TW200411179A - HTS-capable method and assay system for determining the interaction between C-reactive protein and components binding to C-reactive protein - Google Patents

Abstract

The present invention relates to an HTS-capable method and assay system for determining the interaction between C-reactive protein (CRP) and Clq and components binding to CRP and Clq, respectively, to determining the concentration of a solution containing CRP and Clq, respectively, and to determining substances which influence the interaction of CRP and Clq, respectively, and components binding thereto, in particular the interaction between CRP and Clq.

Description

200411179 A7 B7 V. Description of the Invention (!)

SELECT_gL The present invention relates to a high-throughput screening method and analysis system for determining the interaction between c-reactive protein (CRP) and Clq and components that bind to CRP and Clq, respectively. The concentration of the Clq solution, and the substances that affect the interaction between the respective CRP and Clq and their binding components can be determined. C-reactive protein (CRP) is an acute phase plasma protein whose concentration in the serum increases rapidly and significantly after infection or tissue damage [Volanakis (2001), Molecular Immunology, 38, 189 ~ 197] 〇CUP in

In the presence of Ca2 + ions, it can bind to phosphatidylcholine (PCh). The latter are common in the polysaccharides of pathogenic bacteria and in the cell membrane of damaged and necrotic cells. CRP bound to PCh can stimulate a series of classical complement cascades by binding to the protein Clq. Complement is part of the immune system and is primarily involved in the antibody-mediated immune defense response. The three physiological functions of complement are defense against bacterial infections, linkage of innate and acquired immunity, and removal of immune complexes and apoptotic cells. Investigate the difference between classical and other methods and the mannose-lectin complement cascade [Walport (2001), A / \ Eng / · 丄 Afed · 344, 1058 ~ 1066]. Classical complement cascade leads to lysis of bacterial cells

Printed by the Intellectual Property Bureau of the Ministry of Economics, R Industrial Consumer Cooperatives • 3- Printed by the Intellectual Property Bureau of the Ministry of Economic Affairs, Consumer Cooperatives 200411179 A7 B7 V. Description of the Invention (2) and the antibody that Clq starts and binds to the surface of the sprint or PCh CRP performs coordination. C-reactive protein is often used as a marker and, in addition, it is used to predict coronary artery disease that most often causes death in developing countries [Rifai and Ridker (2001), CAewiXr, 47, 403 ~ 411]. According to the latest research [Jia 丨 al et al. (2001), Circiz / tii / o / i 103, 1933 ~ 1935] it is considered that when CRP concentration is reduced or the function of CRP-mediated is blocked, for example, complement is activated by binding to CI q , Can effectively prevent and treat coronary artery disease. Therefore, firstly, a simple method for measuring the concentration of CRP and / or Clq in plasma and secondly, the identification of substances acting on the interaction of CRP and / or Clq with other components, especially the controlled interaction between CRP and C1 q Effect, that is, in an agitated or inhibited state. CRP and Clq binding assays have been described, for example, in Jiang et al. (1991), J. Immunol. 146, 2324 ~ 2330 and Agrawal et al. (200i), J, Jmmunol L 166, 3998 ~ 4004. All enzyme immunoassays (ELIS Α) were used. Both are very time consuming and consumable, and are a multi-phase, uncoordinated approach. Therefore, it usually requires immobilizing the reactants into a solid phase before proceeding. In addition, it needs to be approved by China National Standard (CNS > A4 size (210x297 mm))

200411179 A7 B7 V. Description of the invention (3) Many cleaning steps

Lebdue et al. (1998) ^ 4/1/1 · C / 7 / t. 35, 745 ~ 753 once described a method for measuring CRP immunoturbidity. In Jiedong, the sample and the anti- -CRP antibody co-culture. The main disadvantages of this method are the turbidimeter and the extremely large number of samples. Therefore, the object of the present invention is to provide a simple method for measuring the concentration of CRP or Clq in plasma, and it can further identify substances acting on the interaction of CRP and / or Clq with other components, especially under the control of the excited or inhibited state The interaction between CRP and Clq, and this method has the advantages of being more sensitive, cost saving, material saving, simpler and / or faster than the previous technology. 6 The purpose of the present invention is achieved by measuring two directly bound components Or via a binding reaction of one or more components bound to CRP or Clq, comprising the following steps: printed by the Shelley Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs (a) starting with the addition of a solution containing CRP or Clq; (b ) Adding at least one donor component or a group of components containing at least one donor component to the CRP or Clq-containing solution, the donor component containing at least one or a group of compounds (donor group) that can emit a signal after the light source is excited, and The donor component can be directly bound to or through one or more of the group components ___ · 5- ___ This paper size applies to the National Standard B (CNS) A4 (210 X 297 mm) 200411179 A7 B7 V. Description of the invention (4) Combination of four components to CRP or Clq; (c) At least one receptor component or a group of components containing at least one receptor component is added to the CRP or Clq-containing solution, the The receptor component contains at least one or a group of compounds (receptor group) capable of receiving and transmitting electromagnetic wave signals emitted according to (b) the compound, and the receptor component can be directly bound to or via one or more components of the group component To CRP or Clq; (d) stimulate at least one or a group of compounds based on (b) (donor group) with a light source; (e) detect the emissions of at least one or a group of compounds based on (c) (acceptor group) The binding reaction was measured by electromagnetic waves. Here, it is preferable that the electromagnetic waves emitted according to (c) are radiated with fluorescent light. The light source of (d) may be, for example, a laser or helium light source or a halogen lamp. The electromagnetic wave according to (e) can be detected using, for example, a photomultiplier. In the present invention, a polypeptide or a polypeptide-containing component is preferably used. In a preferred embodiment of the present invention, at least one component thereof is a single or multiple antibodies. If a group of ingredients is used, the ingredients are preferably capable of binding to CRP or Clq or mutual spatial succession. The combination is a series of binding reactions, an example of which is continuous —6 — This paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm)

Printed by Shelley Consumer Cooperative, Intellectual Property Bureau, Ministry of Economic Affairs, 200411179 A7 V. Antibody binding (5) of invention description (primary antibody, secondary antibody, etc. here according to 0>) and (c) Components that directly bind to CRP or Clq It is preferred to directly bind to a specific binding region or to a specific epitope of CRP or ciq. There are different binding sites depending on (b) the components bound to CRP or Clq and (c) the components bound to CRP or Clq. In another preferred embodiment of the method of the present invention, one of the components directly bound to CRP or ciq is a natural binding substance, which is particularly favored with ciq when it is CRP, and vice versa. If the donor or acceptor is directly bound to CRP or C1q ', the donor or acceptor component at this time is Clq or CRP containing a donor or acceptor group. When a group of components capable of forming a binding series is used, Clq or CRP is the first component bound to CRP or C1q, whereby it can bind a donor or acceptor component to CRP or Clq. The remaining components in this group are preferably antibody molecules. Therefore, for example, when starting to add a solution containing CRP, the donor or acceptor component itself in this specific example contains C1 q, or the donor or acceptor component is bound to CRP via C1 q. In this case, the donor or acceptor component may contain an anti-Clq antibody or a secondary antibody capable of binding to the anti-Clq antibody. Therefore, except for those containing anti-CRP antibodies or antibodies that can bind to anti-CRP antibodies or a paper size, Zhongyuan National Standards (CNS > A4 specifications (210 X 297 mm))

Printed by Shelley Consumer Cooperatives, Intellectual Property Bureau, Ministry of Economic Affairs, 200411179 A7 V. Description of the Invention (6) Other components (acceptor or donor components) other than the secondary antibodies can directly counter the latter. In a preferred embodiment, at least one or a group of compounds according to (b) (donor group) and / or (c) (acceptor group) are located on a particle. 'The preferred average diameter of the particle is between Between 15 and 250 nanometers, it is best at about 200 nanometers. Therefore, the donor or acceptor component contains the particles, which in this example are made of a polymeric material. The invention is a high-throughput screening (HTS) method, which can be completed in a single step at a uniform concentration. In a preferred embodiment, the method of the present invention is performed according to the principle of “mixing and measuring”, and the measurement results are quite time-saving and highly accurate. The printing method of the present invention can be measured by the Consumer Cooperative of Intellectual Property Bureau of the Ministry of Economy CRP bound to Clq, so it is possible to identify substances that affect the binding, and the concentration of CRP or Clq in the composite medium, which takes less than 10% of the time compared to the previously described method. This novel method does not require fixing It forms a solid phase reactant, so its main feature is that it can detect two kinds of bound proteins in solution. In addition, it can measure proteins directly taken from biological sources without modifying CRP or Clq, such as combining a fluorophor ). Therefore, the advantage of this method is protein-8. This paper size is applicable to Chinese National Standard (CNS) A4 specifications (2〗 0 × 297 mm) 200411179 A7 B7 V. Description of invention (ο The quality can maintain its original state and avoid Denaturation occurs during immobilization and / or protein modification. Another advantage of the method of the present invention compared with conventional methods is that the sample volume can be greatly reduced. It A sample volume of less than 10 microliters can be used, thus reducing the cost of materials. In addition, it can use highly diluted solutions to make the CRP or Clq volatility less than 1 nanomole, or even less than 100 picomoles ( PM). It is also a very sensitive method. As mentioned above, it is the first method to screen high-throughput substances that affect complement activation when CRP binds to Clq. In a preferred embodiment of the present invention For example, the signal can be transmitted from at least one or a group of compounds according to (a) (donor group) to at least one or a group of compounds according to (b) (acceptor group) via a single oxygen. In this case, the comparison The donor group of plutonium contains compounds that can be converted from dilactate to monooxygen under the printing of the Shellfish Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economics, and the preferred acceptor group contains at least one first compound A compound and a second compound that absorbs energy non-radiatively and fluoresceively stimulates by monooxygen. The formed monooxygen can diffuse from the donor group to the acceptor group and react with the existing chemiluminescent substance. The released energy is transferred to the fluorophore, and the photomultiplier can be used to detect the paper size of the paper. The standard boots of the 0 team NS) A4 specifications (× 297 mm) 200411179 A7 B7 Five Intellectual Property Bureau of the Ministry of Economic Affairs A description of the invention printed by Shelley Consumer Cooperative (0). Fluorescence emitted. Since monooxygen is in an unstable state and will decay in aqueous solution, the preprocessing of the detectable signal is to make the space close to the donor and acceptor microspheres. ◊Due to the decay time of monooxygen, energy cannot be effectively transferred by monooxygen at a long distance, so the binding component used in this method when binding occurs is to select a donor and acceptor group with a distance of less than 200 nanometers. The optimal donor group and acceptor group in this specific example are located on the particles, and the preferred average diameter of the particles is between 150 and 250 nanometers, and the largest is about 200 nanometers. The comparative particles used here are those which enable the final concentration of the donor-carrying particles to be 1 to 40 µg / ml, and the final concentration of the acceptor-carrying particles to be 1 to 80 µg / ml. The binding capacity of the particles per microgram / ml can be, for example, from about 0.1 nanomolar to 1 nanomolar. The best particles for this method are Alpha-Screen-Beads using Perkin Emma Life Sciences. In this specific example, the donor group is stimulated with laser radiation at a wavelength of 680 nanometers, and the radiation of the acceptor group is detected at a wavelength between 520 and 600 nanometers. Therefore, the method in this case is to first add donor and acceptor microspheres that carry the donor and acceptor population. Then, the component enabling binding to itself or to CRP or Clq via further components is bound to the donor and acceptor microspheres. As mentioned above, the composition can be -10. This paper size applies the Chinese National Standard (CNS) A4 specification (210x297 mm) 200411179 A7 B7 V. Description of the invention (9)

Clq or CRP may be an anti-CRP or anti-Clq antibody or another secondary antibody that directly fights the anti-CRP or anti-Clq antibody. In yet another preferred embodiment of the method of the present invention, the signal is transmitted from at least one or a group of compounds according to (b) (donor group) via a non-radioactive energy transmission method, and more particularly via a fluorescence resonance energy transmission method. To at least one or a group of compounds according to (b) (receptor group). In an example of the method, at least one or a group of compounds according to (a) (donor group) may include a sulfonium salt-containing compound, and at least one or a group of compounds according to (b) (acceptor group) may contain isophyllin. Protein (allophycocyanine) method [Grepin et al. (2000), D / scovery Tbfifay, 5, 212]. Alternatively, the donor and acceptor group may be dyes suitable for non-radiative energy transmission ^ It may further utilize all compounds known to those skilled in the art that are suitable for non-radiative energy transmission, especially fluorescence resonance energy transmission [See, for example, Pope et al. (1999), Drug jD / scover less 4,350]. In this specific example, since energy transmission cannot be performed at a relatively long distance, it is preferable to select a binding component using a donor and acceptor group with a distance of less than 10 nanometers after binding. In a preferred embodiment of the present invention, the method of the present invention is measurable. -11- The paper size is applicable to China National Standard (CNS) A4 (210 X 297 mm).

Printed by the Intellectual Property Bureau of the Ministry of Economic Affairs, Consumer Cooperatives, 200411179 A7 B7 Printed by the Intellectual Property Bureau of the Ministry of Economic Affairs, Printed by the Shellfish Consumer Cooperatives. 5. Description of the Invention (H >) CRP or Clq concentration containing CRP or Clq solution, which is more gold Plasma or serum or plasma or serum released in an appropriate physiological buffer or water. The method can be used, for example, for diagnostic purposes', especially to determine the state of organ inflammation and / or to determine the risk of cardiac arrest and / or stroke. Therefore, the present invention relates to an analysis system capable of high-throughput screening (HTS-capable), which can measure the binding reaction between CRP or Clq and components bound to CRP or Clq, and includes the following components: (a) at least one Donor component or a group of components containing at least one donor component, which is a compound (donor group) containing at least one or a group of compounds capable of emitting a signal upon activation of a light source, and the donor component can be directly or through one or A plurality of components from the group of components are bound to CRP or Clq; (b) at least one receptor component or a group of components containing at least one receptor component, the receptor component being containing at least one or a group capable of receiving and transmitting according to (b) A component of one or a group of compounds from which a radio wave signal is emitted by the compound, and the acceptor component can bind to CRP or Clq directly or via one or more components from the group. Here are the preferred high-throughput screening analysis systems: -12- This paper size is applicable to the 0 National Standard (CNS) A4 specification (210 X 297 mm)

200411179 A7 B7 V. Other ingredients of the description of the invention (η): (c) serum or plasma, or blood diluted with physiological buffer or water; (d) a light source that stimulates at least one or a group of compounds according to (a); ( e) A detection system based on the electromagnetic waves emitted by (b). In another specific example of the present invention, in order to observe the effect of the test substance on the binding reaction, before step (a) and / or step (b) and / or step (C) and / or step (d ) Before adding at least one test substance. According to this method, the substance acting on the interaction between CRP or Clq and a conjugate can be measured, especially the substance acting on the interaction between CRP and Clq in the mode of inhibition or activation. In order to determine whether a substance has the required In this high-throughput screening method, a database for screening test substances is preferred. Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs. 'The present invention relates to an analysis system for comba flux screening, which can measure the interaction between CRP or ciq and components bound to CRP or C1 q in the model. Activating substance, which includes the following components: (a) at least one donor component or a group of components containing | at least one donor component, so that the donor component contains at least one or a t group-13- This paper is applicable to the country of China Standard (CNS) A4 specification (210 x 297 mm) 200411179 A7 V. Description of the invention (I2) A compound (donor group) capable of emitting a beacon after the light source is activated, and the donor component can be directly or through one or more The component from the group component binds to CRP or Clq; (b) at least one receptor component or a group of components containing at least one receptor component, the receptor component is containing at least one or a group capable of receiving and transmitting a compound according to (b) A component of one or a group of compounds of the emitted electromagnetic wave signal, and the acceptor component is capable of binding to CRP or Clq directly or via one or more components from the group component; (c) at least one Test compounds. Here, the high-throughput screening analysis system preferably contains the following other components: (d) a light source that stimulates at least one or a group of compounds according to (a); (sentence testing based on electromagnetic waves emitted by (b) A kind of detection 4

Printing system of the Consumer Cooperative of the Intellectual Property Office of the Ministry of Economic Affairs; (f) A solution containing CRP or C1 q. The high-throughput screening analysis system used in the present invention preferably performs the method of the present invention described above. Therefore, the parts and components of a specific specific example in the analysis system correspond to the present invention described above.

200411179 A7 B7 V. Description of the invention (η) The users in the specific examples of the method. Figure 1 is an illustration of the method described in the illustrative example. Here, the donor component is an anti-Clq antibody, and the donor population is located on a particle bound to the anti-Clq antibody. In this example, its donor component can be bound to CRP via Clq. The receptor component in this example is an anti-rabbit secondary antibody, and its receptor group is located on a particle bound to the anti-rabbit secondary antibody. In this example, its receptor component can be bound to CRP via an anti-CRP antibody from a rabbit. Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs This paper is in accordance with Chinese National Standard (CNS) A4 (210X 297 mm) 200411179 A7 B7 V. Description of the invention (ι〇 光

Member of the Intellectual Property Bureau of the Ministry of Economic Affairs, X Consumer Cooperative, prints a specific example: a binding analysis test to determine the binding of CRP to Clq. This test uses the Compal Biosciences company ’s Alpha screening detective set of red (Alphascreen Detection Kit), the donor microsphere contains a compound that can be converted into trioxin after being excited by a certain wavelength, and the acceptor microsphere contains a compound that can be stimulated by monooxygen and also can receive non-radiative energy transmission _ 16_ This paper size applies to China National Standard (CNS) A4 (210 X 297 mm) 200411179 A7 B7 Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs i5) compounds. All of these compounds are hosted in a hydrocolloid matrix. The microspheres used have been pre-coated by the manufacturer, ie the donor microspheres are streptavidin and the acceptor microspheres are anti-rabbit antibodies. Table 1: Overview of materials used. Reagent / Plate molecular weight or concentration supplier, catalog number micro-quantitative disk, 384iQPS, white Greiner 784075 〇 Reactive protein, human, recombinant from E. coli 1 mg / ml, 115 kd (5mer) Calbiochem 236608 Clq ^ human 1 mg / ml, 410kd Calbiochem 204S76 anti_011 >, rabbit 3.57 mg / ml, 165kd Calbiochem 235752 Alfa immunoglobulin detection kit (protein A) Padcard BioScience 6760617 Alfa rabbit immunoglobulin debt Test set Packard BioScience 6760607 anti-Clq, goat multiple strains Jinqing Calbiochem 234390 antifungal avidin protein multiple strains serum Sigma S639G enzyme-linked sulfur biomarker set Pieree 21430 sodium carbonate 58 44 g / Merck 101540 gas Calcium (CaCl2x 2) 407.02g / Merck 102382 Tris (hydroxymethyl) -aminomethane (Tris) 157.6g / Merck Merck 108219 Bovine serum albumin (BSA) Sigma P788S Base 329 · 7 g / mol Sigma P0378 Phosphate buffered saline (without Mg2 + and Ca2 +) (PBS) Gibco BRL 14200-067 -17- This paper Standards apply to Chinese National Standard B (CNS > A4 Regulations (210 x 297 Male®)

200411179 A7 B7 V. Description of the invention (16)

Slide-A-Lyzer Mini Dialysis Tube, 10,000 MWCO Pierce P.69570 Merck KgaA, 1.02931 Riedel deHaen, 32250 Pluronic F-68 10% strength solution Sigma, P5556 Hydrochloric acid 1 mole concentration Merck, 109075 Sodium hydroxide solution 1 Molar concentration Merck, 109137 • Preparation of pure water reaction buffer Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs The following is the reaction buffer used in the Alpha screening detection system: 20 millimoles Tris -Hydrochloric acid, ρΗ7 · 2; 15G 亳 Moral sodium vaporization; 5 亳 Moral calcium gasification; 1 亳 Moral phosphorylated choline; 0.1% BSA 〇Tris-HCl buffer solution preparation method is 3.15 Grams of Tris-hydrochloric acid and 8.77 grams of sodium carbonate were dissolved in water, the pH was adjusted to 7.2 with 1 mole of sodium hydroxide, and then water was added to 1 liter. Store the buffer at room temperature. The reaction agricultural solution was prepared by adding 36.7 g of calcium carbonate, 12.9 mg of phosphorylated choline and 50 mg of BSA to 50 ml of Tris-hydrochloric acid buffer. Analysis program

For this analysis, 2 microliters of donor microspheres / anti-CRP solution was added, followed by 2 microliters of C1 q, 2 microliters of CRP, and 2 microliters of acceptor microspheres / anti-CRP. The final concentration of the final product __- 18- _ This paper size applies to the Chinese National Standard (CNS) A4 specification (2) 0x297 mm. 2004Π179 Α7 Β7 V. Description of the invention (17): CRP: 1 nanomole; Clq: 10 nanomoles; anti-CRP: 7.3 nanomoles; anti-Clq: 1: 1,500; donor microspheres: 20 micrograms / ml; acceptor microspheres: 40 micrograms / ml. In the negative control group, the reaction buffer solution was used instead of the CRP solution, the Clq solution, or both. After 2 hours of incubation at room temperature, the data were interpreted with an AlphaQuest analyzer. Results The measured radiation intensities at 1 nanomole CRP concentration and 10 nanomole c 1 q concentration are as follows (record their photomultiplier counts): No CRP and No Clq: 952 Order 1 1 With CRP and No Clq: 1,255 1 1 1 1 a Without CRP and with Clq: 1,114 1 1 4 With CRP and with Clq ·· 80,376 1 1 • It can be seen that the value of the 'negative control group' is significantly lower, and can be obtained when the solution containing C1 q and CRP is mixed Received a strong letter printed by the Intellectual Property Bureau of the Ministry of Economic Affairs employee consumer cooperatives. __ -19- This paper size applies to China National Standard (CNS) A4 (210 X 297 mm)

Claims (1)

200411179 6. Scope of Patent Application 1. A method for determining the binding reaction between two directly bound components or via one or more components bound to a CRP component, comprising the following steps: (a) adding a solution containing CRP first; (b) At least one donor component or a group of components containing at least one donor component is added to the CRP-containing solution, the donor component contains at least one or a group of compounds (donor group) that can emit a signal after the light source is excited, and the donor The component may be directly bound to the CRP or via one or more components of the group component; (c) at least one receptor component or a group of components containing at least one receptor component is added to the CRP-containing solution, the receptor component containing at least One or a group of compounds (receptor group) capable of receiving and transmitting electromagnetic wave signals emitted according to (b), and the receptor component can be directly bound to or through one or more components of the group component to the CRP; Ministry of Economic Affairs Printed by the Intellectual Property Bureau's Consumer Cooperative (d) using light sources to stimulate at least one or a group of compounds under (b) (donor group); (e) The binding reaction is determined by detecting at least one or a group of electromagnetic waves emitted by the compounds of (c) (receptor group). 2 · —A method for determining the binding reaction between two directly bound components or via one or more components bound to the Clq component, which is -20-This paper size is applicable to China National Standard (CNS) M specification (210 X 297 mm) 200411179 A8B8C8D8 Sixth, the Intellectual Property Bureau of the Ministry of Economy's Intellectual Property Cooperative printed a patent application covering the following steps: (a) Start by adding a solution containing Clq; (b) Add at least one donor component or at least one donor into the Clq-containing solution A group of components of the component, the donor component containing at least one or a group of compounds (donor group) that emits a signal upon activation of a light source, and the donor component may be directly bound to or via one or more components of the group component C 1 q; (c) At least one receptor component or a group of components containing at least one receptor component is added to the Clq-containing solution, and the receptor component contains at least one or a group capable of receiving and transmitting the compounds emitted according to (b) Electromagnetic signal compounds (receptor group), and the receptor component can be directly bound to or via one or more components of the group component Clq; (d) stimulate at least one or a group of compounds based on (b) (donor group) with a light source; (e) measure by detecting electromagnetic waves emitted by at least one or a group of compounds based on (C) (acceptor group) Its binding reaction. 3. The method according to item 1 or 2 of the scope of patent application, wherein the electromagnetic wave is fluorescent light. 4 · If the method of applying for the scope of patent] or item 2 is used, its -21-This paper size is applicable to the Chinese National Standard (CNS) A4 Regulation (210x297). 200411179 Six House Fan Li specially requested A8B8C8D8 t according to (d) The first source is laser or light. 5. The method according to item 1 or 2 of the patent application park, wherein the detection according to (e) is the use of a photomultiplier. 6 · If you apply for a patent, please! The method of item, wherein the components of a group of components according to (b) and / or (c) may be combined to the CRP or combined with each other in space. 7. The method according to item 2 of the scope of patent application, wherein the components in a group of components according to (b) and / or (c) can be combined to Clq or mutually combined in space. 8. The method according to any one of claims 1 to 7, wherein the component is a polypeptide or comprises a polypeptide. 9. The method according to item 8 of the patent application park, wherein at least one component is an antibody. 10. The method according to item 9 of the patent application park, wherein the antibody is a single or multiple antibodies. Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs. 11. If the method in the first scope of the patent application is applied, at least one of the ingredients is a natural binding substance of CRP. 12-The method according to item u of the patent application, wherein the natural binding substance of CRP is Clq. 13 · If you apply for the method of item 12 in the scope of patent application, where __- 22-This paper size is applicable to Chinese national standard (CNS > A4 specification (210 x 297 mm) 200411179 A8 B8 C8 ——————— I ~ 1 jX _ ° VI. Scope of Patent Application-This group contains Clq and an anti-ciq antibody according to item 1 (a > or 1 (b) of the scope of patent application. 14 · Method of applying item 2 of the patent scope At least one of the components is a natural binding substance of Clq. 15. The method of claim 14 in the scope of patent application, wherein the natural binding substance is CRP. 16 The method of claim 15 in the scope of patent application, wherein the group is based on a patent application The components in the scope of item 2 (a) or 2 (b) contain CRP and an anti-CRP antibody. 17 · If the method of applying for the scope of the patent application is in the scope of [2] or [2], the signal is from at least one or a group according to (b) (Donor group) The compounds are transferred to at least one or a group of compounds according to (b) (acceptor group) through non-radioactive energy transmission. Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 18 · 17 Item's method, where the The radioactive energy transfer method is a fluorescence resonance energy transmission method. 19 · If the method of the scope of patent application or item 2 is used, the signal is transmitted through monooxygen. 20 · If the method of the scope of patent application item 19, At least one or a group of them is in accordance with the scope of patent application No. 1 (b) or 2 (b) ___-23 -_ This paper size is applicable to the Chinese National Standard (CNS) A4 specification (210 x 297 public *) 200411179 Compounds (donor group) under the laser excitation, and at least one or a group of compounds according to item 1 (b) or 2 (b) of the scope of the patent application ( Receptor group) contains at least one first compound that can be stimulated by monooxygen and a second compound that non-radioactively absorbs and fluoresces the absorbed energy that is stimulated by monooxygen. 21. The method according to item 20 of the patent application, wherein The donor group and / or acceptor group are located on the particle. 22 The method according to item 21 of the patent application, wherein the average diameter of the particle is about 200 nanometers. Any one of Method for determining the concentration of a CRp-containing solution of unknown CRp content. 24. The method according to any one of the claims 1 to 22, which is used for determining the concentration of a Clq-containing solution of unknown Clq content. 25. For example, the method of claim 23 or 24, wherein the solution is serum, plasma, or J & L slurry or J & L serum diluted with an appropriate physiological buffer or water. 26. The method according to the scope of application for patent No. 25, which is a method for measuring the state of inflammation of an organ and / or measuring the stop of its heartbeat and /-24-200411179 A8 B8 C8 --------------- -D8_ Six, patent application method-or stroke risk method. 27. The method according to any one of the aforementioned patent application parks, which is a high-throughput screening method that is performed in a single step at a uniform concentration. 28. The method according to item 1 of the patent application scope, which comprises adding at least one test substance before step (a) and / or step (b) and / or step (c) and / or step (d) In order to observe the effect of the test substance on the binding reaction between CRP and CRP-binding components. 29. The method according to item 2 of the patent application, which comprises adding at least one test substance before step (a) and / or step (b) and / or step (c) and / or step (d). Observe the effect of the test substance on the binding reaction between Clq and Clq-binding components. 0 Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 30. If the method of the scope of patent application No. 28 or 29, it is used to determine A substance that acts on a binding reaction in an activated mode. 31. An analysis system capable of high-throughput screening (11 d 8-〇3 卩 & 1) 16), which can measure the binding reaction between CRP and the components bound to CRP, including the following components: (a ) At least one donor component or a group containing at least one donor component -25-This paper size is applicable to Chinese National Standard (CNS) A4 (210x297 mm) 200411179 A8B8C8D8 Sixth Ministry of Economic Affairs Intellectual Property Bureau Employees Consumer Cooperatives Scope of Patent Application The donor component is a compound (donor group) containing at least one or a group of compounds capable of emitting a signal upon activation of a light source, and the donor component can be bound to the CRP directly or via one or more components from the group. (b) at least one receptor component or a group of components containing at least one receptor component, which is a component containing at least one or a group of compounds or a group of compounds capable of receiving and emitting electromagnetic wave signals emitted according to (b) a compound And the receptor component can bind to CRP directly or via one or more components from the group component. 32 · —A high-throughput screening analysis system that can measure the binding reaction between Clq and components bound to Clq, including the following components: (a) at least one donor component or a group containing at least one donor component The donor component is a compound (donor group) containing at least one or-group that can emit a signal after the light source is activated, and the donor component can be bound to Clq directly or via one or more components from the group component (b) at least one receptor component or a group of components containing at least one receptor component, which is a component containing at least one or a group of compounds or a group of compounds capable of receiving and transmitting electromagnetic wave signals emitted according to (b) a compound ， And this subject -26-This paper size applies Chinese National Standard (CNS) A4 regulations (210 X 297 public love) 200411179 A8B8CSD8 Six, patent application body composition can be directly or through one or more ingredients from this group of ingredients Binding to Clq. 33. The high-throughput screening and analysis system according to the scope of patent application No. 31 or 32, which includes the following additional components: serum or plasma, or serum or plasma diluted with physiological buffer or water; (d) at least one kind of activation Or a light source of a group of compounds based on (a) (donor group), (e) a detection system that detects electromagnetic waves emitted based on (b). 34 · —A high-throughput screening analysis system for determining activated substances acting on the interaction between CRP and components bound to CRP in the regulation mode, which includes the following components: Consumption by employees of the Intellectual Property Bureau of the Ministry of Economic Affairs The cooperative prints (a) at least one donor component or a group of components containing at least one donor component, which is a compound (donor group) containing at least one or a compound that can emit a signal after being narrated by a light source. The donor component can receive and emit electromagnetic wave signals emitted by one or a group of compounds according to (a), and conversely, the donor component can be bound to the CRP directly or via one or more components from the group component; -27-this paper Standards are applicable to Chinese National Standards (CNS > A4 specifications (210 X 297 mm) A8B8C8D8 200411179 Printed by the Consumers ’Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs Scope of patent application (b) At least one receptor component or a group of at least one receptor component Component, the receptor component contains at least one or a group of compounds capable of receiving and transmitting according to (b) one or a group of compounds (receptor group) A component of the electromagnetic wave signal, and the acceptor component can bind to the CRP directly or via one or more components from the group component; (c) at least one test compound. 35.-a high-throughput screening analysis system, An activating substance for determining the interaction between Clq and a component bound to Clq in the regulation mode, which includes the following components: (a) at least one donor component or a group of components containing at least one donor component; A body composition is a compound (donor group) containing at least one or a group of compounds capable of emitting a signal upon activation of a light source, the donor component being capable of receiving and transmitting electromagnetic wave signals emitted by one or a group of compounds of (a), and the donor The component is capable of binding to Clq directly or via one or more components from the group of components; (b) at least one receptor component or a group of components containing at least one receptor component, the receptor component comprising at least one or a group capable of receiving and A component that emits an electromagnetic wave signal emitted by one or a group of compounds (receptor group) according to (b), and -28-this paper standard applies Chinese National Standard (CNS) A4 specification (210 X 297 mm) Printed by the Consumers' Cooperative of Intellectual Property Bureau of the Ministry of Economic Affairs 200411179 VI. Application scope of the patent The receptor component can be combined directly or via one or more components from the group. Clq; (c) at least one test compound 36. The high-throughput screening analysis system according to item 34 or 35 of the scope of patent application, which includes the following additional components: (d) stimulates at least one or a group of components according to (a) The light source of the compound (donor group); (e) A detection system for detecting electromagnetic waves emitted according to (b) 0 -29-This paper applies the national standard (CNS) A4 specification (210 X 297 mm) ) 200411179 (1), this _representative drawing is the first (none) (two), the component representative symbols of this representative illustration are simply explained: None If there is a chemical formula in this case, Jiangxi reveals the most capable of showing the 4 signs of invention. None Page 2-1