CA2489021A1 - Determining the interaction between a c-reactive protein and constituents that bind to a c-reactive protein - Google Patents
Determining the interaction between a c-reactive protein and constituents that bind to a c-reactive protein Download PDFInfo
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Abstract
The invention relates to an HTS-capable method and testing system for determining the interaction between a C-reactive protein (CRP) or C1q and constituents that bind to a CRP or C1q in order to determine the concentrati on of a solution containing CRP or C1q and to identify substances that influenc e the interaction between CRP or C1q and constituents that bind thereto, particularly the interaction between CRP and C1q.
Description
Description HTS-capable method and testing system for determining the interaction between a C-reactive protein and constituents that bind to a C-reactive protein The present invention relates to an HTS-capable method and assay system for determining the interaction between C-reactive protein (CRP) and C1 q and components binding to CRP and C1q, respectively, to determining the concentration of a solution containing CRP and C1q, respectively, and to determining substances which influence the interaction of CRP and C1q, respectively, and components binding thereto.
The C-reactive protein (CRP) is an acute-phase plasma protein whose serum concentration increases rapidly and greatly after an infection or tissue injury (Volanakis (2001 ), Molecular Immunology 38, 189-197). CRP binds to phosphocholine (PCh) in the presence of Ca2+ ions. The latter is very common in polysaccharides of pathogenic organisms and in cell membranes of damaged and necrotic cells. CRP bound to PCh can activate the classical complement cascade by binding to the protein C1q.
The complement is part of the immune system and primarily involved in the antibody-mediated immune defenses. The three physiological functions of the complement are the defense against bacterial infections, the connection of congenital and acquired immunity and the removal of immunocomplexes and apoptotic cells. A distinction is made between the classical, the alternative and the mannose-lectin complement cascades (Walport (2001 ), N Engl J Med 344, 1058-1066). The classical complement cascade leads to the lysis of bacterial cells and starts with C1q associating with an antibody binding to the cell surface or with PCh-bound CRP.
The C-reactive protein (CRP) is an acute-phase plasma protein whose serum concentration increases rapidly and greatly after an infection or tissue injury (Volanakis (2001 ), Molecular Immunology 38, 189-197). CRP binds to phosphocholine (PCh) in the presence of Ca2+ ions. The latter is very common in polysaccharides of pathogenic organisms and in cell membranes of damaged and necrotic cells. CRP bound to PCh can activate the classical complement cascade by binding to the protein C1q.
The complement is part of the immune system and primarily involved in the antibody-mediated immune defenses. The three physiological functions of the complement are the defense against bacterial infections, the connection of congenital and acquired immunity and the removal of immunocomplexes and apoptotic cells. A distinction is made between the classical, the alternative and the mannose-lectin complement cascades (Walport (2001 ), N Engl J Med 344, 1058-1066). The classical complement cascade leads to the lysis of bacterial cells and starts with C1q associating with an antibody binding to the cell surface or with PCh-bound CRP.
C-reactive protein is used as a marker and, in addition, as a predictor for coronary heart disease, the most common cause of mortality in industrialized countries (Rifai and Ridker (2001 ), Clinical Chemistry 47, 403-411 ).
Owing to new studies (Jialal et al (2001 ), Circulation 103, 1933-1935), it is assumed that lowering the CRP level or blocking the CRP-mediated effector functions, for example complement activation due to binding to C1 q, may be useful for the prevention and treatment of coronary heart disease.
It is therefore desirable firstly to provide a method which allows determining the concentration of CRP and/or C1q in blood plasma in a simple manner and, secondly, allows identification of substances which act on the interaction of CRP andlor C1q with other components, in particular on the interaction between CRP and C1 q, in a modulating, i.e. activating or inhibiting, way.
Methods for determining the binding of CRP to C1q have been described, for example, by Jiang et al. (1991 ), J. Immunol. 146, 2324-2330 and Agrawal et al.
(2001 ), J. Immunol. 166, 3998-4004. Both publications make use of an ELISA
method. Both are very time- and material-consuming, multistage inhomogeneous methods. Thus, in order to carry out the method, one of the reactants always needs initially to be immobilized to a solid phase. Moreover, numerous washing steps are required.
Lebdue et al. (1998), Ann Clin Biochem 35, 745-753 describe a method for immunonephelometric detection of CRP. To this end, the sample is incubated with anti-CRP antibodies loaded with polystyrene particles. Said method has a crucial disadvantage in that a nephelometer is required and a relatively large quantity of sample must be used in order to carry it out.
It was therefore the object of the present invention to provide a method which allows the concentration of CRP or C1q in blood plasma to be determined in a simple manner and which furthermore allows the identification of substances which act on the interaction of CRP or C1 q with other components, in particular on the interaction between C1q and CRP, in a modulating, activating or inhibiting manner, and said method is distinguished advantageously compared to the prior art by being more sensitive, cost-effective, material-saving, simpler and/or more rapidly carried out than the previously described methods.
The object is achieved by a method for determining a binding event of two components binding directly or via one or more components to CRP or C1 q, which comprises the following steps:
(a) initially introducing a CRP- or C1q-containing solution, (b) adding at least one donor component or a group of components containing at least one donor component to said CRP- or C1 q-containing solution, said donor component being a component which comprises at least one compound or group of compounds capable of emitting a signal after excitation by a light source (donor group) and said donor component being capable of binding directly or via one or more components from said group of components to CRP or C1q, (c) adding at least one acceptor component or a group of components containing at least one acceptor component to said CRP- or C1 q-containing solution, said acceptor component being a component which comprises at least one compound or group of compounds capable of receiving and emitting in the form of electromagnetic radiation the signal emitted by the compound or group of compounds according to (b) (acceptor group) and said acceptor component being capable of binding directly or via one or more components of said group of components to CRP or C1 q, (d) exciting the at least one compound or group of compounds according to (b) (donor group) by means of a light source, (e) detecting the electromagnetic radiation emitted by the at least one compound or group of compounds according to (c) (acceptor group) in order to detect said binding event.
The emitted electromagnetic radiation according to (c) is here preferably fluorescence radiation. The light source according to (d) may be, for example, a laser or a lamp, for example a helium or halogen lamp. The electromagnetic radiation according to (e) may be detected, for example, with the aid of a photomuitiplier.
The components to be used according to the invention preferably are or comprise polypeptides. In a particularly preferred embodiment of the invention, at least one of the components is an antibody which may be a monoclonal or polyclonal antibody.
If a group of components is used, said components are preferably capable of binding to CRP or C1q or to one another in spatial succession. Said binding takes place in the form of a binding cascade, an example of which is the successive binding of antibodies (primary antibody, secondary antibody, etc.). The components binding directly to CRP or C1q according to (b) and (c) are here preferably components which bind in each case to a particular binding region or to a particular epitope of the CRP or C1q, said CRP- or C1q-binding component according to (b) binding to a different binding site than the CRP- or C1q-binding component according to (c).
In another preferred embodiment of the method of the invention, one of the components binding directly to CRP or C1 q is a naturally occurring binding partner of the CRP or C1q, which, in case of the CRP is particularly preferably C1q and vice versa. If the donor or acceptor component is bound directly to CRP or C1q, then, in this case, either the donor or the acceptor component is accordingly either C1 q or CRP comprising a donor or acceptor group. When using a group of components capable of forming a binding cascade, C1 q or CRP is accordingly the first component binding to CRP or C1 q via which it is possible to bind either the donor or the acceptor component to CRP or C1q. The remaining components of the group of components are particularly preferably antibody molecules.
Thus, for example, when initially introducing a CRP-containing solution, then, in this embodiment, preferably either the donor or the acceptor component itself comprises C1 q or the donor or acceptor component is bound via C1 q to CRP. In this case, the donor or acceptor component may comprise an anti-C1 q antibody or a secondary antibody capable of binding to said anti-C1 q antibody. Accordingly, the in each case other component (acceptor or donor component) then comprises an anti-CRP
antibody or an antibody capable of binding to the anti-CRP antibody or a tertiary antibody directed against the latter.
Owing to new studies (Jialal et al (2001 ), Circulation 103, 1933-1935), it is assumed that lowering the CRP level or blocking the CRP-mediated effector functions, for example complement activation due to binding to C1 q, may be useful for the prevention and treatment of coronary heart disease.
It is therefore desirable firstly to provide a method which allows determining the concentration of CRP and/or C1q in blood plasma in a simple manner and, secondly, allows identification of substances which act on the interaction of CRP andlor C1q with other components, in particular on the interaction between CRP and C1 q, in a modulating, i.e. activating or inhibiting, way.
Methods for determining the binding of CRP to C1q have been described, for example, by Jiang et al. (1991 ), J. Immunol. 146, 2324-2330 and Agrawal et al.
(2001 ), J. Immunol. 166, 3998-4004. Both publications make use of an ELISA
method. Both are very time- and material-consuming, multistage inhomogeneous methods. Thus, in order to carry out the method, one of the reactants always needs initially to be immobilized to a solid phase. Moreover, numerous washing steps are required.
Lebdue et al. (1998), Ann Clin Biochem 35, 745-753 describe a method for immunonephelometric detection of CRP. To this end, the sample is incubated with anti-CRP antibodies loaded with polystyrene particles. Said method has a crucial disadvantage in that a nephelometer is required and a relatively large quantity of sample must be used in order to carry it out.
It was therefore the object of the present invention to provide a method which allows the concentration of CRP or C1q in blood plasma to be determined in a simple manner and which furthermore allows the identification of substances which act on the interaction of CRP or C1 q with other components, in particular on the interaction between C1q and CRP, in a modulating, activating or inhibiting manner, and said method is distinguished advantageously compared to the prior art by being more sensitive, cost-effective, material-saving, simpler and/or more rapidly carried out than the previously described methods.
The object is achieved by a method for determining a binding event of two components binding directly or via one or more components to CRP or C1 q, which comprises the following steps:
(a) initially introducing a CRP- or C1q-containing solution, (b) adding at least one donor component or a group of components containing at least one donor component to said CRP- or C1 q-containing solution, said donor component being a component which comprises at least one compound or group of compounds capable of emitting a signal after excitation by a light source (donor group) and said donor component being capable of binding directly or via one or more components from said group of components to CRP or C1q, (c) adding at least one acceptor component or a group of components containing at least one acceptor component to said CRP- or C1 q-containing solution, said acceptor component being a component which comprises at least one compound or group of compounds capable of receiving and emitting in the form of electromagnetic radiation the signal emitted by the compound or group of compounds according to (b) (acceptor group) and said acceptor component being capable of binding directly or via one or more components of said group of components to CRP or C1 q, (d) exciting the at least one compound or group of compounds according to (b) (donor group) by means of a light source, (e) detecting the electromagnetic radiation emitted by the at least one compound or group of compounds according to (c) (acceptor group) in order to detect said binding event.
The emitted electromagnetic radiation according to (c) is here preferably fluorescence radiation. The light source according to (d) may be, for example, a laser or a lamp, for example a helium or halogen lamp. The electromagnetic radiation according to (e) may be detected, for example, with the aid of a photomuitiplier.
The components to be used according to the invention preferably are or comprise polypeptides. In a particularly preferred embodiment of the invention, at least one of the components is an antibody which may be a monoclonal or polyclonal antibody.
If a group of components is used, said components are preferably capable of binding to CRP or C1q or to one another in spatial succession. Said binding takes place in the form of a binding cascade, an example of which is the successive binding of antibodies (primary antibody, secondary antibody, etc.). The components binding directly to CRP or C1q according to (b) and (c) are here preferably components which bind in each case to a particular binding region or to a particular epitope of the CRP or C1q, said CRP- or C1q-binding component according to (b) binding to a different binding site than the CRP- or C1q-binding component according to (c).
In another preferred embodiment of the method of the invention, one of the components binding directly to CRP or C1 q is a naturally occurring binding partner of the CRP or C1q, which, in case of the CRP is particularly preferably C1q and vice versa. If the donor or acceptor component is bound directly to CRP or C1q, then, in this case, either the donor or the acceptor component is accordingly either C1 q or CRP comprising a donor or acceptor group. When using a group of components capable of forming a binding cascade, C1 q or CRP is accordingly the first component binding to CRP or C1 q via which it is possible to bind either the donor or the acceptor component to CRP or C1q. The remaining components of the group of components are particularly preferably antibody molecules.
Thus, for example, when initially introducing a CRP-containing solution, then, in this embodiment, preferably either the donor or the acceptor component itself comprises C1 q or the donor or acceptor component is bound via C1 q to CRP. In this case, the donor or acceptor component may comprise an anti-C1 q antibody or a secondary antibody capable of binding to said anti-C1 q antibody. Accordingly, the in each case other component (acceptor or donor component) then comprises an anti-CRP
antibody or an antibody capable of binding to the anti-CRP antibody or a tertiary antibody directed against the latter.
5 In a preferred embodiment, the at least one compound or group of compounds according to (b) (donor group) and/or according to (c) (acceptor group) are localized on particles, with the average diameter of said particles preferably being between 150 and 250 nm, particularly preferably at approximately 200 nm. Accordingly, the donor or acceptor components then comprise said particles which, in this case, may be made of a polymeric material.
The method of the invention is an HTS (high throughput screening)-capable method which may be carried out homogeneously in the form of a one-step method.
In a preferred embodiment, the method of the invention is carried out according to the "mix and measure" principle, resulting in considerable time-savings and higher accuracy of the measured data.
The method of the invention allows the binding of CRP to C1q to be measured, and thus the identification of substances influencing said binding, and the concentration of CRP or C1q in complex media to be measured in less than 10 percent of the time which was required when using previously described inhomogeneous methods.
A particular characteristic feature of the novel method is the possibility of monitoring binding between the two proteins in solution, since none of the reactants need be immobilized on a solid phase. Moreover, it is possible to measure the proteins obtained from biological sources directly, without the need for modifying CRP
or C1q, for example by binding of a fluorophor. The method therefore has the particular advantage that the protein can be subjected in its native state to the assay, thus removing the risk of denaturation which would be present in the case of immobilizing and/or modifying said protein.
The method of the invention is an HTS (high throughput screening)-capable method which may be carried out homogeneously in the form of a one-step method.
In a preferred embodiment, the method of the invention is carried out according to the "mix and measure" principle, resulting in considerable time-savings and higher accuracy of the measured data.
The method of the invention allows the binding of CRP to C1q to be measured, and thus the identification of substances influencing said binding, and the concentration of CRP or C1q in complex media to be measured in less than 10 percent of the time which was required when using previously described inhomogeneous methods.
A particular characteristic feature of the novel method is the possibility of monitoring binding between the two proteins in solution, since none of the reactants need be immobilized on a solid phase. Moreover, it is possible to measure the proteins obtained from biological sources directly, without the need for modifying CRP
or C1q, for example by binding of a fluorophor. The method therefore has the particular advantage that the protein can be subjected in its native state to the assay, thus removing the risk of denaturation which would be present in the case of immobilizing and/or modifying said protein.
Another advantage of the method of the invention is the considerably reduced sample volume of the method of the invention compared to conventional methods.
Thus it is possible to use volumes of less than 10 NI, thus minimizing material costs.
Moreover, it is possible to use highly diluted solutions with CRP or C1q concentrations of less than 1 nM, even less than 100 pM. It is, in addition, a very sensitive method. Furthermore, owing to the comments above, this is the first method which makes possible a high throughput screening for substances which influence complement activation due to binding of CRP to C1q.
In a preferred embodiment of the method of the invention, the signal is transferred from the at least one compound or group of compounds according to (a) (donor group) to the at least one compound or group of compounds according to (b) (acceptor group) via singlet oxygen.
In this case, the donor group preferably comprises a compound which can convert triplet oxygen to singlet oxygen after excitation by a laser, and the acceptor group comprises at least one first compound excitable by singlet oxygen and a second compound capable of absorbing in an emissionless manner and emitting in the form of fluorescence radiation the energy absorbed by the group excitable by singlet oxygen.
The singlet oxygen formed may diffuse from the donor group to the acceptor group and react with the chemiluminescent substances present there. The energy released in the process is transferred to the fluorophores which finally emit said energy in the form of fluorescence radiation which can be detected using a photornultiplier.
A
precondition for a detectable signal is the spatial proximity of donor and acceptor beads, since singlet oxygen is unstable and decays in aqueous solution.
Therefore, the binding components to be used in this method are chosen in such a way that, when the binding event occurs, the distance between the donor and acceptor groups is preferably less than 200 nm, since at a larger distance an effective energy transfer by means of singlet oxygen is not possible, due to the decay time of singlet oxygen.
Thus it is possible to use volumes of less than 10 NI, thus minimizing material costs.
Moreover, it is possible to use highly diluted solutions with CRP or C1q concentrations of less than 1 nM, even less than 100 pM. It is, in addition, a very sensitive method. Furthermore, owing to the comments above, this is the first method which makes possible a high throughput screening for substances which influence complement activation due to binding of CRP to C1q.
In a preferred embodiment of the method of the invention, the signal is transferred from the at least one compound or group of compounds according to (a) (donor group) to the at least one compound or group of compounds according to (b) (acceptor group) via singlet oxygen.
In this case, the donor group preferably comprises a compound which can convert triplet oxygen to singlet oxygen after excitation by a laser, and the acceptor group comprises at least one first compound excitable by singlet oxygen and a second compound capable of absorbing in an emissionless manner and emitting in the form of fluorescence radiation the energy absorbed by the group excitable by singlet oxygen.
The singlet oxygen formed may diffuse from the donor group to the acceptor group and react with the chemiluminescent substances present there. The energy released in the process is transferred to the fluorophores which finally emit said energy in the form of fluorescence radiation which can be detected using a photornultiplier.
A
precondition for a detectable signal is the spatial proximity of donor and acceptor beads, since singlet oxygen is unstable and decays in aqueous solution.
Therefore, the binding components to be used in this method are chosen in such a way that, when the binding event occurs, the distance between the donor and acceptor groups is preferably less than 200 nm, since at a larger distance an effective energy transfer by means of singlet oxygen is not possible, due to the decay time of singlet oxygen.
Particular preference is given in this embodiment to donor group and acceptor group localized on particles, said particles preferably having an average diameter of between 150 and 250 nm, particularly preferably of approximately 200 nm. The preferred use of said particles here is such that the final concentration of donor group-carrying particles is 1-40 Ng/ml and the final concentration of acceptor group-carrying particles is 1-80 Nglml. The binding capacity of said particles may be, for example, from approximately 0.1 nM to 1 nM per pg/ml particles.
Particular preference is given to using Alpha-Screen-Beads from Perkin-Elmer Life Sciences as particles in the method. In this embodiment, the donor group is excited by irradiating at a wavelength of 680 nm, using a laser, and the radiation emitted by the acceptor group can be detected at a wavelength between 520 and 600 nm.
Accordingly, it is possible, in this case, for the method to be carried out in such a way that donor and acceptor beads carrying donor and acceptor groups are introduced initially. Components capable of binding either themselves or via further components to CRP or C1q are then bound to said donor and acceptor beads. As already mentioned above, these components may be either C1q or CRP or may be an anti-CRP or anti-C1q antibody or else may be a secondary antibody directed against said anti-CRP or anti-C1q antibody.
In a further preferred embodiment of the method of the invention, the signal is transferred from the at least one compound or group of compounds according to (b) (donor group) to the at least one compound or group of compounds according to (b) (acceptor group) via emissionless energy transfer, particularly preferably via fluorescence resonance energy transfer.
In this case it is possible to carry out the method, for example, in such a way that the at least one compound or group of compounds according to (a) (donor group) comprises a europium salt-containing compound and that the at least one compound or group of compounds according to (b) (acceptor group) may comprise allophycocyanine (Grepin et al. (2000), Drug Discovery Today 5, 212).
Alternatively, the donor and acceptor groups may be dyes suitable for emissionless energy transfer. It is furthermore possible to use all compounds known to the skilled worker which are suitable for emissionless energy transfer, in particular for fluorescence resonance energy transfer (see, for example, Pope et al. (1999), Drug Discovery Today 4, 350).
In this embodiment, preference is given to choosing the binding components to be used in such a way that the distance between the donor and acceptor groups after binding is less than 10 nm, since an effective emissionless energy transfer is not possible at a larger distance.
In an embodiment preferred according to the invention, the method of the invention is used for determining the concentration of a CRP- or C1 q-containing solution with unknown CRP or C1q content, said solution being preferably blood plasma or blood serum or being blood plasma or blood serum diluted by means of a suitable physiological buffer or by means of water. Said method may be used, for example, for diagnostic purposes, in particular for determining the state of inflammation of an organism andlor for determining the risk of cardiac arrest andlor stroke.
The present invention therefore likewise relates to an HTS-capable, diagnostic assay system for determining a binding event between CRP or C1q and a component binding to CRP or C1q, comprising the following components:
(a) at least one donor component or group of components containing at least one donor component, said donor component being a component which comprises at least one compound or group of compounds capable of emitting a signal after excitation by a fight source (donor group) and said donor component being capable of binding directly or via one or more components from said group of components to CRP or C1 q, (b) at least one acceptor component or group of components containing at least one acceptor component, said acceptor component being a component which comprises at least one compound or group of compounds capable of receiving and emitting in the form of electromagnetic radiation the signal emitted by the compound or group of compounds according to (b) and said acceptor component being capable of binding directly or via one or more components of said group of components to CRP or C1 q.
The HTS-capable assay system here preferably comprises the following additional components:
(c) blood serum or blood plasma, or blood diluted in physiological buffer or water, (d) a light source for exciting the at least one compound or group of compounds according to (a), (e) a detection system for detecting the emitted electromagnetic radiation according to (b).
In another preferred embodiment of the method of the invention, at least one test substance is added prior to step (a) andlor prior to step (b) andlor prior to step (c) and/or prior to step (d), in order to observe, whether said test substance influences the binding event. In this way, it is possible to determine substances which act on the interaction between CRP or C1q and a binder, in particular on the interaction between CRP and Clq, in a modulating, inhibiting or activating manner. This preferably involves screening a library of test substances in the HTS method in order to determine a substance having the desired properties.
The present invention therefore likewise relates to an HTS-capable assay system for determining active substances which act on the interaction between CRP or C1q and a component binding to CRP or C1 q in a modulating manner, comprising the following components:
(a) at least one donor component or group of components containing at least one donor component, said donor component being a component which comprises at least one compound or group of compounds capable of emitting a signal after excitation by a light source (donor group) and said donor component being capable of binding directly or via one or more components from said group of components to CRP or Clq, (b) at least one acceptor component or group of components containing at least one acceptor component, said acceptor component being a component which comprises at least one compound or group of compounds capable of receiving and emitting in the form of electromagnetic radiation the signal emitted by the compound or group of compounds according to (b) and said acceptor component being capable of binding directly or via one or more components of said group of components to CRP or C1 q, 5 (c) at least one test compound.
Said HTS-capable assay system here comprises preferably the following additional components:
(d) a light source for exciting the at least one compound or group of compounds 10 according to (a), (e) a detection system for detecting the emitted electromagnetic radiation according to (b), (f) a CRP- or C1 q-containing solution.
The HTS-capable assay systems of the invention are used here preferably for carrying out the above-described methods of the invention. Particular embodiments of the parts and components of the assay systems therefore correspond to the abovementioned particular embodiments employed in the methods of the invention.
Diagram 1 is a diagrammatic representation of the method described in the exemplary embodiment. Here, the donor component used was an anti-C1q antibody, the donor group being located on particles bound to said anti-C1 q antibody.
In the present case, the donor component can bind via C1 q to the CRP. The acceptor component in the present case is an anti-rabbit secondary antibody, the acceptor group being located on particles bound to said anti-rabbit secondary antibody.
In the present case, the acceptor component can bind via an anti-CRP antibody from rabbit to the CRP.
Particular preference is given to using Alpha-Screen-Beads from Perkin-Elmer Life Sciences as particles in the method. In this embodiment, the donor group is excited by irradiating at a wavelength of 680 nm, using a laser, and the radiation emitted by the acceptor group can be detected at a wavelength between 520 and 600 nm.
Accordingly, it is possible, in this case, for the method to be carried out in such a way that donor and acceptor beads carrying donor and acceptor groups are introduced initially. Components capable of binding either themselves or via further components to CRP or C1q are then bound to said donor and acceptor beads. As already mentioned above, these components may be either C1q or CRP or may be an anti-CRP or anti-C1q antibody or else may be a secondary antibody directed against said anti-CRP or anti-C1q antibody.
In a further preferred embodiment of the method of the invention, the signal is transferred from the at least one compound or group of compounds according to (b) (donor group) to the at least one compound or group of compounds according to (b) (acceptor group) via emissionless energy transfer, particularly preferably via fluorescence resonance energy transfer.
In this case it is possible to carry out the method, for example, in such a way that the at least one compound or group of compounds according to (a) (donor group) comprises a europium salt-containing compound and that the at least one compound or group of compounds according to (b) (acceptor group) may comprise allophycocyanine (Grepin et al. (2000), Drug Discovery Today 5, 212).
Alternatively, the donor and acceptor groups may be dyes suitable for emissionless energy transfer. It is furthermore possible to use all compounds known to the skilled worker which are suitable for emissionless energy transfer, in particular for fluorescence resonance energy transfer (see, for example, Pope et al. (1999), Drug Discovery Today 4, 350).
In this embodiment, preference is given to choosing the binding components to be used in such a way that the distance between the donor and acceptor groups after binding is less than 10 nm, since an effective emissionless energy transfer is not possible at a larger distance.
In an embodiment preferred according to the invention, the method of the invention is used for determining the concentration of a CRP- or C1 q-containing solution with unknown CRP or C1q content, said solution being preferably blood plasma or blood serum or being blood plasma or blood serum diluted by means of a suitable physiological buffer or by means of water. Said method may be used, for example, for diagnostic purposes, in particular for determining the state of inflammation of an organism andlor for determining the risk of cardiac arrest andlor stroke.
The present invention therefore likewise relates to an HTS-capable, diagnostic assay system for determining a binding event between CRP or C1q and a component binding to CRP or C1q, comprising the following components:
(a) at least one donor component or group of components containing at least one donor component, said donor component being a component which comprises at least one compound or group of compounds capable of emitting a signal after excitation by a fight source (donor group) and said donor component being capable of binding directly or via one or more components from said group of components to CRP or C1 q, (b) at least one acceptor component or group of components containing at least one acceptor component, said acceptor component being a component which comprises at least one compound or group of compounds capable of receiving and emitting in the form of electromagnetic radiation the signal emitted by the compound or group of compounds according to (b) and said acceptor component being capable of binding directly or via one or more components of said group of components to CRP or C1 q.
The HTS-capable assay system here preferably comprises the following additional components:
(c) blood serum or blood plasma, or blood diluted in physiological buffer or water, (d) a light source for exciting the at least one compound or group of compounds according to (a), (e) a detection system for detecting the emitted electromagnetic radiation according to (b).
In another preferred embodiment of the method of the invention, at least one test substance is added prior to step (a) andlor prior to step (b) andlor prior to step (c) and/or prior to step (d), in order to observe, whether said test substance influences the binding event. In this way, it is possible to determine substances which act on the interaction between CRP or C1q and a binder, in particular on the interaction between CRP and Clq, in a modulating, inhibiting or activating manner. This preferably involves screening a library of test substances in the HTS method in order to determine a substance having the desired properties.
The present invention therefore likewise relates to an HTS-capable assay system for determining active substances which act on the interaction between CRP or C1q and a component binding to CRP or C1 q in a modulating manner, comprising the following components:
(a) at least one donor component or group of components containing at least one donor component, said donor component being a component which comprises at least one compound or group of compounds capable of emitting a signal after excitation by a light source (donor group) and said donor component being capable of binding directly or via one or more components from said group of components to CRP or Clq, (b) at least one acceptor component or group of components containing at least one acceptor component, said acceptor component being a component which comprises at least one compound or group of compounds capable of receiving and emitting in the form of electromagnetic radiation the signal emitted by the compound or group of compounds according to (b) and said acceptor component being capable of binding directly or via one or more components of said group of components to CRP or C1 q, 5 (c) at least one test compound.
Said HTS-capable assay system here comprises preferably the following additional components:
(d) a light source for exciting the at least one compound or group of compounds 10 according to (a), (e) a detection system for detecting the emitted electromagnetic radiation according to (b), (f) a CRP- or C1 q-containing solution.
The HTS-capable assay systems of the invention are used here preferably for carrying out the above-described methods of the invention. Particular embodiments of the parts and components of the assay systems therefore correspond to the abovementioned particular embodiments employed in the methods of the invention.
Diagram 1 is a diagrammatic representation of the method described in the exemplary embodiment. Here, the donor component used was an anti-C1q antibody, the donor group being located on particles bound to said anti-C1 q antibody.
In the present case, the donor component can bind via C1 q to the CRP. The acceptor component in the present case is an anti-rabbit secondary antibody, the acceptor group being located on particles bound to said anti-rabbit secondary antibody.
In the present case, the acceptor component can bind via an anti-CRP antibody from rabbit to the CRP.
Diagram 1 Light (520 - 620 nm) Acceptor Goat anti-rabbit Light Zabbit anti-CRP
(680 nm) RP
Biotin C1q Donors S~rQ~~v~d~~ Goat anti-C1q Exemplary embodiment: binding assay for determining binding of CRP to C1 q The experiment was carried out using the Alphascreen Detection Kit from Packard Bioscience which comprises donor and acceptor beads, said donor beads comprising compounds which, after excitation at a defined wavelength, can convert triplet oxygen to singlet oxygen, and said acceptor beads comprising compounds excitable by singlet oxygen, and also compounds capable of receiving in the form of an emissionless energy transfer and then emitting in the form of fluorescence radiation energy from the excited compounds. The compounds mentioned are embedded here in each case in a hydrogel matrix. The beads used had already been precoated by the manufacturer, namely the donor beads with streptavidin and the acceptor beads with anti-rabbit antibody.
(680 nm) RP
Biotin C1q Donors S~rQ~~v~d~~ Goat anti-C1q Exemplary embodiment: binding assay for determining binding of CRP to C1 q The experiment was carried out using the Alphascreen Detection Kit from Packard Bioscience which comprises donor and acceptor beads, said donor beads comprising compounds which, after excitation at a defined wavelength, can convert triplet oxygen to singlet oxygen, and said acceptor beads comprising compounds excitable by singlet oxygen, and also compounds capable of receiving in the form of an emissionless energy transfer and then emitting in the form of fluorescence radiation energy from the excited compounds. The compounds mentioned are embedded here in each case in a hydrogel matrix. The beads used had already been precoated by the manufacturer, namely the donor beads with streptavidin and the acceptor beads with anti-rabbit antibody.
Table 1: Overview of the materials used Reagents/plates Molecular weight Supplier, catalog or number concentration Microplate, 384K, PS, white Greiner 784075 C-reactive protein, human,1 mglml, 115 kd Calbiochem 236608 recombinant from E. coli (5mer) c1 q, human 1 mg/ml, 410 kd Calbiochem 204876 Anti-CRP, rabbit 3.57 mg/ml, 165 Calbiochem 235752 kd AIphaScreen IgG detection Packard BioScience kit 6760617 (Protein A) AIphaScreen Rabbit IgG Packard BioScience detection kit 6760607 Anti-c1 q, goat Polyclonal serum Calbiochem 234390 Anti-streptavidin Polyclonal serum Sigma S6390 EZ-Link Sulfo-NHS-LC- Pierce 21430 biotinylation kit Sodium chloride 58.44 g/mol Merck 101540 Calcium chloride (CaCl2 147.02 glmol Merck 102382 x 2 H20) Tris(hydroxymethyl)- 157.6 g/mol Merck 108219 aminomethanehydrochloride (Tris) Bovine serum albumin (BSA) Sigma P7888 Phosphoryl choline chloride329.7 g/mol Sigma P0378 Phosphate-buffered saline Gibco BRL
(Mgz+ 14200-067 and Ca2+-free) (PBS) Slide-A-Lyzer mini dialysis Pierce P.69570 units, 10,000 MWCO
Dimethyl sulfoxide (DMSO) Merck KGaA, 1.02931 Reagentslplates Molecular weight Supplier, catalog or number concentration Ethanol Riedel de Haen, Pluronic F-68 10 % strength solutionSigma, P5556 Hydrochloric acid 1 M Merck, 109057 Sodium hydroxide solution 1 M Merck, 109137 Milli-Q-H20 Preparation of the reaction buffer The following reaction buffer was used in the Alpha-Screen-System: 20 mM Tris-HCI, pH 7.2, 150 mM NaCI, 5 mM CaCl2, 1 mM phosphoryl choline, 0.1 % BSA. A stock solution of Tris-NaCI buffer was prepared by dissolving 3.15 g of Tris-HCI and 8.77 g of NaCI in H20, adjusting the pH to 7.2 by means of 1 M NaOH and then adding to 1 I. The buffer is stored at room temperature. The reaction buffer was prepared by adding 36.7 mg of CaCl2, 12.9 mg of phosphoryl choline and 50 mg of BSA to 50 ml of Tris-NaCI buffer.
Biotinylation of the anti-C1 q antibody In order to enable the anti-C1 q antibody to bind to the streptavidin-coated donor beads, the former needed to be biotinylated first. To this end, 100 NI of anti-C1 q polyclonal serum were dialyzed twice against 200 ml of PBS at room temperature for 1 hour. After adding 20 NI of 1.5 mM sulfo-NHS solution (in Milli-Q water), the solution was left standing at room temperature for 30 minutes and then dialyzed again twice against PBS, in order to remove excess biotinylation reagent.
Assay procedure The assay was carried out by initially introducing 2 NI of donor bead/anti-CRP
solution and then adding 2 NI of C1 q, 2 NI of CRP and 2 NI of acceptor bead/anti-CRP. This resulted in the following final concentrations: CRP: 1 nM; C1 q: 10 nM;
anti-CRP: 7.3 nM; anti-C1 q: 1:1500; donor beads: 20 Ng/ml; acceptor beads:
(Mgz+ 14200-067 and Ca2+-free) (PBS) Slide-A-Lyzer mini dialysis Pierce P.69570 units, 10,000 MWCO
Dimethyl sulfoxide (DMSO) Merck KGaA, 1.02931 Reagentslplates Molecular weight Supplier, catalog or number concentration Ethanol Riedel de Haen, Pluronic F-68 10 % strength solutionSigma, P5556 Hydrochloric acid 1 M Merck, 109057 Sodium hydroxide solution 1 M Merck, 109137 Milli-Q-H20 Preparation of the reaction buffer The following reaction buffer was used in the Alpha-Screen-System: 20 mM Tris-HCI, pH 7.2, 150 mM NaCI, 5 mM CaCl2, 1 mM phosphoryl choline, 0.1 % BSA. A stock solution of Tris-NaCI buffer was prepared by dissolving 3.15 g of Tris-HCI and 8.77 g of NaCI in H20, adjusting the pH to 7.2 by means of 1 M NaOH and then adding to 1 I. The buffer is stored at room temperature. The reaction buffer was prepared by adding 36.7 mg of CaCl2, 12.9 mg of phosphoryl choline and 50 mg of BSA to 50 ml of Tris-NaCI buffer.
Biotinylation of the anti-C1 q antibody In order to enable the anti-C1 q antibody to bind to the streptavidin-coated donor beads, the former needed to be biotinylated first. To this end, 100 NI of anti-C1 q polyclonal serum were dialyzed twice against 200 ml of PBS at room temperature for 1 hour. After adding 20 NI of 1.5 mM sulfo-NHS solution (in Milli-Q water), the solution was left standing at room temperature for 30 minutes and then dialyzed again twice against PBS, in order to remove excess biotinylation reagent.
Assay procedure The assay was carried out by initially introducing 2 NI of donor bead/anti-CRP
solution and then adding 2 NI of C1 q, 2 NI of CRP and 2 NI of acceptor bead/anti-CRP. This resulted in the following final concentrations: CRP: 1 nM; C1 q: 10 nM;
anti-CRP: 7.3 nM; anti-C1 q: 1:1500; donor beads: 20 Ng/ml; acceptor beads:
40 Ng/ml. In the negative control, reaction buffer replaced the CRP solution, the C1q solution or both of these solutions. After incubating at room temperature for 2 hours, the data were read out by means of an AIphaQuest reader.
Results The measured intensities of the emitted radiation at a CRP concentration of 1 nM and a C1q concentration of 10 nM were as follows (photomultiplier counts are listed):
In the absence of CRP and absence of C1 q: 952 In the presence of CRP and absence of C1q: 1255 In the absence of CRP and presence of C1q: 1114 In the presence of CRP and presence of C1q: 80376 As can be seen, the negative controls do not provide a positive result, while a strong signal is visible when the C1 q- and CRP-containing solutions are combined.
Results The measured intensities of the emitted radiation at a CRP concentration of 1 nM and a C1q concentration of 10 nM were as follows (photomultiplier counts are listed):
In the absence of CRP and absence of C1 q: 952 In the presence of CRP and absence of C1q: 1255 In the absence of CRP and presence of C1q: 1114 In the presence of CRP and presence of C1q: 80376 As can be seen, the negative controls do not provide a positive result, while a strong signal is visible when the C1 q- and CRP-containing solutions are combined.
Claims (36)
1. A method for determining a binding event of two components binding directly or via one or more components to CRP, which comprises the following steps:
(a) initially introducing a CRP-containing solution, (b) adding at least one donor component or a group of components containing at least one donor component to said CRP-containing solution, said donor component being a component which comprises at least one compound or group of compounds capable of emitting a signal after excitation by a light source (donor group) and said donor component being capable of binding directly or via one or more components from said group of components to CRP, (c) adding at least one acceptor component or a group of components containing at least one acceptor component to said CRP-containing solution, said acceptor component being a component which comprises at least one compound or group of compounds capable of receiving and emitting in the form of electromagnetic radiation the signal emitted by the compound or group of compounds according to (b) (acceptor group) and said acceptor component being capable of binding directly or via one or more components of said group of components to CRP, (d) exciting the at least one compound or group of compounds according to (b) (donor group) by means of a light source, (e) detecting the electromagnetic radiation emitted by the at least one compound or group of compounds according to (c) (acceptor group) in order to detect said binding event.
(a) initially introducing a CRP-containing solution, (b) adding at least one donor component or a group of components containing at least one donor component to said CRP-containing solution, said donor component being a component which comprises at least one compound or group of compounds capable of emitting a signal after excitation by a light source (donor group) and said donor component being capable of binding directly or via one or more components from said group of components to CRP, (c) adding at least one acceptor component or a group of components containing at least one acceptor component to said CRP-containing solution, said acceptor component being a component which comprises at least one compound or group of compounds capable of receiving and emitting in the form of electromagnetic radiation the signal emitted by the compound or group of compounds according to (b) (acceptor group) and said acceptor component being capable of binding directly or via one or more components of said group of components to CRP, (d) exciting the at least one compound or group of compounds according to (b) (donor group) by means of a light source, (e) detecting the electromagnetic radiation emitted by the at least one compound or group of compounds according to (c) (acceptor group) in order to detect said binding event.
2. A method for determining a binding event of two components binding directly or via one or more components to C1q, which comprises the following steps:
(a) initially introducing a C1q-containing solution, (b) adding at least one donor component or a group of components containing at least one donor component to said C1q-containing solution, said donor component being a component which comprises at least one compound or group of compounds capable of emitting a signal after excitation by a light source (donor group) and said donor component being capable of binding directly or via one or more components from said group of components to C1q, (c) adding at least one acceptor component or a group of components containing at least one acceptor component to said C1q-containing solution, said acceptor component being a component which comprises at feast one compound or group of compounds capable of receiving and emitting in the form of electromagnetic radiation the signal emitted by the compound or group of compounds according to (b) (acceptor group) and said acceptor component being capable of binding directly or via one or more components of said group of components to C1 q, (d) exciting the at least one compound or group of compounds according to (b) (donor group) by means of a light source, (e) detecting the electromagnetic radiation emitted by the at least one compound or group of compounds according to (c) (acceptor group) in order to detect said binding event.
(a) initially introducing a C1q-containing solution, (b) adding at least one donor component or a group of components containing at least one donor component to said C1q-containing solution, said donor component being a component which comprises at least one compound or group of compounds capable of emitting a signal after excitation by a light source (donor group) and said donor component being capable of binding directly or via one or more components from said group of components to C1q, (c) adding at least one acceptor component or a group of components containing at least one acceptor component to said C1q-containing solution, said acceptor component being a component which comprises at feast one compound or group of compounds capable of receiving and emitting in the form of electromagnetic radiation the signal emitted by the compound or group of compounds according to (b) (acceptor group) and said acceptor component being capable of binding directly or via one or more components of said group of components to C1 q, (d) exciting the at least one compound or group of compounds according to (b) (donor group) by means of a light source, (e) detecting the electromagnetic radiation emitted by the at least one compound or group of compounds according to (c) (acceptor group) in order to detect said binding event.
3. The method as claimed in claim 1 or 2, wherein the electromagnetic radiation is fluorescence radiation.
4. The method as claimed in claim 1 or 2, wherein the light source according to (d) is a laser or a lamp.
5. The method as claimed in claim 1 or 2, wherein detection according to (e) is carried out with the aid of a photomultiplier.
6. The method as claimed in claim 1, wherein the components of the group of components according to (b) and/or (c) can bind to CRP or to one another, in spatial succession.
7. The method as claimed in claim 2, wherein the components of the group of components according to (b) and/or (c) can bind to C1q or to one another, in spatial succession.
8. A method as claimed in any of claims 1 to 7, wherein the components are polypeptides or comprise a polypeptide.
9. The method as claimed in claim 8, wherein at least one of the components is an antibody.
10. The method as claimed in claim 9, wherein the antibody is a monoclonal or polyclonal antibody.
11. The method as claimed in claim 1, wherein at least one of the components is a natural binding partner of the CRP.
12. The method as claimed in claim 11, wherein the natural binding partner of the CRP is C1q.
13. The method as claimed in claim 12, wherein the group of components according to claim 1(a) or claim 1(b) comprises C1q and an anti-C1q antibody.
14. The method as claimed in claim 2, wherein at least one of the components is a natural binding partner of the C1q.
15. The method as claimed in claim 14, wherein the natural binding partner is CRP.
16. The method as claimed in claim 15, wherein the group of components according to claim 2(a) or claim 2(b) comprises CRP and an anti-CRP antibody.
17. The method as claimed in claim 1 or 2, wherein the signal is transferred from the at least one compound or group of compounds according to (b) (donor group) to the at least one compound or group of compounds according to (c) (acceptor group) by means of emissionless energy transfer.
18. The method as claimed in claim 17, wherein the emissionless energy transfer is a fluorescence resonance energy transfer.
19. The method as claimed in claim 1 or 2, wherein the signal is transferred via singlet oxygen.
20. The method as claimed in claim 19, wherein the at least one compound or group of compounds according to claim 1 (b) or 2(b) (donor group) comprises a compound which is able to convert triplet oxygen to singlet oxygen after excitation by a laser and wherein the at least one compound or group of compounds according to claim 1 (b) or 2(b) (acceptor group) comprise at least one first compound excitable by singlet oxygen and comprises at least one second compound capable of absorbing in an emissionless manner and emitting in the form of fluorescence radiation the energy absorbed by said compound excitable by singlet oxygen.
21. The method as claimed in claim 20, wherein the donor group and/or the acceptor group are localized on particles.
22. The method as claimed in claim 21, wherein the particles have an average diameter of approximately 200 nm.
23. The method as claimed in any of claims 1 to 22, which is used for determining the concentration of a CRP-containing solution with an unknown CRP content.
24. The method as claimed in any of claims 1 to 22, which is used for determining the concentration of a C1q-containing solution with an unknown C1q content.
25. The method as claimed in claim 23 or 24 wherein the solution is blood serum, blood plasma, or blood serum or blood plasma diluted by means of a physiological buffer or by means of water.
26. The method as claimed in claim 25, which is a diagnostic method for determining the state of inflammation of an organism and/or for determining the risk of cardiac arrest and/or stroke.
27. The method as claimed in any of the preceding claims, which is an HTS-capable homogeneous method which may be carried out as a one-step method.
28. The method as claimed in claim 1, comprising the additional step of adding at feast one test substance prior to step (a) and/or prior to step (b) and/or prior to step (c) and/or prior to step (d), in order to observe whether said test substance influences the binding event between CRP and CRP-binding component.
29. The method as claimed in claim 2, comprising the additional step of adding at least one test substance prior to step (a) and/or prior to step (b) and/or prior to step (c) and/or prior to step (d), in order to observe whether said test substance influences the binding event between C1q and C1q-binding component.
30. The method as claimed in claim 28 or 29, which is used for determining substances which act on the binding event in a modulating, inhibiting or activating manner.
31. An HTS-capable, diagnostic assay system for determining a binding event between CRP and a component binding to CRP, comprising the following components:
(a) at least one donor component or group of components containing at least one donor component, said donor component being a component which comprises at least one compound or group of compounds capable of emitting a signal after excitation by a light source (donor group) and said donor component being capable of binding directly or via one or more components from said group of components to CRP, (b) at least one acceptor component or group of components containing at least one acceptor component, said acceptor component being a component which comprises at least one compound or group of compounds capable of receiving and emitting in the form of electromagnetic radiation the signal emitted by the compound or group of compounds according to (b) (acceptor group) and said acceptor component being capable of binding directly or via one or more components of said group of components to CRP.
(a) at least one donor component or group of components containing at least one donor component, said donor component being a component which comprises at least one compound or group of compounds capable of emitting a signal after excitation by a light source (donor group) and said donor component being capable of binding directly or via one or more components from said group of components to CRP, (b) at least one acceptor component or group of components containing at least one acceptor component, said acceptor component being a component which comprises at least one compound or group of compounds capable of receiving and emitting in the form of electromagnetic radiation the signal emitted by the compound or group of compounds according to (b) (acceptor group) and said acceptor component being capable of binding directly or via one or more components of said group of components to CRP.
32. An HTS-capable, diagnostic assay system for determining a binding event between C1q and a component binding to C1q, comprising the following components:
(a) at least one donor component or group of components containing at least one donor component, said donor component being a component which comprises at least one compound or group of compounds capable of emitting a signal after excitation by a light source (donor group) and said donor component being capable of binding directly or via one or more components from said group of components to C1q, (b) at least one acceptor component or group of components containing at least one acceptor component, said acceptor component being a component which comprises at least one compound or group of compounds capable of receiving and emitting in the form of electromagnetic radiation the signal emitted by the compound or group of compounds according to (b) (acceptor group) and said acceptor component being capable of binding directly or via one or more components of said group of components to C1q.
(a) at least one donor component or group of components containing at least one donor component, said donor component being a component which comprises at least one compound or group of compounds capable of emitting a signal after excitation by a light source (donor group) and said donor component being capable of binding directly or via one or more components from said group of components to C1q, (b) at least one acceptor component or group of components containing at least one acceptor component, said acceptor component being a component which comprises at least one compound or group of compounds capable of receiving and emitting in the form of electromagnetic radiation the signal emitted by the compound or group of compounds according to (b) (acceptor group) and said acceptor component being capable of binding directly or via one or more components of said group of components to C1q.
33. The HTS-capable assay system as claimed in claim 31 or 32, comprising the following additional components:
(c) blood serum or blood plasma, or blood serum or blood plasma diluted in physiological buffer or by means of water, (d) a light source for exciting the at least one compound or group of compounds according to (a) (donor group), (e) a detection system for detecting the emitted electromagnetic radiation according to (b).
(c) blood serum or blood plasma, or blood serum or blood plasma diluted in physiological buffer or by means of water, (d) a light source for exciting the at least one compound or group of compounds according to (a) (donor group), (e) a detection system for detecting the emitted electromagnetic radiation according to (b).
34. An HTS-capable assay system for determining active substances which act on the interaction between CRP and a component binding to CRP in a modulating manner, comprising the following components:
(a) at least one donor component or group of components containing at feast one donor component, said donor component being a component which comprises at least one compound or group of compounds capable of emitting a signal after excitation by a light source (donor group), and said donor component being capable of receiving and emitting in the form of electromagnetic radiation the signal emitted by the compound or group of compounds according to (a), and said donor component being capable of binding directly or via one or more components from said group of components to CRP, (b) at least one acceptor component or group of components containing at least one acceptor component, said acceptor component being a component which comprises at least one compound or group of compounds capable of receiving and emitting in the form of electromagnetic radiation the signal emitted by the compound or group of compounds according to (b) (acceptor group) and said acceptor component being capable of binding directly or via one or more components of said group of components to CRP, (c) at least one test compound.
(a) at least one donor component or group of components containing at feast one donor component, said donor component being a component which comprises at least one compound or group of compounds capable of emitting a signal after excitation by a light source (donor group), and said donor component being capable of receiving and emitting in the form of electromagnetic radiation the signal emitted by the compound or group of compounds according to (a), and said donor component being capable of binding directly or via one or more components from said group of components to CRP, (b) at least one acceptor component or group of components containing at least one acceptor component, said acceptor component being a component which comprises at least one compound or group of compounds capable of receiving and emitting in the form of electromagnetic radiation the signal emitted by the compound or group of compounds according to (b) (acceptor group) and said acceptor component being capable of binding directly or via one or more components of said group of components to CRP, (c) at least one test compound.
35. An HTS-capable assay system for determining active substances which act on the interaction between C1q and a component binding to C1q in a modulating manner, comprising the following components:
(a) at least one donor component or group of components containing at least one donor component, said donor component being a component which comprises at least one compound or group of compounds capable of emitting a signal after excitation by a light source (donor group), and said donor component being capable of receiving and emitting in the form of electromagnetic radiation the signal emitted by the compound or group of compounds according to (a), and said donor component being capable of binding directly or via one or more components from said group of components to C1 q, (b) at least one acceptor component or group of components containing at least one acceptor component, said acceptor component being a component which comprises at least one compound or group of compounds capable of receiving and emitting in the form of electromagnetic radiation the signal emitted by the compound or group of compounds according to (b) (acceptor group) and said acceptor component being capable of binding directly or via one or more components of said group of components to C1q, (c) at least one test compound.
(a) at least one donor component or group of components containing at least one donor component, said donor component being a component which comprises at least one compound or group of compounds capable of emitting a signal after excitation by a light source (donor group), and said donor component being capable of receiving and emitting in the form of electromagnetic radiation the signal emitted by the compound or group of compounds according to (a), and said donor component being capable of binding directly or via one or more components from said group of components to C1 q, (b) at least one acceptor component or group of components containing at least one acceptor component, said acceptor component being a component which comprises at least one compound or group of compounds capable of receiving and emitting in the form of electromagnetic radiation the signal emitted by the compound or group of compounds according to (b) (acceptor group) and said acceptor component being capable of binding directly or via one or more components of said group of components to C1q, (c) at least one test compound.
36. The HTS-capable assay system as claimed in claim 34 or 35, comprising the following additional components:
(d) a light source for exciting the at least one compound or group of compounds according to (a) (donor group), (e) a detection system for detecting the emitted electromagnetic radiation according to (b).
(d) a light source for exciting the at least one compound or group of compounds according to (a) (donor group), (e) a detection system for detecting the emitted electromagnetic radiation according to (b).
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE10226011A DE10226011A1 (en) | 2002-06-12 | 2002-06-12 | HTS-capable method and test system for determining the interaction between c-reactive protein and components that bind to C-reactive protein |
| DE10226011.7 | 2002-06-12 | ||
| PCT/EP2003/006038 WO2003107000A2 (en) | 2002-06-12 | 2003-06-10 | Hts-capable method and testing system for determining the interaction between a c-reactive protein and constituents that bind to a c-reactive protein |
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| CA2489021A1 true CA2489021A1 (en) | 2003-12-24 |
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| CA002489021A Abandoned CA2489021A1 (en) | 2002-06-12 | 2003-06-10 | Determining the interaction between a c-reactive protein and constituents that bind to a c-reactive protein |
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| Country | Link |
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| EP (1) | EP1516187A2 (en) |
| JP (1) | JP2005529348A (en) |
| CN (1) | CN1745300A (en) |
| AR (1) | AR040259A1 (en) |
| AU (1) | AU2003250344A1 (en) |
| BR (1) | BR0311709A (en) |
| CA (1) | CA2489021A1 (en) |
| DE (1) | DE10226011A1 (en) |
| MX (1) | MXPA04012232A (en) |
| NO (1) | NO20050037L (en) |
| RU (1) | RU2005100042A (en) |
| TW (1) | TW200411179A (en) |
| WO (1) | WO2003107000A2 (en) |
| ZA (1) | ZA200409199B (en) |
Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102721813A (en) * | 2012-07-09 | 2012-10-10 | 沃克(天津)生物科技有限公司 | Homogeneous luminous immunoassay assay kit for prostate specific antigen and detection method therefor |
| CN103852465A (en) * | 2012-11-30 | 2014-06-11 | 通用电气公司 | Method and apparatus for detecting marker protein |
| JP2018533017A (en) * | 2015-10-02 | 2018-11-08 | ティージーアール バイオサイエンシス ピーティーワイ エルティーディー． | Specimen detection with multiple substrates |
| CN111398580B (en) * | 2017-11-03 | 2023-06-13 | 科美诊断技术股份有限公司 | Method for detecting target IgM antibody in sample |
| CN109752332A (en) * | 2017-11-07 | 2019-05-14 | 重庆中元汇吉生物技术有限公司 | A kind of C1Q detection kit |
| CN108490194A (en) * | 2018-02-11 | 2018-09-04 | 北京科美生物技术有限公司 | Detect the homogeneous immunological detection reagent suit and its preparation method and application of follicular stimulating hormone |
| CN108445238A (en) * | 2018-02-11 | 2018-08-24 | 北京科美生物技术有限公司 | Detect the homogeneous immunological detection reagent suit and its preparation method and application of interstitialcellstimulating hormone (ICSH) |
| CN108445216B (en) * | 2018-02-11 | 2021-10-15 | 科美诊断技术股份有限公司 | Human anti-mullerian hormone determination kit and preparation method and application thereof |
| CN108169497A (en) * | 2018-02-11 | 2018-06-15 | 北京科美生物技术有限公司 | Human prolactin detection kit and preparation method and application |
| CN108445239B (en) * | 2018-02-11 | 2021-11-02 | 科美诊断技术股份有限公司 | Homogeneous phase immunoassay reagent kit for detecting beta human chorionic gonadotropin, and preparation method and application thereof |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU633488B2 (en) * | 1988-04-04 | 1993-02-04 | Northwestern University | Binding of immune complexes by modified forms of c-reactive protein |
| US6140490A (en) * | 1996-02-01 | 2000-10-31 | Nexstar Pharmaceuticals, Inc. | High affinity nucleic acid ligands of complement system proteins |
| US5283238A (en) * | 1992-04-24 | 1994-02-01 | Immtech International, Inc. | Methods of treating cancer using modified C-reactive protein |
| CA2170873A1 (en) * | 1993-09-03 | 1995-03-09 | Dariush Davalian | Fluorescent oxygen channeling immunoassays |
| US6303325B1 (en) * | 1998-05-29 | 2001-10-16 | Dade Behring Inc. | Method for detecting analytes |
-
2002
- 2002-06-12 DE DE10226011A patent/DE10226011A1/en not_active Withdrawn
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2003
- 2003-06-10 MX MXPA04012232A patent/MXPA04012232A/en not_active Application Discontinuation
- 2003-06-10 WO PCT/EP2003/006038 patent/WO2003107000A2/en not_active Application Discontinuation
- 2003-06-10 TW TW092115617A patent/TW200411179A/en unknown
- 2003-06-10 RU RU2005100042/15A patent/RU2005100042A/en not_active Application Discontinuation
- 2003-06-10 CA CA002489021A patent/CA2489021A1/en not_active Abandoned
- 2003-06-10 JP JP2004513768A patent/JP2005529348A/en active Pending
- 2003-06-10 CN CNA03813442XA patent/CN1745300A/en active Pending
- 2003-06-10 BR BR0311709-0A patent/BR0311709A/en not_active IP Right Cessation
- 2003-06-10 EP EP03759923A patent/EP1516187A2/en not_active Withdrawn
- 2003-06-10 AU AU2003250344A patent/AU2003250344A1/en not_active Abandoned
- 2003-06-10 AR ARP030102067A patent/AR040259A1/en unknown
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2004
- 2004-11-17 ZA ZA200409199A patent/ZA200409199B/en unknown
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2005
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Also Published As
| Publication number | Publication date |
|---|---|
| AR040259A1 (en) | 2005-03-23 |
| BR0311709A (en) | 2005-03-15 |
| CN1745300A (en) | 2006-03-08 |
| WO2003107000A3 (en) | 2004-05-27 |
| MXPA04012232A (en) | 2005-02-25 |
| TW200411179A (en) | 2004-07-01 |
| DE10226011A1 (en) | 2004-01-08 |
| ZA200409199B (en) | 2005-05-18 |
| AU2003250344A1 (en) | 2003-12-31 |
| JP2005529348A (en) | 2005-09-29 |
| EP1516187A2 (en) | 2005-03-23 |
| WO2003107000A2 (en) | 2003-12-24 |
| RU2005100042A (en) | 2006-07-20 |
| NO20050037L (en) | 2005-01-04 |
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