A kit for determining serum amyloid A, its preparation and application

Abstract

The present invention provides a kit for determining serum amyloid A, and its preparation and application. The kit of the present invention optimally screens the buffer systems, components and concentrations of inorganic salts in Reagent 1 and Reagent 2, particularly adds polyanions and DSS, and uses a quenching agent in the preparation process of anti-SAA antibody-coated latex particles. Through the optimal screening of these components, the prepared kit significantly improves the specific recognition and binding ability to serum amyloid A, and enhances performance indicators such as the sensitivity, linear range, anti-interference ability, stability and repeatability of the kit. Among them, in the linear range of 2 mg / L to 500 mg / L, the detected values have good linearity, accuracy and usability, thereby enhancing the comprehensive performance of the immunoassay of serum amyloid A.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemical immunoassay. First, it relates to a kit for determining serum amyloid A, and specifically relates to a preparation method of a kit for determining serum amyloid A and its application in biochemical immunoassay. Background Art

[0002] Serum amyloid A (SAA) is a polymorphic protein family encoded by multiple genes, the precursor of tissue amyloid A, and belongs to acute-phase reaction proteins. SAA in plasma participates in various physiological or pathological reactions in the body and has multiple functions, specifically including: binding to plasma high-density lipoprotein (HDL) to promote the reverse transport of cholesterol to the liver for metabolism; as an acute-phase reaction protein, it can recruit immune cells to the inflammatory site and participate in the inflammatory reaction; some studies have shown that SAA may also induce the expression of enzymes that degrade the extracellular matrix; in addition, the degradation products of SAA can deposit in different organs in the form of amyloid A fibrils, which is an important feature of the histological changes in chronic inflammation. Under normal conditions, SAA in the human body is mainly expressed by hepatocytes; when the body undergoes an acute-phase reaction, cytokines (such as IL-1, IL-6, TNF, etc.) will be released, and these factors will stimulate hepatocytes, macrophages, fibroblasts, and some adipocytes to express a large amount of acute-phase SAA, causing the plasma SAA concentration to increase sharply by 100 - 1000 times. Moreover, the half-life of acute-phase SAA is short, only about 50 minutes. It is a very sensitive acute-phase reaction protein and an important clinical infection and inflammation indicator. Although both SAA and CRP are acute-phase reaction proteins, the high sensitivity and short half-life of SAA may be superior to CRP in some aspects and can be used to evaluate acute-phase reactions; studies have also found that cells in tumor cells and atherosclerotic plaques can also synthesize acute-phase SAA, which may be related to its participation in amyloidosis and the formation of atherosclerosis. As an early indicator of organ transplant rejection and various tumors, SAA can be used for the prognosis evaluation of tumors, atherosclerosis, and cardiovascular events; in addition, SAA can increase in the early stage of both bacterial and viral infections, and combined with CRP, it helps to identify the type of infection and evaluate the antibacterial efficacy.

[0003] At present, the existing methods for the determination of serum SAA include colloidal gold method (detection range 5 - 500 mg / L, low precision), immunoprecipitation method (complicated operation), magnetic particle chemiluminescence method (high determination cost), and latex immunoturbidimetry. Although the existing latex immunoturbidimetry for the determination of SAA has the advantages of high accuracy / sensitivity / specificity, good stability, convenience, speed, and low cost, there are still certain defects in using latex immunoturbidimetry to determine serum SAA. Especially in the preparation process of coating SAA antibody on latex particles, the sensitized latex particles obtained by physical adsorption method have poor stability and the antibody is easy to fall off; while the commonly used chemical coupling method, such as EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) method, although it can improve the stability, the steps are cumbersome, and in the preparation process, in order to remove uncrosslinked antibodies and impurities and ensure the purity of the reagent, generally centrifugation and then ultrasonic dispersion steps are required, and this treatment step is likely to cause antibody inactivation or detachment; therefore, due to the instability of SAA antibody coating in the preparation process, the accuracy and stability of the determination results will be affected. Generally, surfactants, protein protectants and other additives are used to improve the stability, but the additives also need to comprehensively consider the compatibility with the formulation components and the adverse effects on antibody activity, etc. In addition, in actual clinical applications, the interference of substances such as chylomicrons in the sample will also lead to inaccurate test results. Therefore, it is necessary to provide a kit for the determination of SAA by latex immunoturbidimetry with high accuracy and high reliability and its preparation method to solve these problems. Summary of the Invention

[0004] In order to overcome the above problems, the present invention provides a kit for the determination of serum amyloid A with significantly improved performance indicators such as sensitivity, linear range, anti-interference ability, stability, and repeatability after component optimization, and a preparation method and application of the kit for the determination of serum amyloid A.

[0005] To achieve the above invention purposes, the present invention is realized through the following technical solutions:

[0006] In a first aspect, the present invention provides a reagent composition for determining serum amyloid A by latex enhanced immunoturbidimetry, comprising reagent 1 and reagent 2 which are independent of each other. The pH of reagent 1 is 6.5 - 8.5 and it contains a first buffer, inorganic salts, a stabilizer, a polyanion, a preservative, a first surfactant, and polyethylene glycol 6000; the pH of reagent 2 is 6.0 - 8.0 and it contains a second buffer, inorganic salts, a stabilizer, a preservative, a second surfactant, DSS, and latex particles coated with anti-SAA antibody. Among them, the main components of the first buffer and the second buffer are different, and the final concentration of the inorganic salts in both reagent 1 and reagent 2 is 2% - 15%; the concentration of the latex particles coated with anti-SAA antibody in reagent 2 is 0.5 - 2.0 mg / mL, and the particle size of the latex particles used to prepare the latex particles coated with anti-SAA antibody is 50 - 180 nm. During the preparation of the latex particles coated with anti-SAA antibody, a thiol reducing agent is used as a quenching agent to terminate the catalytic activity of unreacted EDC.

[0007] According to a preferred embodiment of the present invention, in the reagent composition, reagent 2 may not include a stabilizer.

[0008] According to a preferred embodiment of the present invention, in the reagent composition, both the first buffer and the second buffer are selected from any one or two of Tris-HCl buffer, phosphate buffer, glycine buffer, HEPES buffer, and aminoacetic acid buffer at a concentration of 15 - 280 mmol / L; the inorganic salts are selected from any one or two of calcium chloride, sodium chloride, and magnesium chloride; the stabilizer is selected from any one or two of 0.1% - 0.5% bovine serum albumin, mannitol, gelatin, and casein; the polyanion is selected from any one of polyanion cellulose, polymaleic acid, and chondroitin sulfate at a concentration of 0.3 - 0.9 mmol / L; the preservative is selected from any one or two of sodium azide, benzoic acid, and ProClin-300; and the final concentration of the preservative in reagent 1 is 0.5% - 3%, and the final concentration of the preservative in reagent 2 is 1% - 5%; the first surfactant is selected from any one or two of Tween 20, Triton X-100, SDS, and B66 at a concentration of 0.05% - 0.2%; the second surfactant is selected from any one or two of Tween 20, Triton X-100, and B66 at a concentration of 0.05% - 0.2%; the final concentration of polyethylene glycol 6000 in reagent 1 is 4% - 8%; the final concentration of DSS in reagent 2 is 50 - 200 mg / L.

[0009] According to a preferred embodiment of the present invention, in the reagent composition, the first buffer is a phosphate buffer; the second buffer is a glycine buffer; the inorganic salts at least include sodium chloride; the stabilizer is bovine serum albumin; and the polyanion is polyanion cellulose.

[0010] Preferably, the concentration of the phosphate buffer in Reagent 1 is 50 mmol / L, and the buffering capacity for pH adjustment is in the range of 6.5 - 8.5.

[0011] Preferably, the inorganic salts in Reagent 1 are 8.5% sodium chloride and 1.5% calcium chloride, which can effectively prevent the interference of inorganic salts with the binding of the enzyme and the substrate, and reduce the viscosity of polyanions.

[0012] Preferably, the stabilizer in both Reagent 1 and Reagent 2 is 0.2% bovine serum albumin.

[0013] Preferably, the preservative in both Reagent 1 and Reagent 2 is 1% sodium azide.

[0014] Preferably, the polyanion in Reagent 1 is 0.5 mmol / L polyanionic cellulose, which can not affect the stability of the reagent and can also reduce the interference of chylomicrons.

[0015] Preferably, the first surfactant in Reagent 1 is 0.1% Tween 20.

[0016] Preferably, the concentration of polyethylene glycol 6000 in Reagent 1 is 5%.

[0017] Preferably, the second buffer in Reagent 2 is 100 mmol / L glycine buffer, and the buffering capacity for pH adjustment is in the range of 6.0 - 8.0.

[0018] Preferably, the inorganic salt in Reagent 2 is 8% sodium chloride.

[0019] Preferably, the second surfactant in Reagent 2 is 0.15% Triton X - 100.

[0020] In a second aspect, the present invention provides a kit for determining serum amyloid A, which comprises the above reagent composition, and also comprises a quality control product and a calibration product. Both the quality control product and the calibration product contain at least two concentration levels of human recombinant amyloid A, and the buffer is a glycine buffer containing human serum.

[0021] According to a preferred embodiment of the present invention, in the kit for determining serum amyloid A, the quality control product contains at least two concentration levels of human recombinant amyloid A, and these two concentration levels are respectively: Level 1: 12 mg / L - 18 mg / L; Level 2: 48 mg / L - 72 mg / L; the calibration product contains at least five concentration levels of human recombinant amyloid A, and these five concentration levels are respectively: 10 mg / L, 60 mg / L, 150 mg / L, 300 mg / L, and 500 mg / L.

[0022] Thirdly, the present invention provides a method for preparing anti-SAA antibody-coated latex particles, comprising the following steps: S1: Suspend the latex particles in 10-20 mM MES buffer and adjust the pH to 5-7; S2: Add EDC and NHS and react at room temperature for 1 hour to catalyze the formation of amide bonds between the carboxyl groups on the surface of the latex particles and the amino groups on the antibody; S3: Quench EDC with a quenching agent at a concentration of 0.05-0.2 M to terminate the catalytic activity of unreacted EDC; S4: Adjust the pH of the solution to 7.2-7.5 to meet the requirements of subsequent antibody conjugation reactions; S5: Add an anti-SAA antibody equimolar to the latex particles and shake and seal the reaction at room temperature for 2 hours to obtain a crude solution containing anti-SAA antibody-coated latex particles; S6: Remove the supernatant from the crude solution containing anti-SAA antibody-coated latex particles by centrifugation or filtration, dilute the target conjugate after the reaction with 10 mM MES buffer, adjust the concentration of the latex particles to 10 g / L, adjust the pH to 6.5, dispense, and store at 4°C for later use.

[0023] According to a preferred embodiment of the present invention, in the method for preparing anti-SAA antibody-coated latex particles, in step S1: the diameter of the latex particles is 50-180 nm and the concentration is 60-100 mg / mL; in step S2: the concentration of EDC is 12-15 mM and the concentration of NHS is 35-50 mM; in step S3: the quenching agent is a thiol reducing agent, and either 2-mercaptoethanol or TCEP can be selected; in step S5: the mass concentration of the anti-SAA antibody equimolar to the latex particles is 1.5-2.5 mg / ml.

[0024] Fourthly, the present invention provides a method for preparing a kit for detecting serum amyloid A, which is used to prepare the above reagent composition. Among them, the preparation process of reagent 1 is as follows: Take the first buffer and add it to a container, and stir at a rotation speed of 200-400 rpm; then add the inorganic salt, stabilizer, preservative, polyanion, first surfactant and polyethylene glycol 6000 in reagent 1 component into the buffer in sequence according to the predetermined concentration ratio and mix well. Finally, adjust the pH of reagent 1 to 6.5-8.5 with a pH regulator, and then reagent 1 is prepared; the preparation process of reagent 2 is as follows: Take the anti-SAA antibody-coated latex particles prepared according to the above method for preparing anti-SAA antibody-coated latex particles, first dilute them with the second buffer to make the final concentration 0.5-2.0 mg / mL, and then add the inorganic salt, and / or stabilizer, second surfactant, preservative and DSS in reagent 2 component into the second buffer in sequence according to the preset concentration and mix well. Finally, adjust the pH to 6.0-8.0, and then reagent 2 is prepared.

[0025] In a fifth aspect, the present invention also claims the use of the above reagent composition, the serum amyloid A assay kit, the method for preparing anti-SAA antibody-coated latex particles, and the method for preparing the serum amyloid A assay kit in the preparation of latex reagent-related products.

[0026] The beneficial effects of the present invention are as follows:

[0027] The present invention provides a serum amyloid A assay kit. The serum amyloid A assay kit of the present invention uses the latex immunoturbidimetry method to measure the content of serum SAA. The principle is that human anti-SAA antibody is coated on latex microparticles. After an immune reaction with SAA in the specimen, aggregated particles are formed. At a certain wavelength, by measuring the turbidity formed by the aggregates, the content of the analyte in the specimen can be obtained corresponding to the standard curve. The present invention optimizes the buffer systems, the components and concentrations of inorganic salts in Reagent 1 and Reagent 2. For example, different buffer systems are selected for Reagent 1 and Reagent 2 respectively, and the concentration of inorganic salts is controlled within the range of 2% - 15%. The performance indicators of the kit preferably configured according to the present invention meet the minimum requirements of the kit performance review, and can be comparable or have little difference from the performance indicators of third-party reagents. Moreover, the sensitivity, anti-interference ability, stability, repeatability of the serum SAA kit prepared according to the present invention and the correlation with the detection of third-party reagents are all relatively excellent.

[0028] The polyanion, DSS, creatively added in the kit of the present invention, and the use of a quenching agent in the preparation process of anti-SAA antibody-coated latex particles further improve the performance of the serum SAA kit. In particular, the stability measurement values of the group with the simultaneous addition of polyanion and DSS are significantly better than those of the group without the addition of polyanion and without the addition of DSS, and at the same time, its stability measurement values are also significantly better than those of third-party reagents. Therefore, the creative addition of polyanion and DSS is beneficial to improving the stability of the kit. The use of a quenching agent in the preparation method of anti-SAA antibody-coated latex particles makes its repeatability measurement values better than those of the control group without the use of a quenching agent. Therefore, the present invention effectively improves the repeatability of reagent detection by using a quenching agent in the preparation process of anti-SAA antibody-coated latex particles.

[0029] The present invention provides a method for preparing a kit for determining serum amyloid A, which optimizes the five main components of buffer, electrolyte, stabilizer, surfactant, and preservative in Reagent 1 and Reagent 2. Moreover, a polyanion is creatively added to Reagent 1, and DSS is creatively added to Reagent 2. During the preparation of latex particles coated with anti-human serum amyloid A antibody, a quenching process is creatively introduced, which improves the stability of the chemical coupling between the latex particles and the anti-human serum amyloid A antibody. The result of layer-by-layer optimization also reduces the ability of chyle interference during the determination process. Finally, the kit prepared by this method has high sensitivity, specificity, and stability during the determination, and the linear range of the determination is further improved.

[0030] In Reagent 2, DSS is newly and creatively added. Here, DSS (4,4-Dimethyl-4-Silapentane-1-Sulfonic acid, 4,4-dimethyl-4-silapentane-1-sulfonic acid, or Disuccinimidyl subera, disuccinimidyl suberate), with the molecular formula: C16H20N2O8, molecular weight: 368.34, and CAS number: 68528-80-3, is generally non-toxic to humans and the environment and is relatively safe. It is often used as a bifunctional cross-linking agent, a standard substance for nuclear magnetic resonance hydrogen spectrum and carbon spectrum, or a pH indicator. As a cross-linking agent, it is often applied to the chemical cross-linking of intracellular proteins before cell lysis and immunoprecipitation. It does not lyse, is insoluble in water, soluble in organic solvents, and has membrane permeability, enabling cross-linking inside cells. The present invention creatively adds DSS to Reagent 2 of the serum SAA kit. Compared with the case where DSS is not used, it can effectively reduce the deviation of the measured value of the sample to be measured during the long-term storage of the kit, that is, adding DSS helps to improve the accuracy of the measured value of the kit, and effectively reduces the relative deviation of the measured value while ensuring the stability of the kit.

[0031] In summary, the present invention provides a kit for determining serum amyloid A, which optimizes the components such as buffer and surfactant in Reagent 1 and Reagent 2 and the preparation process of latex particles coated with anti-SAA antibody. The optimized kit for determining serum amyloid A can be successfully applied to the latex-enhanced immunoturbidimetric method for determining serum amyloid A, thereby overall improving the performance indicators of the kit such as sensitivity, linear range, anti-interference ability, stability, and repeatability. The detection value has good linearity, accuracy, and usability within the linear range of 2 mg / L to 500 mg / L, and it can be used on an automatic biochemical analyzer with a high degree of automation. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1This is the linear evaluation result graph of the serum amyloid A detection kit of the present invention. Among them, a represents Example 1, and b represents Example 3; the abscissa is the theoretical value, with the unit of mg / L, and the ordinate is the measured value, with the unit of mg / L.

[0033] Figure 2 This is the stability evaluation result graph of the serum amyloid A detection kit of the present invention. Among them, A represents Example 3, B represents a third-party reagent, and C represents Example 4; the abscissa is the storage time, with the whole month as a measurement unit, with the unit of month, and the ordinate is the concentration value of quantitative determination, with the unit of mg / L.

[0034] Figure 3 This is the correlation evaluation result graph of the serum amyloid A detection kit of the present invention. The abscissa represents the measured value of Example 3, with the unit of mg / L, and the ordinate represents the measured value of the third-party reagent, with the unit of mg / L. Detailed implementation mode

[0035] The exemplary embodiments of the present invention will be described in detail below with reference to the accompanying drawings, where the same or similar reference numerals represent the same concept. In the following detailed description, for the purpose of explanation, many specific details are set forth to provide a comprehensive understanding of the embodiments disclosed herein. In the following detailed description, for the purpose of explanation, many specific details are set forth to provide a comprehensive understanding of the embodiments disclosed herein. The following embodiments are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention.

[0036] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used can be obtained from commercial sources unless otherwise specified. Among them, human recombinant amyloid A (product number: 610070) was purchased from Medix Biochemica; anti-human amyloid A monoclonal antibody (product number: 100279) was purchased from Medix Biochemica; polyanionic cellulose (product number: P875778) was purchased from Shanghai Macklin Biochemical Co., Ltd.; DSS (product number: C426020010) was purchased from Sinopharm Chemical Reagent Co., Ltd.; latex particles were purchased from Jiehe Tai (Beijing) Biotechnology Co., Ltd.; the activators EDC (product number: N808856) and NHS (product number: N811124) were purchased from Shanghai Macklin Biochemical Co., Ltd.; the automatic biochemical analyzer was purchased from Hitachi, Japan, model 7180; the third-party reagent was the SAA assay kit (latex immunoturbidimetry method) purchased from Beijing Strong Biotechnologies Inc. However, obviously, one or more embodiments can also be implemented without these specific details. For those not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. The molecular biology experimental methods not specifically described in the following examples are all carried out according to the specific methods listed in "Molecular Cloning: A Laboratory Manual" (Third Edition) by J. Sambrook, or according to the kit and product instructions.

[0037] The application object measured in the implementation of the present invention refers to in vitro biological samples of body fluids collected from the human body or animal body. After the sample collection work is completed, the biological sample has been separated from the living human body or animal body. For example, blood samples (whole blood / serum / plasma), body fluids, tissues, excreta, isolated cultures (blood cultures, sputum cultures, etc.), etc. all belong to in vitro inanimate biological samples; the measurement process is completed in vitro. The direct purpose of the measurement is to determine whether there is a target protein (serum amyloid A) or amino acids from its degradation products in the sample. The measurement result helps assist doctors in judging the medical consultation information. This measurement belongs to the measurement of components or contents in inanimate biological samples in vitro. There is no process of directly obtaining a disease diagnosis result or health status in the measurement. Therefore, the present invention does not belong to a disease diagnosis method and meets the basic requirements of the Patent Law for patent protection objects. The following describes the specific embodiments of the present invention.

[0038] The present invention provides a kit for detecting serum amyloid A with simple components and high stability, as well as its preparation method and application. The present invention optimizes the five main components in Reagent 1 and Reagent 2, namely buffer, electrolyte, stabilizer, surfactant, and preservative. Moreover, a polyanion is creatively added to Reagent 1, and DSS is creatively added to Reagent 2. During the preparation of the latex particles coated with anti-human serum amyloid A antibody, a quenching process is creatively introduced to improve the stability of the chemical coupling between the latex particles and the anti-human serum amyloid A antibody. The results of layer-by-layer optimization also reduce the ability of chyle interference during the detection process. Finally, the kit has high sensitivity, specificity, and stability during the detection, and the linear detection range is further improved. The prepared kit for detecting serum amyloid A uses latex-enhanced immunoturbidimetry to detect the content of serum SAA in the sample to be detected. The detection principle is that human anti-SAA antibody is coated on the latex particles. After a specific immune reaction occurs with SAA in the sample to be detected, aggregated particles are formed. At a certain wavelength, by measuring the turbidity formed by the aggregates, the content of the analyte in the specimen can be obtained according to the standard curve of the standard product.

[0039] The preparation process of the kit for detecting serum amyloid A of the present invention mainly includes: preparing Reagent 1, preparing the latex particles coated with anti-human serum amyloid A antibody (hereinafter referred to as anti-SAA antibody-coated latex particles), and preparing Reagent 2. Finally, according to the specification requirements, Reagent 1, Reagent 2, quality control products, and standard products are configured in a kit.

[0040] Among them, the process of preparing Reagent 1 is as follows: Add the first buffer into a container and stir at a rotation speed of 200-400 rpm. Then, add the components of Reagent 1, namely electrolyte, stabilizer, (polyanion), first surfactant, preservative, and polyethylene glycol 6000, into the buffer in sequence according to the predetermined concentration ratio and mix well. Finally, adjust the pH to the range of 6.5-8.5.

[0041] The preparation process of SAA antibody-coated latex particles is as follows: First, latex particles with a diameter of 50 - 180 nm and a concentration of 60 - 100 mg / mL are suspended in 10 - 20 mM MES buffer. After adjusting the pH to 5 - 7, 12 - 15 mM 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) and 35 - 50 mM N-hydroxysuccinimide (NHS) are added, and the reaction is carried out at room temperature for 1 hour. The purpose of this step is to use EDC and NHS as cross-linking agents to catalyze the formation of amide bonds between the carboxyl groups on the surface of latex particles and the amino groups on the antibody. Second, a quenching agent with a concentration of 0.05 - 0.2 M is used to quench EDC. The purpose of this step is to terminate the catalytic activity of unreacted EDC and prevent it from continuing to catalyze non-specific cross-linking reactions. Next, the pH of the solution is adjusted to 7.2 - 7.5 to meet the requirements of subsequent antibody conjugation reactions. An equimolar amount of anti-human serum amyloid A antibody (abbreviated as anti-SAA antibody) is added. According to the molar mass of latex particles: the molar mass of anti-SAA antibody = 40:1, the mass concentration of the added anti-SAA antibody is 1.5 - 2.5 mg / ml, and the reaction is shaken and blocked at room temperature for 2 hours. The purpose is to enable the antibody to fully conjugate with the active groups on the surface of latex particles. Finally, the supernatant of the cross-linked latex particles is removed by centrifugation or filtration, etc. The target conjugate after the reaction is diluted with 10 mM MES buffer, and the concentration of latex particles is adjusted to 10 g / L, and the pH is adjusted to 6.5. Among them, thiol reducing agents (such as 2-mercaptoethanol, 2-Hydroxy-1-ethanethiol, and tris(2-carboxyethyl)phosphine, abbreviated as TCEP) are specifically verified as the quenching agent in this life. The purpose is to use the reaction between thiol reducing agents and the active groups of EDC to make EDC lose its catalytic ability. Based on this purpose, it can also be reasonably predicted that quenching agents such as ethanolamine and glycine replacing 2-mercaptoethanol may produce similar effects. The technical effects of different quenching agents still depend on further verification of specific experiments.

[0042] The process of preparing Reagent 2 is as follows: The obtained SAA antibody-coated latex particles are diluted with a second buffer to a final concentration of 0.5 - 2.0 mg / mL. According to the components of Reagent 2, such as electrolyte, stabilizer, second surfactant, preservative, and / or DSS, etc., they are added to the buffer in sequence according to the preset concentration and mixed evenly. Finally, the pH is adjusted to the range of 6.0 - 8.0.

[0043] In addition, the volume ratio of Reagent 1 to Reagent 2 of the present invention is preferably 4:1. The quality control product and the standard product are prepared from human serum or a liquid with a similar serum matrix of human recombinant amyloid A, wherein the content of human serum is not less than 5%. The specific preparation process of the quality control product is as follows: Human recombinant amyloid A is added to glycine buffer to prepare quality control products at two levels, high value and low value. Among them, the target value range of the low value level is 12 - 18 mg / L, and the target value range of the high value level is 48 - 72 mg / L. The specific preparation process of the standard product is as follows: Human recombinant amyloid A is added to 15 - 100 mmol / L glycine buffer and mixed evenly to prepare 5 concentration series, and the concentrations are 10 mg / L, 60 mg / L, 150 mg / L, 300 mg / L, and 500 mg / L respectively. Then 5 - 10% human serum is added. In addition, the standard product series also includes a human serum standard product without human recombinant amyloid A, that is, the concentration of human recombinant amyloid A is 0 mg / L.

[0044] The preparation process and components of the serum SAA kit finally used to verify the performance indicators of the kit of the present invention are as follows. The components, preparation process, and performance evaluation of Reagent 1 and Reagent 2 are mainly exemplified.

[0045] Example 1: Serum SAA Kit 1

[0046] The main components, concentrations, and preparation process of Reagent 1 are as follows: The first buffer is 100 mmol / L HEPES buffer; the inorganic salts are 8% calcium chloride and 6% sodium chloride; the stabilizer is 0.1% mannitol; the preservative is 0.5% ProClin - 300; the polyanion is 0.3 mmol / L polymaleic acid; the first surfactant is 0.05% Triton X - 100; and 8% polyethylene glycol 6000. Take the first buffer and add it to a container, stir at a speed of 200 rpm, and then add the components of Reagent 1, namely inorganic salts, stabilizer, preservative, polyanion, first surfactant, and polyethylene glycol 6000, into the buffer in sequence according to the predetermined concentration ratio and mix evenly. Finally, use a pH regulator to adjust the pH of Reagent 1 to a pH value of 6.5, and thus Reagent 1 is prepared.

[0047] The preparation process of SAA antibody-coated latex particles is as follows: First, latex particles with a diameter of 50 - 80 nm and a concentration of 60 mg / mL are suspended in 10 mM MES buffer. After adjusting the pH to 5, 12 mM 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) and 35 mM N-hydroxysuccinimide (NHS) are added, and the reaction is carried out at room temperature for 1 hour. Second, 0.05 M quenching agent (2-mercaptoethanol) is used to quench EDC. Next, after adjusting the pH of the solution to 7.2, 1.5 mg / ml anti-SAA antibody equimolar to the latex particles is added, and the reaction is shaken and blocked at room temperature for 2 hours. Finally, the supernatant of the cross-linked latex particles is removed by centrifugation or filtration, etc., and the target conjugate after the reaction is diluted with 10 mM MES buffer to make the concentration of anti-SAA antibody latex particles reach 10 g / L, and the pH is adjusted to 6.5.

[0048] The main components, concentrations and preparation process of Reagent 2 are as follows: The second buffer is 15 mmol / L phosphate buffer; the inorganic salt is 2% calcium chloride; the stabilizer is 0.5% bovine serum albumin; the second surfactant is 0.05% Tween 20; the preservative is 1% ProClin-300; it also includes 150 mg / L DSS; the obtained SAA antibody-coated latex particles are diluted with the second buffer to make the final concentration 0.5 mg / mL. According to the components of Reagent 2, namely inorganic salt, stabilizer, second surfactant, preservative and DSS, they are added to the second buffer in sequence according to the preset concentration and mixed evenly. Finally, the pH is adjusted to 6.0, and Reagent 2 is thus prepared.

[0049] Example 2: Serum SAA Kit 2

[0050] The main components, concentrations and preparation process of Reagent 1 are as follows: The first buffer is 280 mmol / L Tris-HCl buffer; the inorganic salt is 7% magnesium chloride; the stabilizer is 0.3% casein; the preservative is 2% benzoic acid; there is no polyanion; the first surfactant is 0.2% SDS; and 7% polyethylene glycol 6000. Take the first buffer and add it to a container, and stir at a speed of 300 rpm; then add the components of Reagent 1, namely inorganic salt, stabilizer, preservative, first surfactant and polyethylene glycol 6000, to the buffer in sequence according to the predetermined concentration ratio and mix evenly. Finally, the pH of Reagent 1 is adjusted to pH 6.5 - 8.5 with a pH regulator, and Reagent 1 is thus prepared.

[0051] The preparation process of SAA antibody-coated latex particles is as follows: First, latex particles with a diameter of 80 - 110 nm and a concentration of 70 mg / mL are suspended in 15 mM MES buffer. After adjusting the pH to 6, 13 mM 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) and 40 mM N-hydroxysuccinimide (NHS) are added, and the reaction is carried out at room temperature for 1 hour. Secondly, 0.15 M quenching agent (2-mercaptoethanol) is used to quench EDC. Next, after adjusting the pH of the solution to 7.3, 1.75 mg / ml anti-SAA antibody equimolar to the latex particles is added, and the reaction is shaken and blocked at room temperature for 2 hours. Finally, the supernatant of the cross-linked latex particles is removed by centrifugation or filtration, etc., and the target conjugate after the reaction is diluted with 10 mM MES buffer to make the concentration of anti-SAA antibody latex particles reach 10 g / L, and the pH is adjusted to 6.5.

[0052] The main components, concentrations, and preparation process of Reagent 2 are as follows: The second buffer is 50 mmol / L glycine buffer; the inorganic salt is 5% sodium chloride; the stabilizer is 0.2% casein; the second surfactant is 0.1% Triton X-100; the preservative is 2% benzoic acid; it also includes 50 mg / L DSS; the obtained SAA antibody-coated latex particles are diluted with the second buffer to make the final concentration 0.9 mg / mL, and according to the components of Reagent 2, namely inorganic salt, stabilizer, second surfactant, preservative, and DSS, they are added to the second buffer in sequence according to the preset concentration and mixed evenly. Finally, the pH is adjusted to 7.0, and Reagent 2 is thus prepared.

[0053] Example 3: Serum SAA Kit 3

[0054] The main components, concentrations, and preparation process of Reagent 1 are as follows: The first buffer is 50 mmol / L phosphate buffer; the inorganic salts are 8.5% sodium chloride and 1.5% calcium chloride; the stabilizer is 0.2% bovine serum albumin; the preservative is 1% sodium azide; the polyanion is 0.6 mmol / L polyanion cellulose; the first surfactant is 01% Tween 201; and 5% polyethylene glycol 6000. Take the first buffer and add it to a container, and stir at a speed of 400 rpm; then add the components of Reagent 1, namely inorganic salt, stabilizer, preservative, polyanion, first surfactant, and polyethylene glycol 6000, to the buffer in sequence according to the predetermined concentration ratio and mix evenly. Finally, use a pH regulator to adjust the pH of Reagent 1 to pH 7.2, and Reagent 1 is thus prepared.

[0055] The preparation process of SAA antibody-coated latex particles is as follows: First, latex particles with a diameter of 110 - 140 nm and a concentration of 80 mg / mL are suspended in 15 mM MES buffer. After adjusting the pH to 6, 14 mM 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) and 45 mM N-hydroxysuccinimide (NHS) are added, and the reaction is carried out at room temperature for 1 hour. Secondly, 0.1 M quenching agent (2-mercaptoethanol) is used to quench EDC. Next, after adjusting the pH of the solution to 7.4, 2 mg / ml anti-SAA antibody equimolar to the latex particles is added, and the reaction is shaken and blocked at room temperature for 2 hours. Finally, the supernatant of the cross-linked latex particles is removed by centrifugation or filtration, etc., and the target conjugate after the reaction is diluted with 10 mM MES buffer to make the concentration of the anti-SAA antibody latex particles reach 10 g / L, and the pH is adjusted to 6.5.

[0056] The main components, concentrations, and preparation process of Reagent 2 are as follows: The second buffer is 100 mmol / L glycine buffer; the inorganic salt is 8% sodium chloride; there is no stabilizer; the second surfactant is 0.15% Triton X-100; the preservative is 1% sodium azide; it also includes 100 mg / L DSS; the obtained SAA antibody-coated latex particles are diluted with the second buffer to make the final concentration 1.3 mg / mL, and according to the components of Reagent 2, namely inorganic salt, the second surfactant, the preservative, and DSS, they are added to the second buffer in sequence according to the preset concentrations and mixed evenly. Finally, the pH is adjusted to 8.0 to obtain Reagent 2.

[0057] Example 4: Serum SAA Kit 4

[0058] The main components, concentrations, and preparation process of Reagent 1 are as follows: The first buffer is 15 mM glycine buffer; the inorganic salt is 10% calcium chloride; the stabilizer is 0.4% gelatin; the preservative is 3% ProClin-300; the polyanion is 0.9 mM chondroitin sulfate; the first surfactant is 0.15% B66; and 4% polyethylene glycol 6000. Take the first buffer and add it to a container, and stir at a speed of 200 rpm; then add the components of Reagent 1, namely inorganic salt, stabilizer, preservative, polyanion, the first surfactant, and polyethylene glycol 6000, to the buffer in sequence according to the preset concentration ratio and mix evenly. Finally, use a pH regulator to adjust the pH of Reagent 1 to pH 7.6 to obtain Reagent 1.

[0059] The preparation process of SAA antibody-coated latex particles is as follows: First, latex particles with a diameter of 140 - 180 nm and a concentration of 90 mg / mL are suspended in 20 mM MES buffer. After adjusting the pH to 7, 15 mM 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) and 40 mM N-hydroxysuccinimide (NHS) are added, and the reaction is carried out at room temperature for 1 hour. Second, 0.1M quencher (TCEP) is used to quench EDC. Next, after adjusting the pH of the solution to 7.5, 2.25 mg / ml anti-SAA antibody equimolar to the latex particles is added, and the reaction is shaken and blocked at room temperature for 2 hours. Finally, the supernatant of the cross-linked latex particles is removed by centrifugation or filtration, etc., and the target conjugate after the reaction is diluted with 10 mM MES buffer to make the concentration of anti-SAA antibody latex particles reach 10 g / L, and the pH is adjusted to 6.5.

[0060] The main components, concentrations and preparation process of reagent 2 are as follows: The second buffer is 150 mmol / L Tris-HCl buffer; the inorganic salt is 11% magnesium chloride; the stabilizer is 0.1% gelatin; the second surfactant is 0.2% B66; the preservative is 3% ProClin-300; there is no DSS. The obtained SAA antibody-coated latex particles are diluted with the second buffer to make the final concentration 1.7 mg / mL. According to the components of reagent 2, namely inorganic salt, stabilizer, second surfactant, preservative, they are added to the second buffer in sequence according to the preset concentration and mixed evenly. Finally, the pH is adjusted to 7.0, and reagent 2 is thus prepared.

[0061] Example 5: Serum SAA Kit 5

[0062] The main components, concentrations and preparation process of reagent 1 are as follows: The first buffer is 200 mmol / L phosphate buffer; the inorganic salts are 7% sodium chloride and 7% magnesium chloride; the stabilizer is 0.2% bovine serum albumin; the preservative is 1.5% benzoic acid; the polyanion is 0.6 mmol / L polyanion cellulose; the first surfactant is 0.2% Tween 20; and 5% polyethylene glycol 6000. Take the first buffer and add it to a container, and stir at a speed of 300 rpm. Then, the components of reagent 1, namely inorganic salt, stabilizer, preservative, polyanion, first surfactant and polyethylene glycol 6000, are added to the buffer in sequence according to the predetermined concentration ratio and mixed evenly. Finally, the pH of reagent 1 is adjusted to pH 8.0 with a pH regulator, and reagent 1 is thus prepared.

[0063] The preparation process of SAA antibody-coated latex particles is as follows: First, latex particles with a diameter of 50-80 nm and a concentration of 100 mg / mL are suspended in 10 mM MES buffer, and the pH is adjusted to 7. Instead of using a quenching agent, 40 mM N-hydroxysuccinimide (NHS) is first added and reacted at room temperature for 3 hours. After centrifuging to remove the supernatant, the precipitate is added to 10 mM MES buffer, the pH is adjusted to 6.0, and it is ultrasonically dispersed. Then, 15 mM 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) is added and reacted for 1 h, and after centrifuging to remove the supernatant, the precipitate is added to 10 mM MES buffer. Next, after adjusting the pH of the solution to 7.4, 2.5 mg / ml anti-SAA antibody equimolar to the latex particles is added, and the reaction is shaken and sealed at room temperature for 2 hours. Finally, the cross-linked latex particles are centrifuged or filtered to remove the supernatant, and the target conjugate after the reaction is ultrasonically dispersed and washed 3 times with 10 mM MES buffer to adjust the concentration of the anti-SAA antibody latex particles to 10 g / L and the pH to 6.5. This coating method does not use a quenching agent and may leave unreacted EDC. To ensure that each step is fully carried out, by adding EDC and NHS step by step, it is beneficial to better control the reaction process of EDC and NHS, but the total reaction time is relatively long (more than 5 hours), and it also involves multiple centrifugation, ultrasonic and washing processes.

[0064] The main components, concentrations and preparation process of Reagent 2 are as follows: The second buffer is 200 mmol / L glycine buffer; the inorganic salt is 15% magnesium chloride; the stabilizer is 0.3% bovine serum albumin; the second surfactant is 0.15% Tween 20; the preservative is 4% sodium azide; it also includes 200 mg / L DSS. The obtained SAA antibody-coated latex particles are diluted with the second buffer to a final concentration of 2.0 mg / mL, and according to the components of Reagent 2, namely inorganic salt, stabilizer, second surfactant, preservative and / or DSS, they are sequentially added to the second buffer and mixed evenly according to the preset concentrations. Finally, the pH is adjusted to 6.0 to obtain Reagent 2.

[0065] Example 6: Serum SAA Kit 6

[0066] The main components, concentrations, and preparation process of Reagent 1 are as follows: The first buffer is a 150 mmol / L glycine buffer; the inorganic salt is 15% sodium chloride; the stabilizer is 0.5% mannitol; the preservative is 2.5% sodium azide; the polyanion is 0.3 mmol / L polymaleic acid; the first surfactant is 0.1% B66; and 6% polyethylene glycol 6000. Take the first buffer and add it to a container, and stir at a speed of 400 rpm; then add the components of Reagent 1, namely the inorganic salt, stabilizer, preservative, polyanion, first surfactant, and polyethylene glycol 6000, into the buffer in sequence according to the predetermined concentration ratio and mix well. Finally, adjust the pH of Reagent 1 to 8.5 with a pH regulator, and Reagent 1 is thus prepared.

[0067] The preparation process of SAA antibody-coated latex particles is as follows: First, suspend latex particles with a diameter of 110 - 140 nm and a concentration of 80 mg / mL in 10 mM MES buffer, adjust the pH to 5, then add 12 mM 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) and 50 mM N-hydroxysuccinimide (NHS), and react at room temperature for 1 hour; secondly, quench EDC with 0.2M quenching agent (2-mercaptoethanol); next, after adjusting the pH of the solution to 7.2, add 2 mg / ml anti-SAA antibody in an equimolar amount to the latex particles, and shake and seal the reaction at room temperature for 2 hours; finally, remove the supernatant from the cross-linked latex particles by centrifugation or filtration and other methods, and dilute the resulting target conjugate with 10 mM MES buffer to make the concentration of anti-SAA antibody latex particles reach 10 g / L, and adjust the pH to 6.5.

[0068] The main components, concentrations, and preparation process of Reagent 2 are as follows: The second buffer is a 280 mmol / L HEPES buffer; the inorganic salt is 8% calcium chloride; the stabilizer is 0.4% mannitol; the second surfactant is 0.1% B66; the preservative is 5% sodium azide; it also includes 80 mg / L DSS; dilute the obtained SAA antibody-coated latex particles with the second buffer to make the final concentration 1.4 mg / mL, and add the components of Reagent 2, namely the inorganic salt, stabilizer, second surfactant, preservative, and / or DSS, into the second buffer in sequence according to the preset concentration and mix well. Finally, adjust the pH to 8.0, and Reagent 2 is thus prepared.

[0069] Example 7: Determination method of serum SAA kit

[0070] The present invention quantitatively determines the serum SAA kits shown in Examples 1 - 6 prepared above by the two - point endpoint method. The determination process is as follows: Set up 3 types of reaction tubes, including a blank tube, a sample tube, and a calibration tube. Among them, add 2 μL of distilled water to the blank tube, 2 μL of the sample to be tested to the sample tube, and 2 μL of the standard product to the calibration tube; First, add 200 μL of Reagent 1 solution to each of the 3 types of reaction tubes, and incubate at 37°C for 5 minutes; Then, add 40 μL of Reagent 2 solution to each reaction tube, mix well for 30 seconds, and use an automatic biochemical analyzer to read the absorbance (A1) corresponding to the 3 types of reaction tubes; Then, place them at 37°C and incubate for 4 minutes, and use the automatic biochemical analyzer to read the absorbance (A2) corresponding to the 3 types of reaction tubes again. The determination principle is the absorbance difference ΔA (that is, a method for calculating the concentration or activity of the analyte by measuring the total change in the concentration of the product or substrate from the start to the equilibrium of the reaction), and the calculation formula is as follows: ΔA = A2 - A1, 。

[0071] Example 8: Performance indicators of the serum SAA kit

[0072] Take the sample to be tested, use the kits prepared in Examples 1 - 6 above, and perform determination and analysis with an automatic biochemical analyzer according to the determination method of Example 7. The determination results are used to verify the specific performance indicators of the reagent. The performance includes sensitivity, linear range, anti - interference ability, stability, repeatability, and the correlation of the determination results evaluated with a third - party reagent. The unit of the test value of the sample to be tested and the control value of the same sample to be tested by the control reagent is mg / L. The sample to be tested is a clinical sample containing different concentrations of serum amyloid A.

[0073] Sensitivity: The sample to be tested is 5 clinical serum samples containing different concentrations of amyloid A. Use the serum SAA kits prepared in Examples 1, 3, 5, and 6 above to perform parallel determinations respectively, and each determination is repeated 10 times. Among them, the sensitivity determination results of Examples 1, 3, 5, and 6 are shown in Table 1 below.

[0074] Table 1 Sensitivity

[0075] ;

[0076] As can be seen from the sensitivity determination results in the above table, as the concentration of the sample to be tested increases, the mean value shows a corresponding increasing trend. It can be seen that the serum SAA kits prepared in Example 1, Example 3, Example 5, and Example 6 have sensitive response capabilities to samples with different concentrations. When the concentration of the sample to be tested is a low value less than 5 mg / L, the overall difference in the coefficient of variation (abbreviated as CV value) is relatively large. However, relatively speaking, the CV values of the measured values corresponding to Example 1, Example 3, and Example 6 are comparable to the control, and even slightly better than the control group. The SD values are all less than 1, indicating that the measured data are relatively stable and reliable, and the SD values under low-concentration samples are also small. Therefore, the measurement results of these 4 kits are more stable and reliable. In particular, the SD value of the kit prepared in Example 3 is relatively the smallest, indicating that its sensitivity is relatively the best.

[0077] Linear range: Human recombinant amyloid A was added to glycine buffer to prepare two calibrated samples with high (500 mg / L) and low (2 mg / L) concentrations. Then, they were mixed in different proportions to further prepare 4 calibrated samples with different concentrations, and thus 6 calibrated samples containing SAA serum with different concentrations were prepared. The theoretical concentrations were 500 mg / L, 350 mg / L, 250 mg / L, 150 mg / L, 50 mg / L, and 2 mg / L respectively. The serum SAA kits prepared in the above examples were used for parallel determination respectively, and each determination was repeated 3 times. The two extreme linear determination results of Example 1 and Example 3 were plotted for more intuitive illustration. Taking the mean value (yi) of the determination results as the dependent variable and the dilution concentration (xi) as the independent variable, a linear regression graph was plotted as Figure 1 shown, and Table 2 exemplarily lists the specific determination results of Example 3.

[0078] Table 2 Linear determination results of Example 3

[0079]

[0080] From Figure 1 Combined with Table 2, it can be seen that the regression equation corresponding to Example 1 is Y = 1.1453x - 14.798 (R 2 = 0.9938), and the regression equation corresponding to Example 3 is Y = 0.9972x + 1.9357 (R 2 = 0.9999). The slopes are both close to 1, indicating that there is a good linear relationship between the measured values of Example 1 and Example 3 and the theoretical values. The coefficient of determination (R 2)( ) is also very close to 1, indicating a very high model fitting degree, further indicating a good linear relationship, and the detection values in the linear range of 2 mg / L to 500 mg / L are relatively reliable. From the comparison of the intercepts, it can be seen that the intercept corresponding to Example 3 is relatively small, indicating that when the measured value is 0, the measurement result of the kit deviates from the theoretical value (0) to a relatively small extent. While the intercept corresponding to Example 1 is relatively large and negative, indicating that when the measured value is 0, the measurement result of the kit is much lower than the theoretical value (0), and there may be a deviation problem during low-concentration measurement. Therefore, during low-concentration measurement, the deviation of the measured value corresponding to Example 3 is relatively small, and the accuracy of the measurement result within the linear range is relatively better than the measured value corresponding to Example 1. Moreover, from the specific values of the linear deviation, it can be seen that the absolute values of the linear deviations corresponding to Example 3 are all less than 10%, indicating a high consistency between the measured values and the theoretical values of Example 3 within the linear range of 2 mg / L to 500 mg / L. Combining with the corresponding linear regression graph, it can be known that the linearity of the detection values of Example 3 in the range of 2 mg / L to 500 mg / L is good and can be used.

[0081] Anti-interference ability: The test samples are from the same human serum sample. Limited by the available dosage of the collected serum sample, only the serum SAA kits prepared in Example 2, Example 3, and Example 5 and the third-party reagent are selected to perform parallel measurements on the test samples respectively. If there is a deviation in the test results due to the presence of chylomicrons (such as lipid components like triglycerides) in the sample, the impact of adding different contents of triglycerides (TG) on the measured value and the theoretical value can be detected to characterize the anti-chylomicron interference ability of the kit to a certain extent. Therefore, different contents of triglycerides are added to the same human serum sample to prepare test samples containing different concentrations of TG, and different kits are used to perform parallel measurements on them respectively. Each measurement is repeated 3 times, and the average value is calculated and the relative deviation is calculated. The measurement results of the anti-interference ability are shown in Table 3.

[0082] Table 3 Anti-interference ability

[0083] ;

[0084] The experimental results show that: when the TG concentration is below 10 mmol / L, the measured values of the 4 kits have a high degree of conformity with the theoretical values of the samples to be tested, and all have a certain anti-interference ability; when the TG concentration is below 20 mmol / L, the relative deviations of the measurement results of Example 3 and Example 5 are both less than 10%, and they have an anti-interference ability that meets the requirements; when the TG concentration is below 30 mmol / L (clinical samples with such a high TG concentration are relatively rare), only the relative deviation of the measurement result of Example 3 is less than 10%, and it has an anti-interference ability that meets the requirements; it can be inferred that: the serum SAA kit prepared in Example 3 has a relatively strong anti-interference ability for clinical samples containing chylomicrons such as triglycerides. Especially in the case of interference with an extreme content of 30 mmol / L of TG, the serum SAA kit prepared in Example 3 still has a strong anti-interference ability, and the accuracy of the measured value is relatively credible.

[0085] Stability evaluation: The kits of Example 2, Example 3, Example 4 and a third-party reagent were each placed at 2 - 8 °C for 0, 2, 4, 8, 12, 16 months, and then the same sample to be tested was measured respectively; the sample to be tested was from the same serum sample, which was sub-packed and stored in a -20 °C refrigerator, and was randomly taken out for the determination experiment of serum SAA during measurement, and each measurement was repeated 3 times. Results: The acceptable deviation of the measured value of the serum SAA kit prepared in Example 2 for the sample to be tested can be stably maintained for 14 months when placed at 2 - 8 °C, while the acceptable deviations of the measured values of the serum SAA kits prepared in Example 3, Example 4 and the third-party reagent for the sample to be tested can be stably maintained for 16 months when placed at 2 - 8 °C. The linear changes of the measured mean values of these 3 kits with the storage time are as Figure 2 shown. It can be seen from the figure that: the measured values of the serum SAA kit prepared in Example 3 change relatively little with the storage time when placed at 2 - 8 °C for 0, 2, 4, 8, 12, 16 months, and its stability is relatively the best; since the DSS is not used in Reagent 2 of the serum SAA kit prepared in Example 4, the deviation of the measured value for the sample to be tested is significantly worse than that of Example 3 with DSS added. It can be seen that adding DSS to Reagent 2 helps to improve the accuracy of the measured value of the kit and effectively reduces the relative deviation of the measured value.

[0086] Repeatability: The sample to be tested was a randomly selected large-dose clinical human serum sample. The serum SAA kits prepared in Example 3, Example 5 and Example 6 of the present invention were used to measure the sample to be tested respectively, and each measurement was repeated 10 times. The average value and coefficient of variation (CV) of the measured values were calculated respectively. The repeatability measurement results are shown in Table 4.

[0087] Table 4 Repeatability measurement results

[0088] ;

[0089] As can be seen from Table 4, the differences among the average values corresponding to Example 3, Example 5, and Example 6 are not significant, and the coefficient of variation (CV) is less than 10% for all of them, and all are considered to be within the acceptable repeatability range. It can be known that the fluctuations among the measured values of the serum SAA kits prepared by Example 3, Example 5, and Example 6 are small, and the repeatability of the three types of kits is relatively good. Further, the coefficient of variation corresponding to Example 3 is the smallest, only 2.4%. It can be seen that the repeatability of the serum SAA kit prepared by Example 3 is significantly better than that of Example 5 and Example 6.

[0090] Correlation: According to the above detection results of repeatability, the serum SAA kit prepared by Example 3 with better repeatability and a third-party reagent were used for parallel detection of the samples to be tested. The samples to be tested were 50 randomly selected clinical serum specimens. Taking the measured values of the same sample to be tested by the two reagents as the abscissa and ordinate respectively, the correlation curve graph of the measured values of the two reagents was plotted as Figure 3 shown, and the regression equation is y = 1.012x - 0.375 (R 2 = 0.996). It can be seen that the measurement results of these two reagents are highly consistent, showing good linear correlation, that is, the coincidence rate of the measured values of the serum SAA kit prepared by Example 3 is good, and it has good accuracy and correlation.

[0091] Based on the performance indicators of the serum SAA kits prepared in 6 examples, it can be seen that: in the present invention, the buffer systems, components and concentrations of inorganic salts in Reagent 1 and Reagent 2 are optimized. For example, different buffer systems are selected for Reagent 1 and Reagent 2 respectively, and the concentration of inorganic salts is controlled within the range of 2% - 15%. As a result, the performance indicators of the kits preferably configured in the present invention meet the minimum requirements of the kit performance review, and are comparable or have little difference from the performance indicators of third - party reagents. In addition, the sensitivity, anti - interference ability, stability, repeatability and correlation with the detection of third - party reagents of the serum SAA kit prepared in Example 3 are relatively excellent. And since the accuracy of the measurement results of Example 1 within the linear range is relatively better than the measured values corresponding to Example 3, it can be inferred that the polyanion, DSS, and the use of a quenching agent in the preparation process of anti - SAA antibody - coated latex particles, which are creatively added in both Example 1 and Example 3 of the present invention, can effectively improve the performance of the serum SAA kit. The stability measurement results show that the stability measurement values corresponding to Example 3 with both polyanion and DSS added are better than those of Example 2 without polyanion added and Example 4 without DSS added, and the stability measurement values corresponding to Example 3 are also significantly better than those of third - party reagents. It can be seen that the creatively added polyanion and DSS in the present invention are beneficial to improving the stability of the kit; the repeatability measurement results show that the repeatability measurement values corresponding to Example 3 and Example 6 using the quenching agent are better than those of Example 5 without using the quenching agent. It can be seen that the use of a quenching agent in the preparation process of anti - SAA antibody - coated latex particles can improve the repeatability of reagent detection.

[0092] The specific embodiments of the present invention have been described in detail, making it easy for those skilled in the art to understand. However, based on all the disclosures, different or similar modifications or substitutions can be made to some of the specific details, and these changes are all within the protection scope of the present invention. The entire scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. A reagent composition, characterized in that, The method is used to determine serum amyloid A by latex enhanced immunoturbidimetry, comprising a reagent 1 and a reagent 2 which are independent of each other, and is characterized in that: The pH of the reagent 1 is 6.5-8.5, and it comprises a first buffer, an inorganic salt, a stabilizer, a polyanion, a preservative, a first surfactant and polyethylene glycol 6000; the pH of the reagent 2 is 6.0-8.0, and it comprises a second buffer, an inorganic salt, a stabilizer, a preservative, a second surfactant, disuccinimidyl suberate and anti-SAA antibody-coated latex particles; wherein the main components of the first buffer and the second buffer are different, and the first buffer is a phosphate buffer; the second buffer is a glycine buffer; the final concentration of the inorganic salt in the reagent 1 and the reagent 2 is 2%-15%; the polyanion is selected from any one of 0.3-0.9 mmol / L polyanionic cellulose, polymaleic acid and chondroitin sulfate; the final concentration of the disuccinimidyl suberate in the reagent 2 is 50-200 mg / L; The concentration of the anti-SAA antibody-coated latex particles in reagent 2 is 0.5-2.0 mg / mL, wherein the particle size of the latex particles for preparing the anti-SAA antibody-coated latex particles is selected from the range of 50-80 nm, 80-110 nm, 110-140 nm, and 140-180 nm, and an anti-SAA antibody is added in an amount equimolar to the latex particles. The process for preparing the anti-SAA antibody-coated latex particles includes using 2-mercaptoethanol as a quencher to terminate the catalytic activity of unreacted EDC. After adding EDC and NHS, quenching is performed first, and then pH adjustment, coupling and blocking are performed in sequence.

2. The reagent composition according to claim 1, wherein The reagent 2 may not include a stabilizer.

3. The reagent composition according to any one of claims 1 to 2, characterized in that The concentration of the first buffer and the second buffer is 15 to 280 mmol / L; The inorganic salt is selected from any one or two of calcium chloride, sodium chloride and magnesium chloride; The stabilizer is selected from any one or two of 0.1% to 0.5% bovine serum albumin, mannitol, gelatin and casein; The preservative is selected from any one or two of sodium azide, benzoic acid and ProClin-300; and the final concentration of the preservative in reagent 1 is 0.5% to 3%, and the final concentration of the preservative in reagent 2 is 1% to 5%; The first surfactant is selected from any one or two of 0.05% to 0.2% Tween 20, Triton X-100, SDS, and B66; The second surfactant is selected from any one or two of 0.05% to 0.2% Tween 20, Triton X-100, and B66; The final concentration of polyethylene glycol 6000 in reagent 1 is 4% to 8%.

4. The reagent composition according to any one of claims 1-2, characterized in that, The inorganic salt contains at least sodium chloride; the stabilizer is bovine serum albumin; and the polyanion is polyanionic cellulose.

5. A kit for detecting serum amyloid A, characterized in that, Comprising the reagent composition described in any one of claims 1-4, further comprising a quality control product and a calibration product, both the quality control product and the calibration product contain at least two concentration levels of human recombinant amyloid A, and the buffer solution is a glycine buffer solution containing human serum.

6. The serum amyloid A assay kit according to claim 5, characterized in that, The quality control product contains at least two concentration levels of human recombinant amyloid A, and these two concentration levels are respectively: Level 1: 12 mg / L to 18 mg / L; Level 2: 48 mg / L to 72 mg / L; The calibration product contains at least five concentration levels of human recombinant amyloid A, and these five concentration levels are respectively: 10 mg / L, 60 mg / L, 150 mg / L, 300 mg / L, and 500 mg / L.

7. A preparation method of a serum amyloid A assay kit, characterized in that, The reagent composition described in claim 1 is prepared, wherein, The preparation process of reagent 1 is as follows: Take the first buffer solution and add it to a container, stir at a rotation speed of 200-400 rpm; then add the inorganic salt, stabilizer, preservative, polyanion, first surfactant, and polyethylene glycol 6000 in the components of reagent 1 into the buffer solution in sequence according to a predetermined concentration ratio and mix well. Finally, adjust the pH of reagent 1 to a pH value of 6.5-8.5 with a pH regulator, and thus reagent 1 is prepared; The preparation process of reagent 2 is as follows: Take the anti-SAA antibody-coated latex particles, first dilute them with the second buffer solution to a final concentration of 0.5-2.0 mg / mL, and then add the inorganic salt, and / or stabilizer, second surfactant, preservative, and disuccinimidyl suberate in the components of reagent 2 into the second buffer solution in sequence according to a preset concentration and mix well. Finally, adjust the pH to 6.0-8.0, and thus reagent 2 is prepared.

8. Use of the reagent composition described in any one of claims 1-4, the serum amyloid A assay kit described in any one of claims 5-6, and the preparation method of the serum amyloid A assay kit described in claim 7 in the preparation of latex reagent-related products.

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