Serum amyloid protein A determination kit and preparation and application thereof

Abstract

The invention provides a serum amyloid A determination kit as well as preparation and application thereof, the kit optimizes and screens components and concentrations of buffer systems and inorganic salts in a reagent 1 and a reagent 2, particularly adds polyanions and DSS, and uses a quenching agent in the preparation process of anti-SAA antibody coated latex particles. According to the kit prepared by optimizing and screening the components, the specific recognition and binding capacity to the serum amyloid protein A is remarkably improved, the performance indexes such as the sensitivity, the linear range, the anti-interference capacity, the stability and the repeatability of the kit are improved, the linearity of a detection value in the linear range of 2mg / L to 500mg / L is good, the detection value is accurate and usable, and the kit is suitable for being used in the field of detection of the serum amyloid protein A. Therefore, the comprehensive performance of immunoassay on the serum amyloid protein A is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemical immunoassay, and firstly to a serum amyloid A assay kit, and also specifically to a preparation method of the serum amyloid A assay kit and an application thereof in biochemical immunoassay. Background Art

[0002] Serum amyloid A (SAA) is a family of polymorphic proteins encoded by multiple genes. It is the precursor of tissue amyloid A and belongs to the acute phase response protein. SAA in plasma participates in a variety of physiological or pathological reactions in the body and has multiple functions, including: binding to plasma high-density lipoprotein (HDL) to promote the reverse transport of cholesterol to the liver for metabolism; as an acute phase response protein, it can recruit immune cells to the inflammatory site and participate in the inflammatory response; 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 be deposited in different organs in the form of amyloid A fibrils, which is an important feature of the pathological histological changes of chronic inflammation. Under normal conditions, SAA in the human body is mainly expressed by liver cells; when the body has an acute phase reaction, it will release cytokines (such as IL-1, IL-6, TNF, etc.), which will stimulate liver cells, macrophages, fibroblasts and some fat cells to express a large amount of acute phase SAA, causing the SAA concentration in plasma to increase sharply by 100-1000 times. In addition, 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 SAA and CRP are both acute phase reactions, SAA is a protein, but its high sensitivity and short half-life may be superior to CRP in some aspects and can be used to evaluate acute phase reactions. Studies have also found that tumor cells and cells in atherosclerotic plaques can also synthesize acute phase SAA, which may be related to its participation in amyloidosis and atherosclerosis formation. As an early indicator of organ transplant rejection and various tumors, SAA can be used to evaluate the prognosis of tumors, atherosclerosis and cardiovascular events. In addition, SAA can be elevated in the early stages of bacterial and viral infections, and its combination with CRP can help identify the type of infection and evaluate the efficacy of antibacterial drugs.

[0003] At present, the methods used to determine serum SAA include colloidal gold method (detection range 5-500 mg / L, low precision), immunoagglutination method (complex operation), magnetic particle chemiluminescence method (high determination cost) and latex immunoturbidimetry. Although the existing latex immunoturbidimetry for determining SAA has the advantages of high accuracy / sensitivity / specificity, good stability, convenience and low cost, there are still some defects in the determination of serum SAA by latex immunoturbidimetry, 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. The commonly used chemical coupling method, such as EDC (1-ethyl-3-[3 -dimethylaminopropyl] carbodiimide hydrochloride) method, although it can improve stability, the steps are cumbersome, and in order to remove uncross-linked antibodies and impurities in the preparation process and ensure the purity of the reagents, it is generally necessary to include a step of ultrasonic dispersion after centrifugation, which is easy to cause antibody inactivation or shedding; therefore, the instability of the SAA antibody coating during the preparation process will affect the accuracy and stability of the measurement results, and generally surfactants, protein protectants and other additives are used to improve stability, but additives also need to comprehensively consider the compatibility with the preparation components and adverse effects on antibody activity, etc. In addition, in actual clinical applications, interference from substances such as chylomicrons in the sample can also lead to inaccurate test results. Therefore, it is necessary to provide a kit for determining SAA by latex immunoturbidimetry with high accuracy and reliability and a preparation method thereof to address these problems. Summary of the invention

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

[0005] To achieve the above-mentioned purpose, the present invention is implemented by the following technical solutions: In the first aspect, the present invention provides a reagent composition for determining serum amyloid A by latex enhanced immunoturbidimetry, comprising a reagent 1 and a reagent 2 which are independent of each other, wherein the pH of the reagent 1 is 6.5-8.5, and the reagent 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 the reagent comprises a second buffer, an inorganic salt, a stabilizer, a preservative, a second surfactant, DSS and anti-SAA antibody-coated latex particles; wherein the main components of the first buffer and the second buffer are different, and the final concentrations of the inorganic salts in the reagent 1 and the reagent 2 are both 2%-15%; the concentration of the anti-SAA antibody-coated latex particles in the 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 50-180 nm, and the process of preparing the anti-SAA antibody-coated latex particles comprises using a thiol reducing agent as a quencher to terminate the catalytic activity of unreacted EDC.

[0006] According to a preferred embodiment of the present invention, in the reagent composition, the reagent 2 may not include a stabilizer.

[0007] According to a preferred embodiment of the present invention, in the reagent composition, the first buffer and the second buffer are selected from any one or two of 15-280 mmol / L Tris-HCl buffer, phosphate buffer, glycine buffer, HEPES buffer and aminoacetic acid buffer; 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%-0.5% bovine serum albumin, mannitol, gelatin and casein; the polyanion is selected from any one of 0.3-0.9 mmol / L polyanionic cellulose, polymaleic acid and chondroitin sulfate; 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 0.05%-0.2% Tween 20, Triton Any one or two of 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%; the final concentration of DSS in reagent 2 is 50 to 200 mg / L.

[0008] 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 salt contains at least sodium chloride; the stabilizer is bovine serum albumin; and the polyanion is polyanionic cellulose.

[0009] Preferably, the concentration of the phosphate buffer in reagent 1 is 50 mmol / L, and the buffering capacity for adjusting the pH is between 6.5 and 8.5.

[0010] Preferably, the inorganic salt in reagent 1 is 8.5% sodium chloride and 1.5% calcium chloride, which can effectively prevent the interference of inorganic salts on the binding of enzyme and substrate and reduce the viscosity of polyanion.

[0011] Preferably, the stabilizer in both reagent 1 and reagent 2 is 0.2% bovine serum albumin.

[0012] Preferably, the preservative in both reagent 1 and reagent 2 is 1% sodium azide.

[0013] Preferably, the polyanion in reagent 1 is 0.5 mmol / L polyanionic cellulose, which may not affect the stability of the reagent and may also reduce chylomicron interference.

[0014] Preferably, the first surfactant in reagent 1 is 0.1% Tween 20.

[0015] Preferably, the concentration of polyethylene glycol 6000 in reagent 1 is 5%.

[0016] Preferably, the second buffer in reagent 2 is a 100 mmol / L glycine buffer, and the buffering capacity for adjusting the pH is 6.0-8.0.

[0017] Preferably, the inorganic salt in reagent 2 is 8% sodium chloride.

[0018] Preferably, the second surfactant in reagent 2 is 0.15% Triton X-100.

[0019] In a second aspect, the present invention provides a serum amyloid A assay kit, comprising the above-mentioned reagent composition, and also comprising a quality control product and a calibrator, wherein the quality control product and the calibrator both contain at least 2 concentration levels of human recombinant amyloid A, and the buffer is a glycine buffer containing human serum.

[0020] According to a preferred embodiment of the present invention, in the serum amyloid A assay kit, the quality control comprises at least 2 concentration levels of human recombinant amyloid A, and the 2 concentration levels are: level 1: 12 mg / L~18 mg / L; level 2: 48 mg / L~72 mg / L; the calibrator comprises at least 5 concentration levels of human recombinant amyloid A, and the 5 concentration levels are: 10 mg / L, 60 mg / L, 150 mg / L, 300 mg / L and 500 mg / L.

[0021] In a third aspect, the present invention provides a method for preparing anti-SAA antibody-coated latex particles, comprising the following steps: S1: suspending latex particles in 10-20 mM MES buffer and adjusting the pH to 5-7; S2: adding EDC and NHS, reacting 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: quenching the EDC with a 0.05-0.2 M quencher to terminate the catalytic activity of the unreacted EDC; S4: adjusting the pH of the solution to 7.2-7.5 to meet the needs of subsequent antibody coupling reactions; S5: adding an anti-SAA antibody in an amount equimolar to the latex particles, shaking the reaction on a shaker at room temperature for 2 hours to obtain a crude solution containing anti-SAA antibody-coated latex particles; S6: removing the supernatant from the crude solution containing the anti-SAA antibody-coated latex particles by centrifugation or filtration, diluting the target conjugate after the reaction with a 10 mM MES buffer, and adjusting the concentration of the latex particles to 10 g / L, adjust the pH to 6.5, divide into portions, and store at 4°C until use.

[0022] 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 quencher is a thiol reducing agent, and either 2-mercaptoethanol or TCEP is selected; in step S5: the mass concentration of the anti-SAA antibody in an equimolar amount to the latex particles is 1.5-2.5 mg / ml.

[0023] In a fourth aspect, the present invention provides a method for preparing a serum amyloid A assay kit for preparing the above-mentioned reagent composition, wherein the preparation process of reagent 1 is: taking a first buffer solution and adding it to a container, stirring at a speed of 200 to 400 rpm; then the inorganic salt, stabilizer, preservative, polyanion, first surfactant and polyethylene glycol 6000 in the reagent 1 component are added to the buffer solution in sequence according to a predetermined concentration ratio and mixed evenly, and finally the pH of the reagent 1 is adjusted to a pH value of 6.5-8.5 with a pH regulator, thereby preparing reagent 1; the preparation process of reagent 2 is: taking the anti-SAA antibody-coated latex particles prepared according to the above-mentioned preparation method of the anti-SAA antibody-coated latex particles, first diluting it with a second buffer solution to make its final concentration of 0.5-2.0 mg / mL, and then adding the inorganic salt, and / or stabilizer, second surfactant, preservative and DSS in the reagent 2 component to the second buffer solution in sequence according to the preset concentration and mixing evenly, and finally adjusting the pH to 6.0-8.0, thereby preparing reagent 2.

[0024] In a fifth aspect, the present invention also seeks to protect the use of the above-mentioned reagent composition, serum amyloid A assay kit, method for preparing anti-SAA antibody-coated latex particles, and method for preparing serum amyloid A assay kit in the preparation of latex reagent-related products.

[0025] The beneficial effects of the present invention are: The present invention provides a serum amyloid A assay kit. The human serum amyloid A assay kit of the present invention adopts latex immunoturbidimetry to determine the serum SAA content. The principle is that human anti-SAA antibodies are coated on latex particles, and after immune reaction with SAA in the specimen, aggregated particles are formed. At a certain wavelength, the turbidity formed by the aggregates is measured, and the content of the analyte in the specimen can be calculated by corresponding to the standard curve. The present invention optimizes the buffer system, the components and concentrations of inorganic salts in reagents 1 and 2. For example, different buffer systems are selected for reagents 1 and 2, and the concentration of inorganic salts is controlled within the range of 2%-15%, so that the performance index of the kit preferably configured by the present invention meets the minimum requirements of the kit performance evaluation, and can be comparable to or have little difference with the performance index of third-party reagents, and the sensitivity, anti-interference ability, stability, repeatability and correlation with third-party reagent detection of the serum SAA kit prepared according to the present invention are relatively excellent.

[0026] The polyanion, DSS, and anti-SAA antibody coated latex particles creatively added in the kit of the present invention use a quencher in the preparation process to further improve the performance of the serum SAA kit. In particular, the stability measurement value of the reagent with both polyanion and DSS added is significantly better than the stability without polyanion and DSS, and its stability measurement value is also significantly better than that of a third-party reagent. Therefore, the creative addition of polyanion and DSS is beneficial to improving the stability of the kit. The use of a quencher in the preparation method of the anti-SAA antibody coated latex particles makes its repeatability measurement value better than that of the control group without the quencher. Therefore, the present invention effectively improves the repeatability of reagent detection by using a quencher in the preparation process of the anti-SAA antibody coated latex particles.

[0027] The invention provides a preparation method of a serum amyloid A assay kit, optimizes five main components of a buffer, an electrolyte, a stabilizer, a surfactant and a preservative in reagents 1 and 2, creatively adds a polyanion to reagent 1, creatively adds DSS to reagent 2, and creatively introduces a quenching process in the preparation process of latex particles coated with anti-human serum amyloid A antibodies, thereby improving the stability of the chemical coupling between the latex particles and the anti-human serum amyloid A antibodies. As a result of layer-by-layer optimization, the interference ability of chylomicron in the assay process is also reduced. Finally, a kit prepared by the method has high sensitivity, specificity and stability in the assay, and the linear range of the assay is further improved.

[0028] In reagent 2, the present invention newly adds DSS, wherein DSS (4,4-Dimethyl-4-Silapentane-1-Sulfonic acid, 4,4-dimethyl-4-silapentane-1-sulfonic acid, or Disuccinimidyl subera, disuccinimidyl suberate), molecular formula: C16H20N2O8, molecular weight: 368.34, CAS number: 68528-80-3, is generally non-toxic to the human body and the environment, relatively safe, and is often used as a bifunctional cross-linking agent, a standard substance for nuclear magnetic hydrogen spectrum and carbon spectrum, or a pH indicator. As a cross-linking agent, it is often used in the chemical cross-linking of intracellular proteins before cell lysis and immunoprecipitation. It does not lyse, is insoluble in water, is soluble in organic solvents, has membrane permeability, and can be cross-linked in cells. The present invention creatively adds DSS to reagent 2 of the serum SAA kit. Compared with not using DSS, the deviation of the measured values ​​of the test samples during the long-term storage of the kit can be effectively reduced. That is, the addition of DSS helps to improve the accuracy of the measured values ​​of the kit, and effectively reduces the relative deviation of the measured values ​​while ensuring the stability of the kit.

[0029] In summary, the present invention provides a serum amyloid A assay kit, optimizes the preparation process of components such as buffer and surfactant in reagent 1 and reagent 2 and anti-SAA antibody-coated latex particles, and the optimized serum amyloid A assay kit can be successfully applied to latex-enhanced immunoturbidimetry for the determination of serum amyloid A, thereby improving the overall performance indicators of the kit such as sensitivity, linear range, anti-interference ability, stability, repeatability, etc. The linearity of the detection value within the linear range of 2 mg / L to 500 mg / L is good, accurate, and available, and it can be used on a fully automatic biochemical analyzer with a high degree of automation. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a linear evaluation result diagram of the serum amyloid A assay kit of the present invention, wherein a represents Example 1, and b represents Example 3; the abscissa is the theoretical value, in mg / L, and the ordinate is the measured value, in mg / L; Figure 2 The stability evaluation result diagram of the serum amyloid A assay kit of the present invention is shown, wherein A represents Example 3, B represents a third-party reagent, and C represents Example 4; the abscissa represents the storage time, with a whole month as a unit of measurement, and the unit is individuals; the ordinate represents the concentration value of the quantitative measurement, and the unit is mg / L; Figure 3This is a correlation evaluation result diagram of the serum amyloid A assay kit of the present invention, wherein the abscissa represents the measured value of Example 3, in mg / L, and the ordinate represents the measured value of a third-party reagent, in mg / L. DETAILED DESCRIPTION

[0031] Exemplary embodiments of the present invention are described in detail below in conjunction with the accompanying drawings, wherein the same or similar representations represent the same concept. In the following detailed description, for ease of explanation, many specific details are set forth to provide a comprehensive understanding of the disclosed embodiments. In the following detailed description, for ease of explanation, many specific details are set forth to provide a comprehensive understanding of the disclosed embodiments. The following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention.

[0032] The experimental methods used in the following examples are conventional methods unless otherwise specified, and the materials and reagents used are commercially available unless otherwise specified. Among them, human recombinant amyloid A (Cat. No. 610070) was purchased from Medix Biochemica; anti-human amyloid A monoclonal antibody (Cat. No. 100279) was purchased from Medix Biochemica; polyanionic cellulose (Cat. No. P875778) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; DSS (Cat. No. C426020010) was purchased from Sinopharm Chemical Reagent Co., Ltd.; latex particles were purchased from Jiehetai (Beijing) Biotechnology Co., Ltd.; activator EDC (Cat. No. N808856) and NHS (Cat. No. N811124) were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; fully automatic biochemical analyzer was purchased from Hitachi, Japan, model 7180; the third-party reagent was the SAA assay kit (latex immunoturbidimetry) purchased from Beijing Jiuqiang Biotechnology Co., Ltd. However, it is obvious that one or more embodiments can be implemented without these specific details. If no specific conditions are specified in the embodiments, the conventional conditions or the conditions recommended by the manufacturer are used. The molecular biology experimental methods not specifically described in the following embodiments are all carried out according to the specific methods listed in the book "Molecular Cloning Laboratory Manual" (3rd edition) by J. Sambrook, or according to the kits and product instructions.

[0033] The application object of the determination in the implementation of the present invention refers to a biological sample of body fluid collected from a human or animal body. After the sample collection work is completed, the biological sample has been separated from the living human or animal body, such as blood samples (whole blood / serum / plasma), body fluids, tissues, excrement, separation cultures (blood cultures, sputum cultures, etc.), etc., all of which are in vitro inanimate biological samples; the determination process is completed in vitro, and the direct purpose of the determination is to determine whether there is a target protein (serum amyloid A) or its amino acids from the degradation products of the target protein in the sample. The result of the determination is helpful to assist doctors in judging the consultation information. The determination belongs to the determination of components or contents in inanimate biological samples in vitro. There is no process of directly obtaining the diagnosis results of the disease or the health status in the determination. Therefore, the present invention does not belong to a method for diagnosing a disease, and meets the basic requirements of the Patent Law for patent protection objects. The specific embodiments of the present invention are described below.

[0034] The present invention provides a serum amyloid A assay kit with simple components and stability and its preparation and application. The present invention optimizes the five main components of buffer, electrolyte, stabilizer, surfactant and preservative in reagent 1 and reagent 2, and creatively adds polyanion to reagent 1 and DSS to reagent 2. In the preparation process of latex particles coated with anti-human serum amyloid A antibody, a quenching process is creatively introduced to improve the stability of chemical coupling between latex particles and anti-human serum amyloid A antibody. The result of layer-by-layer optimization also reduces the ability of chylomicron interference in the determination process. Finally, the sensitivity, specificity and stability of the kit in the determination are all very high, and the linear range of the determination is further improved. The prepared serum amyloid A assay kit adopts latex enhanced immunoturbidimetry to determine the serum SAA content in the sample to be tested. The determination principle is that human anti-SAA antibodies are coated on latex particles, and after specific immune reaction occurs with SAA in the sample to be tested, aggregated particles are formed. At a certain wavelength, the turbidity formed by the aggregates is measured, and the content of the measured substance in the sample can be calculated by corresponding to the standard curve of the standard product.

[0035] The preparation process of the serum amyloid A determination kit of the present invention mainly includes: preparing reagent 1, preparing anti-human serum amyloid A antibody-coated latex particles (abbreviated as anti-SAA antibody-coated latex particles) and preparing reagent 2, and finally configuring reagent 1, reagent 2, quality control products and standard products in a kit according to specification requirements.

[0036] The process of preparing reagent 1 is as follows: adding the first buffer solution into the container, stirring at a speed of 200-400 rpm, and then adding the components of reagent 1, electrolyte, stabilizer, (polyanion), first surfactant, preservative and polyethylene glycol 6000, into the buffer solution in sequence according to a predetermined concentration ratio and mixing, and finally adjusting the pH to within the range of 6.5-8.5.

[0037] The preparation process of anti-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, and after the pH is adjusted to 5-7, 12-15 mM 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) and 35-50 mM N-hydroxysuccinimide (NHS) are added and reacted 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 the latex particles and the amino groups on the antibody; secondly, EDC is quenched with a 0.05-0.2 M quencher. The purpose of this step is to terminate the catalytic activity of unreacted EDC and prevent it from continuing to catalyze nonspecific cross-linking reactions; next, the pH of the solution is adjusted to 5-7. Adjust to 7.2-7.5 to meet the needs of the subsequent antibody coupling reaction; add anti-human serum amyloid A antibody (abbreviated as anti-SAA antibody) in an amount equimolar to the latex particles. 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. Shake and block the reaction on a shaker at room temperature for 2 hours to allow the antibody to fully couple with the active groups on the surface of the latex particles; finally, remove the supernatant from the cross-linked 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, and adjust the pH to 6.5. Among them, the original quencher specifically verified the thiol reducing agents (such as 2-mercaptoethanol, 2-Hydroxy-1-ethanethiol and tris(2-carboxyethyl)phosphine, Tris(2-carboxyethyl)phosphine, abbreviated as TCEP). The purpose was to use thiol reducing agents to react with the active groups of EDC to make EDC lose its catalytic ability. Based on this purpose, it can also be reasonably predicted that ethanolamine, glycine and other quenchers replacing 2-mercaptoethanol may produce similar effects. The technical effects of different quenchers still depend on further verification of specific experiments.

[0038] The process of preparing reagent 2 is as follows: dilute the obtained anti-SAA antibody-coated latex particles with the second buffer to a final concentration of 0.5-2.0 mg / mL, add the electrolyte, stabilizer, second surfactant, preservative and / or DSS components of reagent 2 to the buffer in sequence according to the preset concentrations and mix well, and finally adjust the pH to a range of 6.0-8.0.

[0039] In addition, the volume ratio of reagent 1 and reagent 2 of the present invention is preferably 4:1, and the quality control and standard are prepared from human serum or a liquid similar to a serum matrix of human recombinant amyloid protein A, wherein the content of human serum is not less than 5%. The specific configuration process of the quality control is: human recombinant amyloid protein A is added to a glycine buffer to prepare a quality control product at two levels, high and low, wherein 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 configuration process of the standard is: human recombinant amyloid protein A is added to a 15-100 mmol / L glycine buffer and mixed to prepare 5 concentration series, the concentrations are: 10 mg / L, 60 mg / L, 150 mg / L, 300 mg / L and 500 mg / L, and then 5-10% human serum is added. In addition, the standard series also includes a human serum standard without human recombinant amyloid protein A, that is, the concentration of human recombinant amyloid protein A is 0 mg / L.

[0040] The preparation process and components of the serum SAA kit of the present invention, which is ultimately used to verify the performance indicators of the kit, are as follows, with emphasis on the components, preparation process and performance evaluation of reagent 1 and reagent 2.

[0041] Example 1: Serum SAA Kit 1 The main components, concentrations and preparation process of reagent 1 are as follows: the first buffer is 100 mmol / L HEPES buffer; the inorganic salt is 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. The first buffer is added to a container and stirred at a speed of 200 rpm, and then the components of reagent 1, inorganic salt, stabilizer, preservative, polyanion, first surfactant and polyethylene glycol 6000, are added to the buffer in sequence according to a predetermined concentration ratio and mixed evenly, and finally the pH of reagent 1 is adjusted to a pH value of 6.5 with a pH regulator, so as to prepare reagent 1.

[0042] The preparation process of anti-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, and 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; secondly, EDC is quenched with 0.05 M quencher (2-mercaptoethanol); next, the pH of the solution is adjusted to 7.2, and 1.5 mg / ml anti-SAA antibody is added in an amount equimolar to the latex particles, and the reaction is blocked on a shaker 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 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.

[0043] 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; and it also includes 150 mg / L DSS; the obtained anti-SAA antibody-coated latex particles are diluted with the second buffer to a final concentration of 0.5 mg / mL, and the inorganic salt, stabilizer, second surfactant, preservative and DSS components of reagent 2 are added to the second buffer in sequence according to the preset concentrations and mixed, and finally the pH is adjusted to 6.0 to prepare reagent 2.

[0044] Example 2: Serum SAA Kit 2 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. The first buffer is added to a container and stirred at a speed of 300 rpm; then the components of reagent 1, inorganic salt, stabilizer, preservative, first surfactant and polyethylene glycol 6000, are added to the buffer in sequence according to a predetermined concentration ratio and mixed evenly, and finally the pH of reagent 1 is adjusted to a pH value of 6.5-8.5 with a pH regulator, so as to prepare reagent 1.

[0045] The preparation process of anti-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, and 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, EDC is quenched with 0.15 M quencher (2-mercaptoethanol); next, the pH of the solution is adjusted to 7.3, and 1.75 mg / ml anti-SAA antibody is added in an amount equimolar to the latex particles, and the reaction is blocked on a shaker 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 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.

[0046] The main components, concentrations and preparation process of reagent 2 are as follows: the second buffer is 50 mmol / L aminoacetic acid 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; and also includes 50 mg / L DSS; the obtained anti-SAA antibody-coated latex particles are diluted with the second buffer to a final concentration of 0.9 mg / mL, and the inorganic salt, stabilizer, second surfactant, preservative and DSS components of reagent 2 are added to the second buffer in sequence according to the preset concentrations and mixed, and finally the pH is adjusted to 7.0 to prepare reagent 2.

[0047] Example 3: Serum SAA Kit 3 The main components, concentrations and preparation process of reagent 1 are as follows: the first buffer is 50 mmol / L phosphate buffer; the inorganic salt is 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 polyanionic cellulose; the first surfactant is 0.1% Tween 201; and 5% polyethylene glycol 6000. The first buffer is added to a container and stirred at a speed of 400 rpm; then the components of reagent 1, inorganic salt, stabilizer, preservative, polyanion, first surfactant and polyethylene glycol 6000 are added to the buffer in sequence according to a predetermined concentration ratio and mixed evenly, and finally the pH of reagent 1 is adjusted to a pH value of 7.2 with a pH regulator, so as to prepare reagent 1.

[0048] The preparation process of anti-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, and 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, EDC is quenched with 0.1 M quencher (2-mercaptoethanol); next, the pH of the solution is adjusted to 7.4, and 2 mg / ml anti-SAA antibody is added in an amount equimolar to the latex particles, and the reaction is blocked on a shaker 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 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.

[0049] 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; and also includes 100 mg / L DSS; the obtained anti-SAA antibody-coated latex particles are diluted with the second buffer to a final concentration of 1.3 mg / mL, and the inorganic salt, second surfactant, preservative and DSS components of reagent 2 are added to the second buffer in sequence according to the preset concentrations and mixed, and finally the pH is adjusted to 8.0 to prepare reagent 2.

[0050] Example 4: Serum SAA Kit 4 The main components, concentrations and preparation process of reagent 1 are as follows: the first buffer is 15mmol / L glycine buffer; the inorganic salt is 10% calcium chloride; the stabilizer is 0.4% gelatin; the preservative is 3% ProClin-300; the polyanion is 0.9mmol / L chondroitin sulfate; the first surfactant is 0.15% B66; and 4% polyethylene glycol 6000. The first buffer is added to a container and stirred at a speed of 200 rpm; then the components of reagent 1, inorganic salt, stabilizer, preservative, polyanion, first surfactant and polyethylene glycol 6000, are added to the buffer in sequence according to a predetermined concentration ratio and mixed evenly, and finally the pH of reagent 1 is adjusted to a pH value of 7.6 with a pH regulator, so as to prepare reagent 1.

[0051] The preparation process of anti-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, and 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; secondly, EDC is quenched with 0.1 M quencher (TCEP); next, the pH of the solution is adjusted to 7.5, and 2.25 mg / ml anti-SAA antibody is added in an amount equimolar to the latex particles, and the reaction is blocked on a shaker 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 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.

[0052] 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 anti-SAA antibody-coated latex particles obtained are diluted with the second buffer to a final concentration of 1.7 mg / mL, and the inorganic salt, stabilizer, second surfactant and preservative components of reagent 2 are added to the second buffer in sequence according to the preset concentrations and mixed, and finally the pH is adjusted to 7.0 to prepare reagent 2.

[0053] Example 5: Serum SAA Kit 5 The main components, concentrations and preparation process of reagent 1 are as follows: the first buffer is 200 mmol / L phosphate buffer; the inorganic salt is 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 polyanionic cellulose; the first surfactant is 0.2% Tween 20; and 5% polyethylene glycol 6000. The first buffer is added to a container and stirred at a speed of 300 rpm; then the components of reagent 1, inorganic salt, stabilizer, preservative, polyanion, first surfactant and polyethylene glycol 6000 are added to the buffer in sequence according to a predetermined concentration ratio and mixed evenly, and finally the pH of reagent 1 is adjusted to a pH value of 8.0 with a pH regulator, so as to prepare reagent 1.

[0054] The preparation process of anti-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 quencher, 40 mM N-hydroxysuccinimide (NHS) is added first and reacted at room temperature for 3 hours; after centrifugation to remove the supernatant, the precipitate is added to 10 mM MES buffer, the pH is adjusted to 6.0, and ultrasonic dispersion is performed; then 15 mM 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) is added to react for 1 h, the supernatant is removed by centrifugation, and the precipitate is added to 10 mM MES buffer; next, the pH of the solution is adjusted to 7.4, and 2.5 mg / ml anti-SAA antibody is added in an amount equimolar to the latex particles, and the reaction is blocked on a shaker at room temperature for 2 hours; finally, the cross-linked latex particles are centrifuged or filtered to remove the supernatant, and 10 The target conjugate after the reaction was ultrasonically dispersed and washed three times with mM MES buffer to make the concentration of anti-SAA antibody latex particles reach 10 g / L, and the pH was adjusted to 6.5. This coating method does not use a quencher, which may leave unreacted EDC. To ensure that each step is fully carried out, the step-by-step addition of EDC and NHS is conducive to better control of the reaction process of EDC and NHS, but the total reaction time is long (more than 5 hours), and it also involves multiple centrifugation, ultrasound and washing processes.

[0055] 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; and it also includes 200 mg / L DSS; the obtained anti-SAA antibody-coated latex particles are diluted with the second buffer to a final concentration of 2.0 mg / mL, and the inorganic salt, stabilizer, second surfactant, preservative and / or DSS components of reagent 2 are added to the second buffer in sequence according to the preset concentrations and mixed, and finally the pH is adjusted to 6.0 to prepare reagent 2.

[0056] Example 6: Serum SAA Kit 6 The main components, concentrations and preparation process of reagent 1 are as follows: the first buffer is 150 mmol / L aminoacetic acid 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. The first buffer is added to a container and stirred at a speed of 400 rpm; then the components of reagent 1, inorganic salt, stabilizer, preservative, polyanion, first surfactant and polyethylene glycol 6000 are added to the buffer in sequence according to a predetermined concentration ratio and mixed evenly, and finally the pH of reagent 1 is adjusted to a pH value of 8.5 with a pH regulator, so as to prepare reagent 1.

[0057] The preparation process of anti-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 10 mM MES buffer, and after adjusting the pH to 5, 12 mM 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) and 50 mM N-hydroxysuccinimide (NHS) are added, and the reaction is carried out at room temperature for 1 hour; secondly, EDC is quenched with 0.2M quencher (2-mercaptoethanol); next, the pH of the solution is adjusted to 7.2, and 2 mg / ml anti-SAA antibody is added in an amount equimolar to the latex particles, and the reaction is blocked on a shaker 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 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.

[0058] The main components, concentrations and preparation process of reagent 2 are as follows: the second buffer is 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; and it also includes 80 mg / L DSS; the obtained anti-SAA antibody-coated latex particles are diluted with the second buffer to a final concentration of 1.4 mg / mL, and the inorganic salt, stabilizer, second surfactant, preservative and / or DSS components of reagent 2 are added to the second buffer in sequence according to the preset concentrations and mixed, and finally the pH is adjusted to 8.0 to prepare reagent 2.

[0059] Example 7: Serum SAA test kit assay method The present invention adopts the two-point endpoint method to perform quantitative determination on the serum SAA kit shown in the above preferred prepared Examples 1 to 6. The determination process is as follows: set up three reaction tubes, including a blank tube, a sample tube and a calibration tube, wherein the blank tube is added with 2 μL of distilled water, the sample tube is added with 2 μL of the sample to be tested, and the calibration tube is added with 2 μL of the standard; first add 200 μL of reagent 1 solution to each of the three reaction tubes and incubate at 37°C for 5 minutes; then add 40 μL of reagent 2 solution to each reaction tube, mix for 30 seconds, and read the absorbance (A1) corresponding to the three reaction tubes with a fully automatic biochemical analyzer; then incubate at 37°C for 4 minutes, and read the absorbance (A2) corresponding to the three reaction tubes again with a fully automatic biochemical analyzer. The determination principle is the absorbance difference ΔA (i.e., the method of calculating the concentration or activity of the substance to be tested by measuring the total change in the concentration of the product or substrate from the start of the reaction to the time when the reaction reaches equilibrium), and the calculation formula is as follows: ΔA=A2-A1, .

[0060] Example 8: Performance indicators of serum SAA kit Take the sample to be tested, use the kit prepared in the above Examples 1 to 6, and perform the test analysis using a fully automatic biochemical analyzer according to the test method of Example 7. The test results are used to verify the specific performance indicators of the reagent, including sensitivity, linear range, anti-interference ability, stability, repeatability, and the correlation of the test results evaluated using third-party reagents. The units of the test value of the sample to be tested and the control value of the control reagent for the same sample to be tested are both mg / L. The samples to be tested are clinical samples containing different concentrations of serum amyloid A.

[0061] Sensitivity: The samples to be tested were 5 clinical serum samples containing different concentrations of amyloid A, which were respectively measured in parallel using the serum SAA kits prepared in the above-mentioned Examples 1, 3, 5, and 6, and each measurement was repeated 10 times. The sensitivity measurement results of Examples 1, 3, 5, and 6 are shown in Table 1 below.

[0062] Table 1 Sensitivity ; From the sensitivity measurement results in the above table, it can be seen that 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 of different concentrations. When the concentration of the sample to be tested is a low value of less than 5 mg / L, the overall difference in the coefficient of variation (CV value) is relatively large, but relatively speaking, the CV values ​​corresponding to the measured values ​​of Example 1, Example 3, and Example 6 are equivalent to the control, and are even slightly better than the control group. The SD values ​​are all less than 1, which shows that the measured data are relatively stable and reliable, and the SD values ​​under low concentration samples are also small, so the measurement results of these 4 kits are more stable and reliable, especially the SD value of the measurement result of the kit prepared in Example 3 is relatively the smallest, which shows that its sensitivity is relatively optimal.

[0063] Linear range: Human recombinant amyloid protein A was added to glycine buffer to prepare two fixed-value samples of high value (500 mg / L) and low value (2 mg / L) of determined concentrations, and then mixed in different proportions to further prepare 4 fixed-value samples of different concentrations, thereby preparing 6 fixed-value samples containing SAA serum of different concentrations, with theoretical concentrations of 500 mg / L, 350 mg / L, 250 mg / L, 150 mg / L, 50 mg / L, and 2 mg / L. The serum SAA kits prepared in the above examples were used for parallel determinations, and each determination was repeated 3 times. The two extreme linear determination results of Example 1 and Example 3 were plotted for more intuitive explanation, and the mean value (yi) of the determination results was used as the dependent variable, and the dilution concentration (xi) was used as the independent variable to draw a linear regression graph as shown in the figure. Figure 1 As shown, Table 2 exemplarily lists the specific measurement results of Example 3.

[0064] Table 2 Linearity measurement results of Example 3

[0065] 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), the regression equation corresponding to Example 3 is Y=0.9972x+1.9357 (R 2 =0.9999), the slopes are close to 1, which shows that there is a good linear relationship between the measured values ​​of Example 1 and Example 3 and the theoretical values, and the determination coefficient (R 2) is also very close to 1, indicating that the model fit is extremely high, further indicating that it has a good linear relationship, and the detection value in the linear range of 2mg / L to 500mg / L is more reliable. The intercept comparison shows that the intercept corresponding to Example 3 is relatively small, indicating that when the measured value is 0, the degree of deviation of the test result of the kit from the theoretical value (0) is small. The intercept corresponding to Example 1 is large and negative, indicating that when the measured value is 0, the test result of the kit is far lower than the theoretical value (0). When the concentration is low, there may be a deviation problem. Therefore, when the concentration is low, the deviation of the measured value corresponding to Example 3 is relatively small, and the accuracy of the measurement result in 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 that the consistency between the measured value and the theoretical value of Example 3 in the linear range of 2mg / L to 500mg / L is high. Combined with the corresponding linear regression graph, it can be seen that the linearity of the detection value of Example 3 in the range of 2mg / L to 500mg / L is good and available.

[0066] Anti-interference ability: The samples to be tested are from the same human serum sample. Due to the limited available dose of the collected serum sample, only the serum SAA kits prepared by Example 2, Example 3, and Example 5 and the third-party reagents are selected to perform parallel measurements on the samples to be tested. Due to the presence of chylomicrons (lipid components such as triglycerides) in the samples, the test result deviation is caused. If the effect of adding different amounts of triglycerides (TG) on the measured value and the theoretical value is detected, the ability of the kit to resist chylomicron interference can be characterized to a certain extent. Therefore, different amounts of triglycerides are added to the same human serum sample to prepare samples to be tested containing different concentrations of TG, and different kits are used to perform parallel measurements on them. Each measurement is repeated 3 times, and the average is calculated and the relative deviation is calculated. The results of the anti-interference ability measurement are shown in Table 3.

[0067] Table 3 Anti-interference capability ; The experimental results show that when the TG concentration is below 10 mmol / L, the measured values ​​of the four kits are highly consistent 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 less than 10%, and the anti-interference ability meets the requirements; and when the TG concentration is below 30 mmol / L (clinical samples with this high TG concentration are relatively rare), only the relative deviations of the measurement results of Example 3 are less than 10%, and the anti-interference ability 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 under the interference condition of the extreme content of TG concentration of 30 mmol / L, the serum SAA kit prepared in Example 3 still has a strong anti-interference ability, and the accuracy of the measured value is relatively reliable.

[0068] Stability evaluation: After placing Example 2, Example 3, Example 4 and the third-party reagent at 2-8°C for 0, 2, 4, 8, 12, and 16 months, the same sample to be tested was tested respectively; the sample to be tested originated from the same serum sample, which was packaged and stored in a -20°C refrigerator. During the test, it was randomly taken out for the serum SAA test, and each test was repeated 3 times. Results: The acceptable deviation of the measured value of the sample to be tested by the serum SAA kit prepared in Example 2 can be stably maintained for 14 months at 2-8°C, while the acceptable deviation of the measured value of the sample to be tested by the serum SAA kit prepared in Example 3 and Example 4 and the third-party reagent can be stably maintained for 16 months at 2-8°C. The linear changes of the measured mean values ​​of the three kits with storage time are as shown in the figure. Figure 2 As shown in the figure, it can be seen that the measured values ​​of the serum SAA kit prepared in Example 3 placed at 2-8°C for 0, 2, 4, 8, 12, and 16 months vary relatively little with the storage time, and the stability is relatively the best; since the serum SAA kit prepared in Example 4 does not use DSS in reagent 2, the deviation of the measured value of the test sample is obviously not as good as that of Example 3 in which DSS is added. It can be seen that adding DSS in reagent 2 helps to improve the accuracy of the measured value of the kit and effectively reduce the relative deviation of the measured value.

[0069] Repeatability: The sample to be tested was a randomly selected large-dose clinical human serum sample. The serum SAA kits prepared in Examples 3, 5 and 6 of the present invention were used to measure the sample to be tested, respectively. Each measurement was repeated 10 times, and the average value and coefficient of variation (CV) of the measured values ​​were calculated. The repeatability measurement results are shown in Table 4.

[0070] Table 4 Repeatability test results ; As can be seen from Table 4, the average values ​​corresponding to Examples 3, 5, and 6 are not much different, and the coefficient of variation (CV) is less than 10%, which is considered to be within an acceptable repeatability range. It can be seen that the fluctuations between the measured values ​​of the serum SAA kits prepared by Examples 3, 5, and 6 are small, and the repeatability of the three types of kits is relatively good. Furthermore, the minimum coefficient of variation corresponding to Example 3 is only 2.4%, which shows that the repeatability of the serum SAA kit prepared by Example 3 is significantly better than that of Examples 5 and 6.

[0071] Correlation: According to the above repeatability test results, the serum SAA kit prepared in Example 3 and the third-party reagent with better repeatability were used to parallel test the samples to be tested. The samples to be tested were 50 randomly selected clinical serum specimens. The measured values ​​of the two reagents on the same sample to be tested were used as the horizontal axis and the vertical axis, respectively, to draw a correlation curve of the measured values ​​of the two reagents as shown in FIG. Figure 3 As shown, the regression equation is y=1.012x-0.375 (R 2 =0.996), it can be seen that the measurement results of the two reagents are highly consistent and show good linear correlation, that is, the measured value of the serum SAA kit prepared by Example 3 has a good coincidence rate and has good accuracy and correlation.

[0072] Comprehensively analyzing the performance indicators of the serum SAA test kits prepared in the six embodiments, it can be seen that the present invention optimizes the buffer system, the components and concentrations of inorganic salts in reagents 1 and 2. For example, different buffer systems are selected for reagents 1 and 2, and the concentration of inorganic salts is controlled within the range of 2%-15%, so that the performance indicators of the test kits preferably configured by the present invention meet the minimum requirements of the performance evaluation of the test kits, and are comparable to or have little difference with 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 test kit prepared by Example 3 are relatively superior, and since the accuracy of the measurement results of Example 1 within the linear range is relatively better than the corresponding measured values ​​of Example 3, it can be inferred that the polyanions, DSS, and the use of quenchers in the preparation process of the anti-SAA antibody-coated latex particles creatively added in Examples 1 and 3 of the present invention can effectively improve the performance of the serum SAA test kit. The stability measurement results show that the stability measurement values ​​corresponding to Example 3 in which both polyanions and DSS were added were better than those of Example 2 in which no polyanions were added and Example 4 in which no DSS was added, and the stability measurement value corresponding to Example 3 was also significantly better than that of the third-party reagent, which shows that the polyanions and DSS creatively added by 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 a quencher were better than those of Example 5 without a quencher, which shows that the use of a quencher in the preparation of anti-SAA antibody-coated latex particles can improve the repeatability of reagent detection.

[0073] The specific embodiments of the present invention have been described in detail so that those skilled in the art will be easily understood. However, based on all the descriptions disclosed, different or similar modifications or replacements may be made to certain specific details, and these changes are within the scope of protection of the present invention. The full scope of the present invention is given by the attached 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, DSS, and anti-SAA antibody-coated latex particles; wherein the main components of the first buffer and the second buffer are different, and the final concentration of the inorganic salt in the reagent 1 and the reagent 2 are both 2%-15%; 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 used to prepare the anti-SAA antibody-coated latex particles is 50-180 nm, and the process of preparing the anti-SAA antibody-coated latex particles includes using a thiol reducing agent as a quenching agent to terminate the catalytic activity of unreacted EDC.

2. The reagent composition according to claim 1, characterized in that 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 first buffer and the second buffer are each selected from any one or two of 15-280 mmol / L Tris-HCl buffer, phosphate buffer, glycine buffer, HEPES buffer and aminoacetic acid buffer; 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 polyanion is selected from any one of 0.3-0.9 mmol / L polyanionic cellulose, polymaleic acid and chondroitin sulfate; 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%; The final concentration of DSS in reagent 2 is 50-200 mg / L.

4. The reagent composition according to claim 1, characterized in that The first buffer is a phosphate buffer; the second buffer is a glycine buffer; the inorganic salt contains at least sodium chloride; the stabilizer is bovine serum albumin; and the polyanion is polyanionic cellulose.

5. A serum amyloid A assay kit, characterized in that: The reagent composition comprises the reagent composition described in any one of claims 1 to 4, and further comprises a quality control product and a calibrator, wherein the quality control product and the calibrator both contain at least 2 concentration levels of human recombinant amyloid protein A, and the buffer is a glycine buffer containing human serum.

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

7. A method for preparing anti-SAA antibody coated latex particles, characterized in that: The following steps are involved: 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: quenching EDC with a quenching agent of 0.05-0.2 M to terminate the catalytic activity of unreacted EDC; S4: adjusting the pH of the solution to 7.2-7.5 to meet the needs of the subsequent antibody coupling reaction; S5: adding an anti-SAA antibody in an amount equimolar to the latex particles, shaking and blocking the reaction on a shaker 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 the 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, divide into portions, and store at 4°C for later use.

8. The method for preparing anti-SAA antibody coated latex particles according to claim 7, characterized in that: 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 quencher is a thiol reducing agent, and any one of 2-mercaptoethanol and TCEP is selected; In step S5: the mass concentration of the anti-SAA antibody in an equimolar amount to the latex particles is 1.5-2.5 mg / ml.

9. A method for preparing a serum amyloid A assay kit, characterized in that: Prepare the reagent composition described in any one of claims 1-2, wherein The preparation process of the reagent 1 is as follows: adding a first buffer solution into a container and stirring at a speed of 200 to 400 rpm; then adding the inorganic salt, stabilizer, preservative, polyanion, first surfactant and polyethylene glycol 6000 in the components of the reagent 1 into the buffer solution in sequence according to a predetermined concentration ratio and mixing; finally adjusting the pH of the reagent 1 to a pH value of 6.5-8.5 with a pH regulator, thereby preparing the reagent 1; The preparation process of reagent 2 is as follows: take the anti-SAA antibody-coated latex particles prepared according to the preparation method of anti-SAA antibody-coated latex particles described in any one of claims 7-8, first dilute them with a second buffer to a final concentration of 0.5-2.0 mg / mL, and then add the inorganic salts, and / or stabilizers, second surfactants, preservatives and DSS in the components of reagent 2 to the second buffer in sequence according to preset concentrations and mix them, and finally adjust the pH to 6.0-8.0 to prepare reagent 2.

10. The reagent composition described in any one of claims 1 to 4, the serum amyloid A assay kit described in any one of claims 5 to 6, the preparation method of anti-SAA antibody-coated latex particles described in any one of claims 7 to 8, and the preparation method of the serum amyloid A assay kit described in claim 9, and their use in the preparation of latex reagent-related products.

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