEDURE FOR THE DETECTION OF MULTIPLE ANALYTICS. |
WO2005062057A1 (en) * | 2003-12-01 | 2005-07-07 | Dade Behring Marburg Gmbh | Conjugates, and use thereof in detection methods |
-
2004
- 2004-08-30 DE DE200410042124 patent/DE102004042124B4/en not_active Expired - Fee Related
-
2005
- 2005-08-29 EP EP05782650A patent/EP1787123A2/en not_active Withdrawn
- 2005-08-29 WO PCT/EP2005/009284 patent/WO2006024476A2/en active Application Filing
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Also Published As
Publication number | Publication date |
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WO2006024476A3 (en) | 2006-10-26 |
DE102004042124A1 (en) | 2006-03-02 |
DE102004042124B4 (en) | 2006-07-06 |
WO2006024476A2 (en) | 2006-03-09 |
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