Abstract:
Disclosed are methods and assay systems for high throughout screening of interactions between C-reactive protein, C1q, and components binding to them.

Description:
[0001]    This application claims the benefit of priority under 35 U.S.C. §119 of German Application No. 10226011.7 filed on Jun. 12, 2002, and of U.S. Provisional Application No. 60/426,945 filed on Nov. 15, 2002, the contents of both of which are hereby incorporated by reference. 
     
    
     
       FIELD OF THE INVENTION  
         [0002]    Embodiments of the present invention relate to high-throughput-screening (HTS) capable methods and assay systems for determining the interaction between C-reactive protein (CRP) and C1q and components binding to CRP and C1q, respectively. The methods and assay systems are useful for, for example, determining the concentration of a solution containing CRP and C1q, respectively, and for determining substances which influence the interaction of CRP and C1q, respectively, and of the components binding thereto.  
         BACKGROUND OF THE INVENTION  
         [0003]    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 Ca 2+  ions. Such ions are 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.  
           [0004]    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.  
           [0005]    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 (Aifai and Aidker (2001), Clinical Chemistry 47,403-411).  
           [0006]    New studies (Jialal et al (2001), Circulation 103, 1933-1935) suggest that lowering the CRP level or blocking the CRP-mediated effector functions, for example complement activation due to binding to C1q, may be useful for the prevention and treatment of coronary heart disease.  
           [0007]    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 and/or C1q with other components, in particular on the interaction between CRP and C1q, in a modulating, i.e. activating or inhibiting, way.  
           [0008]    Disclosed herein is a method which makes possible a high throughput screening for substances which influence complement activation due to binding of CRP to C1q. 
       
    
    
     BRIEF DESCRIPTION OF THE FIGURES  
       [0009]    [0009]FIG. 1 illustrates an assay for determining the binding of CRP to C1q according to one embodiment of the invention. A donor component is located on particles bound to anti-C1q antibody which in turn is bound to C1q. An acceptor component is located on particles bound to goat anti-rabbit antibody; the goat anti-rabbit antibody is bound to rabbit anti-CRP antibody which in turn is bound to CRP. Light that impinges on the acceptor component causes it to convert ambient oxygen to singlet oxygen. Binding between CRP and C1q brings the donor and acceptor components sufficiently close together to allow the singlet oxygen to reach the acceptor component, causing the component to release light. The light is detectable with standard photodetection equipment, such as a photomultiplier. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0010]    According to one embodiment of the invention, one determines a binding event of two components binding directly or via one or more components to CRP or C1q by performing the following steps:  
         [0011]    (a) initially introducing a CRP- or C1q-containing solution;  
         [0012]    (b) adding at least one donor component or a group of components containing at least one donor component to the CRP- or C1q-containing solution, the donor component comprising at least one compound or group of compounds capable of emitting a signal after excitation by a light source (donor group) and the donor component being capable of binding directly or via one or more components from the group of components to CRP or C1q;  
         [0013]    (c) adding at least one acceptor component or a group of components containing at least one acceptor component to the CRP- or C1q-containing solution, the acceptor component comprising 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 the acceptor component being capable of binding directly or via one or more components of the group of components to CRP or C1q;  
         [0014]    (d) exciting the at least one compound or group of compounds according to (b) (donor group) by means of a light source;  
         [0015]    (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 the binding event.  
         [0016]    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 photomultiplier.  
         [0017]    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, the components are preferably capable of binding to CRP or C1q or to one another in spatial succession. The 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, the CRP- or C1q-binding component according to (b) binding to a different binding site than the CRP- or C1q-binding component according to (c).  
         [0018]    In another preferred embodiment of the method of the invention, one of the components binding directly to CRP or C1q is a naturally occurring binding partner of the CRP or C1q, which, in case of the CRP is 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 C1q or CRP comprising a donor or acceptor group. When using a group of components capable of forming a binding cascade, C1q or CRP is accordingly the first component binding to CRP or C1q 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 preferably antibody molecules.  
         [0019]    Thus, for example, when initially introducing a CRP-containing solution, preferably either the donor or the acceptor component itself comprises C1q or the donor or acceptor component is bound via C1q to CRP. In this case, the donor or acceptor component may comprise an anti-C1q antibody or a secondary antibody capable of binding to the anti-C1q 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.  
         [0020]    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 the particles preferably being between 150 and 250 nm, preferably at approximately 200 nm. Accordingly, the donor or acceptor components then comprise the particles which, in this case, may be made of a polymeric material.  
         [0021]    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. 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 needs to 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 protein can therefore (although need not) 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 the protein.  
         [0022]    One may employ a reduced sample volume of the method of the invention compared to conventional methods. Thus it is possible to use volumes of less than 10 μl. Moreover, it is possible to use highly diluted solutions with CRP or C1q concentrations of less than 1 nM, and even less than 100 pM.  
         [0023]    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 (b) (donor group) to the at least one compound or group of compounds according to (c) (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.  
         [0024]    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 the energy in the form of fluorescence radiation which can be detected using a photomultiplier. 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.  
         [0025]    Particular preference is given in this embodiment to donor group and acceptor group localized on particles, the particles preferably having an average diameter of between 150 and 250 nm, preferably of approximately 200 nm. The preferred use of the particles here is such that the final concentration of donor group-carrying particles is 1-40 μg/ml and the final concentration of acceptor group-carrying particles is 1-80 μg/ml. The binding capacity of the particles may be, for example, from approximately 0.1 nM to 1 nM per μg/ml particles.  
         [0026]    Particular preference is given to using AlphaScreen 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 the 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 the anti-CRP or anti-C1q antibody.  
         [0027]    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, 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 (b) (donor group) comprises a europium salt-containing compound and that the at least one compound or group of compounds according to (c) (acceptor group) may comprise allophycocyanine (Grepin et al. (2000), Drug Discovery Today 5,212).  
         [0028]    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 C1q-containing solution with unknown CRP or C1q content, the 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. The method may be used, for example, for diagnostic purposes, in particular for determining the state of inflammation of an organism and/or for determining the risk of cardiac arrest and/or stroke.  
         [0029]    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:  
         [0030]    (a) at least one donor component or group of components containing at least one donor component, the donor component comprising at least one compound or group of compounds capable of emitting a signal after excitation by a light source (donor group) and the donor component being capable of binding directly or via one or more components from the group of components to CRP or C1q; and  
         [0031]    (b) at least one acceptor component or group of components containing at least one acceptor component, the acceptor component comprising 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 the acceptor component being capable of binding directly or via one or more components of the group of components to CRP or C1q.  
         [0032]    The HTS-capable assay system here preferably comprises the following additional components:  
         [0033]    (c) blood serum or blood plasma, or blood diluted in physiological buffer or water;  
         [0034]    (d) a light source for exciting the at least one compound or group of compounds according to (a); and  
         [0035]    (e) a detection system for detecting the emitted electromagnetic radiation according to (b).  
         [0036]    In another preferred embodiment of the method of the invention, at least one test substance is added 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 the 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 C1q, 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.  
         [0037]    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 C1q in a modulating manner, comprising the following components:  
         [0038]    (a) at least one donor component or group of components containing at least one donor component, the donor component comprising at least one compound or group of compounds capable of emitting a signal after excitation by a light source (donor group) and the donor component being capable of binding directly or via one or more components from the group of components to CRP or C1q; and  
         [0039]    (b) at least one acceptor component or group of components containing at least one 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 (a) and the acceptor component being capable of binding directly or via one or more components of the group of components to CRP or C1q;  
         [0040]    (c) at least one test compound.  
         [0041]    The HTS-capable assay system here comprises preferably the following additional components:  
         [0042]    (d) a light source for exciting the at least one compound or group of compounds according to (a);  
         [0043]    (e) a detection system for detecting the emitted electromagnetic radiation according to (b); and  
         [0044]    (f) a CRP- or C1q-containing solution.  
         [0045]    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.  
       EXAMPLES  
       [0046]    Binding assay for determining binding of CRP to C1q FIG. 1 shows the method according to the embodiment described below. Here, the donor component used was an anti-C1q antibody, the donor group being located on particles bound to the anti-C1q antibody. In the present case, the donor component can bind via C1q 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 the anti-rabbit secondary antibody. In the present case, the acceptor component can bind via an anti-CRP antibody from rabbit to the CRP.  
         [0047]    The experiment was carried out using the Alphascreen Detection Kit from Packard Bioscience which comprises donor and acceptor beads, the donor beads comprising compounds which, after excitation at a defined wavelength, can convert triplet oxygen to singlet oxygen, and the 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, the donor beads with streptavidin and the acceptor beads with anti-rabbit antibody.  
                             TABLE 1                           Overview of the materials used                MOLECULAR WEIGHT OR   SUPPLIER, CATALOG       REAGENTS/PLATES   CONCENTRATION   NUMBER               Microplate, 384K, PS, white       Greiner 784075       C-reactive protein, human,   1 mg/ml, 115 kd (5mer)   Calbiochem 236608       recombinant from  E. coli         C1q, human   1 mg/ml, 410 kd   Calbiochem 204876       Anti-CRP, rabbit   3.57 mg/ml, 165 kd   Caibiochem 235752       AlphaScreen IgG detection       Packard BioScience       kit (Protein A)       6760617       AlphaScreen Rabbit IgG       Packard BioScience       detection kit       6760607       Anti-C1q, 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 (CaCl 2  × 2   147.02 g/mol   Merck 102382       H 2 O)       Tris(hydroxymethyl)-   157.6 g/mol   Merck 108219       aminomethanehydrochloride       (Tris)       Bovine serum albumin (BSA)       Sigma P7888       Phosphoryl choline chloride   329.7 g/mol   Sigma P0378       Phosphate-buffered saline       Gibco BRL 14200-067       (Mg 2+  + and Ca 2+  -free) (PBS)       Slide-A-Lyzer mini dialysis       Pierce P .69570       units 10,000 MWCO       Dimethyl sulfoxide (DMSO)       Merck KGaA, 1.02931       Ethanol       Riedel de Haen, 32250       Pluronic F-68   10% strength solution   Sigma, P5556       Hydrochloric acid   1 M   Merck, 109057       Sodium hydroxide solution   1 M   Merck, 109137       Milli-Q-H20                  
 
       Preparation of the Reaction Buffer  
       [0048]    The following reaction buffer was used in the Alpha-Screen-System: 20 mM Tris-HCl, pH 7.2, 150 mM NaCl, 5 mM CaCl 2 , 1 mM phosphoryl choline, 0.1% BSA. A stock solution of Tris-NaCl buffer was prepared by dissolving 3.15 g of Tris-HCl and 8.77 g of NaCl in H 2 O, adjusting the pH to 7.2 by means of 1 M NaOH and then adding H2O to 1 l. The buffer is stored at room temperature. The reaction buffer was prepared by adding 36.7 mg of CaCl 2 , 12.9 mg of phosphoryl choline and 50 mg of BSA to 50 ml of Tris-NaCl buffer.  
         
       [0049]    Biotinylation of the Anti-C1q Antibody  
         [0050]    In order to enable the anti-C1q antibody to bind to the streptavidin-coated donor beads, the former were biotinylated first. To this end, 100μl of anti-C1q polyclonal serum were dialyzed twice against 200 ml of PBS at room temperature for 1 hour. After adding 20 μl 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  
       [0051]    The assay was carried out by initially introducing 2 μl of donor bead/anti-CRP solution and then adding 2 μl of C1q, 2 μl of CRP and 2 μl of acceptor bead/anti-CRP. This resulted in the following final concentrations: CRP: 1 nM; C1q: 10 nM; anti-CRP: 7.3 nM; anti-C1q: 1:1500; donor beads: 20 μg/ml; acceptor beads: 40 μg/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 AlphaQuest reader.  
       Results  
       [0052]    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):  
         [0053]    In the absence of CRP and absence of C1q: 952  
         [0054]    In the presence of CRP and absence of C1q: 1255  
         [0055]    In the absence of CRP and presence of C1q: 1114  
         [0056]    In the presence of CRP and presence of C1q: 80376  
         [0057]    As can be seen, the negative controls did not provide a positive result, while a strong signal is visible when the C1q- and CRP-containing solutions are combined.