Method for improving linear range of latex-enhanced turbidimetry kit and latex-enhanced turbidimetry kit

Abstract

The application provides a method for improving the linear range of a latex-enhanced turbidimetry kit and the latex-enhanced turbidimetry kit, and relates to the technical field of analysis and detection. The application finds that sodium deoxycholate is added to reagent 1 of the existing latex-enhanced turbidimetry kit. Experimental verification shows that the correlation coefficient of the existing latex-enhanced turbidimetry kit can be effectively improved, and the upper limit of the detection linearity of the latex-enhanced turbidimetry kit is improved to 500 mg / L, thereby effectively alleviating the problem that the linear range of the reagent of the existing latex-enhanced turbidimetry kit is narrow, and 90% of the sample amount in the clinic cannot be covered, so as to affect the accuracy of the clinical detection result.

Description

Technical Field

[0001] This invention relates to the field of analytical testing technology, and in particular to a method for improving the linear range of a latex-enhanced turbidimetric assay kit and the latex-enhanced turbidimetric assay kit itself. Background Technology

[0002] Latex-enhanced turbidimetry is a method for detecting antigen content. It works by reacting the antigen to be detected in the sample with specific antibodies coated on latex microspheres in the detection reagent, forming immune complexes. This causes the latex microspheres to connect into a network, thereby increasing the turbidity of the reaction solution. Within a certain range, the turbidity is positively correlated with the concentration of the antigen to be detected in the sample. Therefore, by measuring the absorbance of the reaction system at a specific wavelength and referring to a calibration curve, the content of the antigen to be detected in the sample can be calculated.

[0003] Compared with other detection methods, latex-enhanced turbidimetry has a high throughput, short processing time, and can be automated, reducing the influence of human factors on the test results, while also possessing good sensitivity and accuracy. Therefore, latex immunoturbidimetry has promising application prospects.

[0004] However, in the development of latex-enhanced turbidimetric assays, when the linear range of the reagents is not wide enough, optimization is generally required at the reagent preparation stage. For example, changing the antibody / antigen manufacturer, type, dosage, or reaction ratio. However, these methods significantly alter the original reagent kit process, introducing highly unstable factors. When the above methods fail, optimization measures are taken at the reagent application stage, such as changing the type of buffer pair for R1, PEG concentration, or salt concentration. These methods are relatively simple and do not directly affect the stability of the reagent, but they often do not have a significant effect. The linear upper limit of most reagent kits on the market can only reach between 200-300 mg / L.

[0005] Therefore, existing latex-enhanced turbidimetric assay kits suffer from a narrow linear range, failing to cover 90% of clinical sample volumes and thus affecting the accuracy of clinical test results. Developing a method to improve the linear range of these kits has become both necessary and urgent.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] The primary objective of this invention is to provide a method for improving the linear range of a latex-enhanced turbidimetric assay kit. This method can effectively improve the correlation coefficient of existing latex-enhanced turbidimetric assay kits and raise the upper limit of the detection linearity of the latex-enhanced turbidimetric assay kit to 500 mg / L, thereby effectively alleviating the problem of the narrow linear range of the reagent in existing latex-enhanced turbidimetric assay kits.

[0008] A second objective of this invention is to provide a latex-enhanced turbidimetric assay kit, which is prepared by the method described above for improving the linear range of the latex-enhanced turbidimetric assay kit.

[0009] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0010] According to one aspect of the present invention, a method for improving the linear range of a latex-enhanced turbidimetric assay kit is provided, the latex-enhanced turbidimetric assay kit comprising reagent 1 and reagent 2, the method comprising adding sodium deoxycholate to reagent 1 of the latex-enhanced turbidimetric assay kit.

[0011] Preferably, the method includes adding sodium deoxycholate to reagent 1 of the latex-enhanced turbidimetric assay kit at a mass ratio of 0.05% to 0.4%;

[0012] Preferably, the method includes adding sodium deoxycholate to reagent 1 of the latex-enhanced turbidimetric assay kit at a mass ratio of 0.05% to 0.2%.

[0013] Preferably, the latex-enhanced turbidimetric assay kit includes any one of the following: serum amyloid protein assay kit, C-reactive protein assay kit, D-dimer assay kit, myoglobin assay kit, ferritin assay kit, or plasma lipoprotein phospholipase A2 assay kit.

[0014] Preferably, the latex-enhanced turbidimetric assay kit is a serum amyloid protein detection kit, wherein sodium deoxycholate is added to reagent 1 at a mass ratio of 0.05% to 0.2%; more preferably, at a mass ratio of 0.05% to 0.15%.

[0015] Alternatively, the latex-enhanced turbidimetric assay kit is a D-dimer detection kit, wherein sodium deoxycholate is added to reagent 1 at a mass ratio of 0.05% to 0.2%; preferably, at a mass ratio of 0.15% to 0.2%.

[0016] Alternatively, the latex-enhanced turbidimetric assay kit is a myoglobin detection kit, wherein sodium deoxycholate is added to reagent 1 at a mass ratio of 0.05% to 0.2%; preferably 0.1% to 0.2% by mass.

[0017] Alternatively, the latex-enhanced turbidimetric assay kit is a C-reactive protein assay kit, wherein sodium deoxycholate is added to reagent 1 at a mass ratio of 0.05% to 0.2%.

[0018] Alternatively, the latex-enhanced turbidimetric assay kit is a ferritin assay kit, wherein sodium deoxycholate is added to reagent 1 at a mass ratio of 0.15% to 0.2%.

[0019] Alternatively, the latex-enhanced turbidimetric assay kit is a plasma lipoprotein phospholipase A2 assay kit, wherein sodium deoxycholate is added to reagent 1 at a mass ratio of 0.05% to 0.2%; preferably 0.1% to 0.2% by mass.

[0020] According to another aspect of the present invention, a latex-enhanced turbidimetric assay kit is also provided, wherein the detection linear range of the latex-enhanced turbidimetric assay kit is 2-500 mg / L; the kit comprises reagent 1, reagent 2 and sodium deoxycholate.

[0021] Preferably, the working concentration of sodium deoxycholate in reagent 1 is 0.05% to 0.4% wt, and more preferably 0.05% to 0.2% wt.

[0022] Preferably, the latex-enhanced turbidimetric assay kit includes any one of the following: serum amyloid protein detection kit, C-reactive protein detection kit, D-dimer detection kit, myoglobin detection kit, ferritin detection kit, or plasma lipoprotein phospholipase A2 detection kit;

[0023] Preferably, the latex-enhanced turbidimetric assay kit is selected from any one of (A1) to (A6);

[0024] (A1) Serum amyloid protein detection kit, with a working concentration of sodium deoxycholate of 0.05% to 0.2% wt; preferably 0.05% to 0.15% wt;

[0025] (A2) D-dimer detection kit, the working concentration of sodium deoxycholate is 0.05% to 0.2% wt; preferably 0.15% to 0.2% wt;

[0026] (A3) Myoglobin assay kit, with sodium deoxycholate at a working concentration of 0.05%–0.2% wt; preferably 0.1%–0.2% wt;

[0027] (A4) C-reactive protein assay kit, sodium deoxycholate working concentration is 0.05% to 0.2% wt;

[0028] (A5) Ferritin assay kit, sodium deoxycholate working concentration is 0.15% to 0.2% wt;

[0029] (A6) Plasma lipoprotein phospholipase A2 assay kit, with sodium deoxycholate at a working concentration of 0.05% to 0.2% wt; preferably 0.1% to 0.2% wt.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] This invention, through research, discovered that adding sodium deoxycholate to reagent 1 of an existing latex-enhanced turbidimetric assay kit can effectively improve the correlation coefficient of the kit and raise its upper limit of linearity to approximately 500 mg / L. This effectively alleviates the problem of the existing latex-enhanced turbidimetric assay kit having a narrow linear range, failing to cover 90% of clinical sample volumes, and thus affecting the accuracy of clinical test results.

[0032] The latex-enhanced turbidimetric assay kit provided by this invention has a high linear range, with a detection linear range of 2-500 mg / L. Attached Figure Description

[0033] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0034] Figure 1 This is a linear range graph of the SAA detection kit containing different amounts of sodium deoxycholate provided in Example 1 of the present invention. Detailed Implementation

[0035] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] According to one aspect of the present invention, a method for improving the linear range of a latex-enhanced turbidimetric assay kit, the latex-enhanced turbidimetric assay kit comprising reagent 1 and reagent 2;

[0037] The method involves adding sodium deoxycholate to reagent 1 of the latex-enhanced turbidimetric assay kit.

[0038] This invention, through research, discovered that adding sodium deoxycholate to reagent 1 of an existing latex-enhanced turbidimetric assay kit can effectively improve the correlation coefficient of the kit and raise its upper limit of linearity to approximately 500 mg / L. This effectively alleviates the problem of the existing latex-enhanced turbidimetric assay kit having a narrow linear range, failing to cover 90% of clinical sample volumes, and thus affecting the accuracy of clinical test results.

[0039] It is speculated that the role of sodium deoxycholate in reagent 1 of the latex-enhanced turbidimetric assay kit of the present invention is to improve the linear range by utilizing the dispersing effect of sodium deoxycholate. Specifically, due to the complex composition of serum samples and the close connections between proteins, the addition of sodium deoxycholate can break these connections, causing the proteins to disperse and significantly increasing the turbidity during the reaction. Therefore, a linear broadening will be observed during instrument testing.

[0040] It should be noted that in actual clinical applications, some latex-enhanced turbidimetric assay kits have relatively wide linear range requirements for clinical testing. For example, patients with severe SAA often have their levels rise to high values ​​of 500-1000 mg / L within a few hours.

[0041] Therefore, when the linear range of a reagent is insufficient, existing technologies typically optimize the reagent preparation process. However, this alters the original reagent kit manufacturing process, introducing significant instability. Another approach is to optimize the reagent application, such as changing the type of buffer pair, PEG concentration, or salt concentration of R1. While this method doesn't directly affect the stability of the reagent, it often proves ineffective. Consequently, most commercially available reagent kits have a linear upper limit of only 200-300 mg / L, thus impacting the accuracy of clinical test results.

[0042] During the research and development process, it was discovered that by simply adding a certain amount of sodium deoxycholate to reagent 1 of the latex-enhanced turbidimetric assay kit, the correlation coefficient of the existing latex-enhanced turbidimetric assay kit can be effectively improved, and the upper limit of the detection linearity of the latex-enhanced turbidimetric assay kit can be increased to about 500 mg / L.

[0043] It should also be noted that the method of improving the linear range of the latex-enhanced turbidimetric assay kit in this invention is different from existing methods for extending the linear range of latex-enhanced turbidimetric assay kits. For example: 1. Increasing the antibody concentration in the system. 2. Changing the proportions of each component in the reaction system. 3. Adjusting the salt concentration in the system.

[0044] It has the following advantages: 1. No increased cost: The material cost of the kit is mainly concentrated on the antibody; without increasing the amount of antibody, the cost will not increase. 2. Does not disrupt the stability of the original system: Adding antibody will introduce instability to the entire system. 3. No need to adjust the pre-determined reaction ratios and parameters. 4. Does not change the original performance; it does not change the salt concentration in the system, as changes in salt concentration will lead to changes in other properties of the kit.

[0045] In a preferred embodiment of the present invention, the method includes adding sodium deoxycholate to reagent 1 of the latex-enhanced turbidimetric assay kit at a mass ratio of 0.05%-0.4%, for example, at a mass ratio of 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, or 0.4%, or any range between two points.

[0046] In a preferred embodiment, the sodium deoxycholate is added at a mass ratio of 0.05%-0.4%. It should be noted that the specific amount of sodium deoxycholate added is related to the specific turbidimetric reaction system, and those skilled in the art can select the addition mass ratio within the range of 0.05%-0.4% according to actual needs.

[0047] In the preferred embodiment described above, the method includes adding sodium deoxycholate to reagent 1 of the latex-enhanced turbidimetric assay kit at a mass ratio of 0.05 to 0.2%.

[0048] In a preferred embodiment of the present invention, the latex-enhanced turbidimetric assay kit includes any one of the following: serum amyloid protein assay kit (SAA), C-reactive protein assay kit (CRP), D-dimer assay kit (DD), myoglobin assay kit (MYO), ferritin assay kit (FER), or plasma lipoprotein phospholipase A2 assay kit (Lp-PLA2).

[0049] As a preferred embodiment, the method for extending the linear range of the reagent kit provided by this invention can improve the linear range of test results in various detection projects and can be widely applied to various latex immunoturbidimetric assays. Examples include: SAA, CRP, DD, MYO, FER, Lp-PLA2, etc.

[0050] In the preferred embodiment described above, the latex-enhanced turbidimetric assay kit is a serum amyloid protein detection kit, wherein sodium deoxycholate is added to reagent 1 at a mass ratio of 0.05% to 0.2%, including but not limited to 0.05%, 0.1%, 0.15%, or 0.2%, or any mass ratio between any two points.

[0051] Alternatively, the latex-enhanced turbidimetric assay kit is a D-dimer detection kit, wherein sodium deoxycholate is added to reagent 1 at a mass ratio of 0.05% to 0.2%, including but not limited to 0.05%, 0.1%, 0.15%, or 0.2%, or any mass ratio between any two points.

[0052] Alternatively, the latex-enhanced turbidimetric assay kit is a myoglobin assay kit, wherein sodium deoxycholate is added to reagent 1 at a mass ratio of 0.05% to 0.2%, including but not limited to 0.05%, 0.1%, 0.15%, or 0.2%, or any mass ratio between any two points.

[0053] Alternatively, the latex-enhanced turbidimetric assay kit is a C-reactive protein assay kit, wherein sodium deoxycholate is added to reagent 1 at a mass ratio of 0.05% to 0.2%, including but not limited to 0.05%, 0.1%, 0.15%, or 0.2%, or any mass ratio between any two points.

[0054] Alternatively, the latex-enhanced turbidimetric assay kit is a ferritin assay kit, wherein sodium deoxycholate is added to reagent 1 at a mass ratio of 0.15% to 0.2%, including but not limited to 0.15%, 0.16%, 0.18%, or 0.2%, or any mass ratio between any two points.

[0055] Alternatively, the latex-enhanced turbidimetric assay kit is a plasma lipoprotein phospholipase A2 assay kit, wherein sodium deoxycholate is added to reagent 1 at a mass ratio of 0.05% to 0.2%; including but not limited to 0.05%, 0.1%, 0.15%, or 0.2%, or any mass ratio between any two points.

[0056] In the preferred embodiment described above, the latex-enhanced turbidimetric assay kit is a serum amyloid protein detection kit, wherein sodium deoxycholate is added to reagent 1 at a mass ratio of 0.05% to 0.15%.

[0057] Alternatively, the latex-enhanced turbidimetric assay kit is a D-dimer detection kit, wherein sodium deoxycholate is added to reagent 1 at a mass ratio of 0.15% to 0.2%;

[0058] Alternatively, the latex-enhanced turbidimetric assay kit is a myoglobin detection kit, wherein sodium deoxycholate is added to reagent 1 at a mass ratio of 0.1% to 0.2%.

[0059] Alternatively, the latex-enhanced turbidimetric assay kit is a plasma lipoprotein phospholipase A2 assay kit, wherein sodium deoxycholate is added to reagent 1 at a mass ratio of 0.1% to 0.2%.

[0060] In a preferred embodiment of the present invention, reagent 2 in the latex-enhanced turbidimetric assay kit is a dispersion of latex particles coated with antibodies.

[0061] Preferably, the antibody coated in reagent 2 corresponds to the detection substance.

[0062] Preferably, reagent 1 in the latex-enhanced turbidimetric assay kit includes a surfactant and a buffer solution that promotes antigen-antibody reactions.

[0063] In the preferred embodiment described above, the antibody-coated latex particles are microspheres of a single particle size.

[0064] Preferably, the antibody comprises rabbit anti-human antibody.

[0065] Preferably, the latex particles coated with rabbit anti-human antibodies have a particle size of 30-200 nm.

[0066] Preferably, the rabbit anti-human antibody includes rabbit anti-human SAA antibody or rabbit anti-human Fer antibody.

[0067] As a preferred implementation, in the SAA project, the linear range is required to be above 0-300 mg / L. In order to achieve this linear range, the particle size of the antibody-coated latex particles is preferably selected to be 30-200 nm.

[0068] In a preferred embodiment of the present invention, the pH value of reagent 1 is 4.5-9.0, preferably 7.5;

[0069] The pH value of reagent 2 is 4.5-9.0, preferably 7.4.

[0070] It should be noted that the pH values ​​of reagents 1 and 2 can be adjusted by those skilled in the art according to the requirements of reactivity. The purpose is to keep the pH of reagents 1 and 2 as close to neutral as possible, and to avoid side reactions caused by excessive acidity or alkalinity.

[0071] Preferably, the latex-enhanced turbidimetric assay kit is an SAA detection kit, comprising reagent 1 and reagent 2;

[0072] (1) The components and concentrations of reagent 1 are as follows:

[0073] Buffer solution containing one or more of the following: acetic acid, phosphate, and citric acid: 100-200mM;

[0074] One or two of sodium chloride or potassium chloride: 3-10 g / L;

[0075] Ammonium sulfate: 1-8 g / L;

[0076] Preservative P300: 0.5-2 g / L;

[0077] Polymerization accelerator PEG-6000 or polyvinylpyrrolidone (PVP): 10-50 g / L;

[0078] EDTA-2Na: 20-200 mg / L;

[0079] The mass ratio of sodium deoxycholate in reagent 1 is 0.05%-0.4%;

[0080] The solvent is purified water, and the pH of reagent 1 is 7.5;

[0081] (2) The components and concentrations of reagent 2 are as follows:

[0082] (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) buffer: 50 mM;

[0083] Rabbit anti-human SAA antibody coated latex particles: 1%;

[0084] Polyoxyethylene sorbitan fatty acid ester: 1-5 g / L;

[0085] Trehalose: 2g / L;

[0086] Glycerin: 1 g / L;

[0087] The solvent is purified water, and the pH of reagent 2 is 7.4.

[0088] Among them, the rabbit anti-human SAA antibody coated latex particles in reagent 2 are single-size microspheres with a particle size between 30-200 nm.

[0089] According to one aspect of the present invention, a latex-enhanced turbidimetric assay kit is also provided, wherein the detection linear range of the latex-enhanced turbidimetric assay kit is 2-500 mg / L; the latex-enhanced turbidimetric assay kit comprises reagent 1, reagent 2 and sodium deoxycholate.

[0090] In an optional embodiment, sodium deoxycholate is premixed in reagent 1.

[0091] In an optional embodiment, sodium deoxycholate is premixed in reagent 1, and the latex-enhanced turbidimetric kit is a kit in which sodium deoxycholate is premixed in reagent 1, prepared according to the aforementioned method for improving the linear range of the latex-enhanced turbidimetric kit.

[0092] In an optional embodiment, the latex-enhanced turbidimetric assay kit includes individually packaged sodium deoxycholate.

[0093] In an optional embodiment, the latex-enhanced turbidimetric assay kit includes individually packaged sodium deoxycholate, and reagents 1 and 2 in the kit are derived from a basic latex-enhanced turbidimetric assay kit with a detection linear range of less than 300 mg / L. In this embodiment, the individually packaged sodium deoxycholate is used to improve the linear range of the basic latex-enhanced turbidimetric assay kit. Furthermore, reagent 1 in the basic latex-enhanced turbidimetric assay kit may optionally contain or not contain sodium deoxycholate. The basic latex-enhanced turbidimetric assay kit can be a commercially available kit or a basic latex-enhanced turbidimetric assay kit with a linear range of less than 300 mg / L formulated according to general knowledge in the art.

[0094] In an optional embodiment, the working concentration of sodium deoxycholate in reagent 1 is 0.05% to 0.4% wt, for example, but not limited to 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, or 0.4% wt, or any range between two points, preferably 0.05% to 0.2% wt. In this document, the working concentration refers to the concentration of sodium deoxycholate in reagent 1 during detection.

[0095] In an optional embodiment, the latex-enhanced turbidimetric assay kit includes any one of the following: serum amyloid protein assay kit, C-reactive protein assay kit, D-dimer assay kit, myoglobin assay kit, ferritin assay kit, or plasma lipoprotein phospholipase A2 assay kit.

[0096] In an optional embodiment, the latex-enhanced turbidimetric assay kit is selected from any one of (A1) to (A6);

[0097] (A1) Serum amyloid protein detection kit, wherein the working concentration of sodium deoxycholate is 0.05% to 0.2% wt, including but not limited to 0.05, 0.1, 0.15 or 0.2% wt, or any range between two points; preferably 0.05% to 0.15% wt;

[0098] (A2) D-dimer detection kit, the working concentration of sodium deoxycholate is 0.05% to 0.2% wt, including but not limited to 0.05, 0.1, 0.15 or 0.2% wt, or any range between two points; preferably 0.15% to 0.2% wt;

[0099] (A3) Myoglobin assay kit, wherein the working concentration of sodium deoxycholate is 0.05% to 0.2% wt, including but not limited to 0.05, 0.1, 0.15 or 0.2% wt, or any range between two points; preferably 0.1% to 0.2% wt;

[0100] (A4) C-reactive protein assay kit, with sodium deoxycholate working concentration of 0.05% to 0.2% wt, including but not limited to 0.05, 0.1, 0.15 or 0.2% wt, or any range between two points;

[0101] (A5) Ferritin assay kit, with sodium deoxycholate working concentration of 0.15% to 0.2% wt, including but not limited to 0.15, 0.16, 0.17, 0.18, 0.19 or 0.2% wt, or any range between two points;

[0102] (A6) Plasma lipoprotein phospholipase A2 assay kit, wherein the working concentration of sodium deoxycholate is 0.05% to 0.2% wt, including but not limited to 0.05, 0.1, 0.15 or 0.2% wt, or any range between two points; preferably 0.1% to 0.2% wt.

[0103] In an optional embodiment, reagents 1 and 2 in the provided latex-enhanced turbidimetric kit are the same as those described in the aforementioned method for improving the linear range of the latex-enhanced turbidimetric kit.

[0104] In an optional embodiment, when reagent 1 contains sodium deoxycholate, the pH of reagent 1 is 4.5-9.0, preferably greater than 6, and more preferably 7.5.

[0105] The technical solution of the present invention will be further described below with reference to the embodiments.

[0106] Example 1

[0107] (a) An SAA detection kit, comprising reagent 1 and reagent 2;

[0108] (1) The components and concentrations of reagent 1 are as follows:

[0109] Buffer solution containing one or more of the following: acetic acid, phosphate, and citric acid: 150mM;

[0110] One or both of sodium chloride and potassium chloride: 3 g / L;

[0111] Ammonium sulfate: 2.5 g / L;

[0112] Preservative P300: 0.5 g / L;

[0113] Polymerization accelerator PEG-6000 or polyvinylpyrrolidone (PVP): 30 g / L;

[0114] EDTA-2Na: 50 mg / L;

[0115] Sodium deoxycholate accounts for 0.15% of the mass of reagent 1;

[0116] The solvent is purified water, and the pH of reagent 1 is 7.5;

[0117] (2) The components and concentrations of reagent 2 are as follows:

[0118] (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) buffer: 50 mM;

[0119] Rabbit anti-human SAA antibody coated latex particles: 1%;

[0120] Polyoxyethylene sorbitan fatty acid ester: 2.4 g / L;

[0121] Trehalose: 2g / L;

[0122] Glycerin: 1 g / L;

[0123] The solvent is purified water, and the pH of reagent 2 is 7.4.

[0124] (3) The preparation methods of reagents 1 and 2 in the SAA detection kit include:

[0125] According to the component amounts of Reagent 1, add each component to the buffer solution in sequence and stir thoroughly. Then add the solvent water and continue stirring until well mixed. Adjust the pH of the solution to 7.5 and filter the solution through a microporous membrane. The resulting filtrate is Reagent 1.

[0126] Activation of microspheres: Take a small amount of carboxyl microspheres and disperse them by ultrasonication. Add an appropriate buffer solution and add 0.1g of activator EDC.

[0127] Antibody conjugation: A certain amount of SAA monoclonal antibody was added to the activated microsphere latex and stirred in a shaker at 37°C for five hours.

[0128] Sealing: Add a certain amount of sealing liquid.

[0129] Dispersion: After adding the stabilizer, take out the latex and place it in an ultrasonic instrument, and sonicate it in an ice bath for 30 minutes.

[0130] Aging: Place the dispersed reagent in an oven at 37°C for 24 hours.

[0131] The aged solution was filtered using a microporous membrane, and the resulting filtrate was reagent 2.

[0132] Mark it and wait for testing.

[0133] (II) Detect the sample to be tested using the above-mentioned SAA detection kit:

[0134] The test conditions for determining SAA in the sample were as follows: temperature: 37℃; cuvette optical path: 1.0 cm; main detection wavelength: 570 nm; no secondary wavelengths.

[0135] The method for determining SAA in the sample is as follows: After mixing the sample with reagent 1, incubate at 37°C for 5 minutes. Then add reagent 2, mix well, incubate at 37°C for 30 seconds, and read the absorbance A1. Continue incubation for 4 minutes and 30 seconds, and read the absorbance A2; ΔA = A2 - A1. The sample volume is 3 μl, reagent 1 volume is 240 μl, and reagent 2 volume is 60 μl.

[0136] The specific test results are shown in the table below:

[0137]

[0138] Figure 1 This is a linear range graph of SAA detection kits containing different amounts of sodium deoxycholate in Reagent 1 of this embodiment; wherein, Figure 1 In each chart, the horizontal axis represents the actual concentration (mg / L); the vertical axis represents the actual measured concentration (mg / L).

[0139] Comparative Example 1

[0140] It should be noted that although this invention hypothesizes that sodium deoxycholate utilizes the dispersing effect of its surfactant to improve the linear range, the applicant selected multiple surfactants for experiments during the research and development process, and set up four gradient concentrations for each.

[0141] However, comparisons of correlation coefficients show that different surfactant types have varying effects on the linear range, but only sodium deoxycholate exhibits a significant improvement, with its correlation coefficient increasing from 0.982 to over 0.999. Simultaneously, the absolute or relative deviation is reduced, resulting in a significant improvement in the linear range. Therefore, the present invention's method of adding sodium deoxycholate to reagent 1 of an existing latex-enhanced turbidimetric assay kit to improve the upper limit of the detection linearity of the kit should not be constrained by theory, as not all surfactants possess the effects of this invention.

[0142] The comparative experiments and data for other surfactants are as follows:

[0143] (1) The selected comparative surfactants are shown in the table below:

[0144]

[0145] (2) Preparation of this comparative sample detection kit:

[0146] Except for replacing sodium deoxycholate in reagent 1 of the SAA detection kit in Example 1 with the aforementioned comparative surfactant, the composition and preparation method of reagents 1 and 2 in the other kits are the same as in Example 1.

[0147] The test samples were then tested using the aforementioned comparison kit, with the specific methods and parameters being the same as in Part (II) of Example 1. The test results are as follows:

[0148] Table: Anionic Polyacrylamide

[0149]

[0150] Table: Sodium Alkylbenzene Sulfonate

[0151]

[0152] Table: Sodium Oleoxyethanesulfonate

[0153]

[0154]

[0155] Table: Cationic Polyacrylamide

[0156]

[0157] Table: Imidazolin sodium

[0158]

[0159] Table: Morpholine Guanidines

[0160]

[0161]

[0162] Table: Triazine Derivatives

[0163]

[0164] Table: Sulfobetaine

[0165]

[0166] Table: Carboxylic acid betaine

[0167]

[0168]

[0169] Table: Phosphobetaine

[0170]

[0171] Table: Octylphenyl polyoxyethylene ether TX-100

[0172]

[0173] Table: OP Emulsifiers

[0174]

[0175] In summary, different surfactant types have varying effects on the linear range, but only sodium deoxycholate shows a significant improvement, with its correlation coefficient increasing from 0.982 to over 0.999. Simultaneously, the absolute or relative deviation is reduced, resulting in a significant improvement in the linear range. Other surfactants cannot achieve the technical effects of Example 1 of this invention.

[0176] Furthermore, research has revealed that the method for extending the linear range of the reagent kit provided by this invention can improve the linear range of test results in various detection projects and can be widely applied to various latex immunoturbidimetric assays. For example: SAA, CRP, DD, MYO, FER, Lp-PLA2. See Examples 2-6 below for details.

[0177] Example 2

[0178] A D-dimer (DD) detection kit currently sold by Wuhan Hanhai New Enzyme Biotechnology Co., Ltd. includes reagent 1 and reagent 2; the batch numbers of the two reagents are as follows: R1 20220726 and R220221121.

[0179] The above R1 was divided into several groups, and sodium deoxycholate of different concentrations (0.05%, 0.10%, 0.15%, 0.20%) was added to each group.

[0180] The test conditions for determining DD in the sample using the detection reagent are as follows: temperature: 37℃; cuvette optical path: 1.0 cm; main detection wavelength: 700 nm; no secondary wavelengths.

[0181] The method for determining DD in the sample is as follows: After mixing the sample with reagent 1, incubate at 37°C for 5 minutes. Then add reagent 2, mix well, incubate at 37°C for 30 seconds, and read the absorbance A1. Continue incubation for 4 minutes and 30 seconds, and read the absorbance A2; ΔA = A2 - A1. The sample volume is 4 μl, reagent 1 volume is 180 μl, and reagent 2 volume is 60 μl.

[0182] The specific test results are shown in the table below:

[0183]

[0184]

[0185] Note: SDC refers to sodium deoxycholate.

[0186] As can be seen from the above, since the linear range of DD in clinical practice is generally only 20 mg / L, in order to continuously improve its linear range, this invention attempts to add a certain amount of sodium deoxycholate to reagent 1, which can significantly increase the correlation coefficient from the original 0.837 to more than 0.9, and the linear range is also significantly improved.

[0187] Example 3

[0188] A myoglobin (MYO) detection kit currently sold by Wuhan Hanhai New Enzyme Biotechnology Co., Ltd. includes reagent 1 and reagent 2; the batch numbers of the two reagents are as follows: R1 20221130 batch and R2 20220923 batch.

[0189] R1 was divided into several groups, and sodium deoxycholate of different concentrations (0.05%-0.10%-0.15%-0.20%) was added to each group.

[0190] The test conditions for determining MYO in the sample using the detection reagent are as follows: temperature: 37℃; cuvette optical path: 1.0 cm; main detection wavelength: 570 nm; no secondary wavelengths.

[0191] The method for determining MYO in the sample is as follows: After mixing the sample with reagent 1, incubate at 37°C for 5 minutes. Then add reagent 2, mix well, incubate at 37°C for 30 seconds, and read the absorbance A1. Continue incubation for 4 minutes and 30 seconds, and read the absorbance A2; ΔA = A2 - A1. The sample volume is 6 μl, reagent 1 volume is 180 μl, and reagent 2 volume is 60 μl.

[0192] The specific test results are shown in the table below:

[0193]

[0194]

[0195] Note: SDC refers to sodium deoxycholate.

[0196] Currently, the linear range of myo in clinical practice is generally around 800 ng / mL. In this invention, we attempted to add a small amount of sodium deoxycholate to its R1 reagent, which significantly improved the correlation coefficient from the original 0.951 to over 0.99, indicating a significant improvement in the linear range.

[0197] Example 4

[0198] A C-reactive protein (CRP) detection kit currently sold by Wuhan HanHai New Enzyme Biotechnology Co., Ltd. includes reagent 1 and reagent 2; the batch numbers of the two reagents are as follows: R1 20220829-w1 batch and R2 20220829-w1 batch.

[0199] R1 was divided into several groups, and sodium deoxycholate of different concentrations (0.05%-0.10%-0.15%-0.20%) was added to each group.

[0200] The test conditions for determining CRP in samples using the detection reagent are as follows: temperature: 37℃; cuvette optical path: 1.0 cm; main detection wavelength: 570 nm; no secondary wavelengths.

[0201] The method for determining CRP in the sample is as follows: After mixing the sample with reagent 1, incubate at 37°C for 5 minutes. Then add reagent 2, mix well, incubate at 37°C for 30 seconds, and read the absorbance A1. Continue incubation for 4 minutes and 30 seconds, and read the absorbance A2; ΔA = A2 - A1. The sample volume is 2 μl, reagent 1 volume is 100 μl, and reagent 2 volume is 100 μl.

[0202] The specific test results are shown in the table below:

[0203]

[0204]

[0205] Note: SDC refers to sodium deoxycholate.

[0206] Currently, the linear range of CRP in clinical practice is generally 500 mg / L. This application attempts to improve the linear range by adding a small amount of sodium deoxycholate to its R1 reagent, and the correlation coefficient is also increased from the original 0.994 to over 0.998.

[0207] Example 5

[0208] A ferritin (Fer) detection kit is currently being developed by Wuhan HanHai New Enzyme Biotechnology Co., Ltd. The kit includes reagent 1 and reagent 2.

[0209] The components and concentrations of Reagent 1 are as follows: a combination buffer of one or more of acetic acid, phosphate, and citric acid: 50 mM; one or two of sodium chloride or potassium chloride: 0.9 g / L; ammonium sulfate: 2.4 g / L; preservative P300: 0.1 g / L; polymerization promoter PEG-6000 or polyvinylpyrrolidone PVP: 5 g / L; EDTA-2Na: 20 mM; solvent is purified water, and the pH of Reagent 1 is 7.5.

[0210] The components and concentrations of Reagent 2 are as follows: (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) buffer: 50 mM; rabbit anti-human Fer antibody-coated latex particles: 1%; polyoxyethylene sorbitan fatty acid ester: 1-5 g / L; trehalose: 2 g / L; glycerol: 1 g / L; the solvent is purified water, and the pH of Reagent 2 is 7.4. The rabbit anti-human Fer antibody-coated latex particles in Reagent 2 are single-size microspheres with a particle size between 30-200 nm.

[0211] The specific preparation method of Fer detection reagent is as follows:

[0212] Activation of microspheres: Take a small amount of carboxyl microspheres and disperse them by ultrasonication. Add an appropriate buffer solution and add 0.1g of activator EDC.

[0213] Antibody conjugation: A certain amount of FER monoclonal antibody was added to the activated microsphere latex and stirred in a shaker at 37°C for five hours.

[0214] Sealing: Add a certain amount of sealing liquid.

[0215] Dispersion: After adding the stabilizer, take out the latex and place it in an ultrasonic instrument, and sonicate it in an ice bath for 30 minutes.

[0216] Aging: Place the dispersed reagent in an oven at 37°C for 24 hours.

[0217] Mark it and wait for testing.

[0218] S1. Preparation of Reagent 1: According to the component amounts of Reagent 1, add each component to the buffer solution in sequence and stir thoroughly. Then add the solvent water and continue stirring. Adjust the pH of the solution to 7.5 and filter the solution using a microporous membrane. The resulting filtrate is Reagent 1.

[0219] R1 was divided into several groups, and sodium deoxycholate of different concentrations (0.05%-0.10%-0.15%-0.20%) was added to each group.

[0220] The test conditions for determining Fer in the sample using the detection reagent are as follows: temperature: 37℃; cuvette optical path: 1.0 cm; dominant detection wavelength: 546 nm; no secondary wavelengths.

[0221] The method for determining Fer in the sample is as follows: After mixing the sample with reagent 1, incubate at 37°C for 5 minutes. Then add reagent 2, mix well, incubate at 37°C for 30 seconds, and read the absorbance A1. Continue incubation for 4 minutes and 30 seconds, and read the absorbance A2; ΔA = A2 - A1. The sample volume is 3 μl, reagent 1 volume is 200 μl, and reagent 2 volume is 50 μl.

[0222] The specific test results are shown in the table below:

[0223]

[0224] Note: SDC refers to sodium deoxycholate.

[0225] Currently, the linear range of FER in clinical practice is generally 800 ng / mL. In this application, we attempted to add a small amount of sodium deoxycholate to its R1 reagent, which improved the correlation coefficient from the original 0.988 to over 0.999, and the relative deviation could be controlled within 5%.

[0226] Example 6

[0227] A plasma lipoprotein phospholipase A2 (Lp-PLA2) detection kit, currently available from Wuhan Hanhai New Enzyme Biotechnology Co., Ltd., includes reagent 1 and reagent 2; the batch numbers of the two reagents are as follows: R1 20221130 batch and R2 20220923 batch.

[0228] R1 was divided into several groups, and sodium deoxycholate of different concentrations (0.05%-0.10%-0.15%-0.20%) was added to each group.

[0229] The test conditions for determining Lp-PLA2 in the sample using the detection reagent are as follows: temperature: 37℃; cuvette optical path: 1.0 cm; main detection wavelength: 600 nm; no secondary wavelengths.

[0230] The method for determining Lp-PLA2 in the sample is as follows: After mixing the sample with reagent 1, incubate at 37°C for 5 minutes. Then add reagent 2, mix well, incubate at 37°C for 30 seconds, and read the absorbance A1. Continue incubation for 4 minutes and 30 seconds, and read the absorbance A2; ΔA = A2 - A1. The sample volume is 3 μl, reagent 1 volume is 240 μl, and reagent 2 volume is 60 μl.

[0231] The specific test results are shown in the table below:

[0232]

[0233] Note: SDC refers to sodium deoxycholate.

[0234] Currently, the linear range of LP-PLA2 in clinical practice is generally 2000 ng / mL. In this application, we attempted to add a small amount of sodium deoxycholate to its R1 reagent, which improved the correlation coefficient from the original 0.91 to 0.99, and the relative deviation can be controlled within 15%.

[0235] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for improving the linear range of a latex-enhanced turbidimetric assay kit, characterized in that, The latex-enhanced turbidimetric assay kit is a serum amyloid protein detection kit, comprising reagent 1 and reagent 2. The method includes adding sodium deoxycholate to reagent 1 of the latex-enhanced turbidimetric assay kit at a mass ratio of 0.05% to 0.2%, wherein: (1) The components and concentrations of reagent 1 are as follows: Buffer solution containing one or more of the following: acetic acid, phosphate, and citric acid: 100~200mM; One or two of sodium chloride or potassium chloride: 3~10g / L; Ammonium sulfate: 1~8 g / L; Preservative P300: 0.5~2g / L; Polymerization accelerator PEG-6000 or polyvinylpyrrolidone (PVP): 10~50g / L; EDTA-2Na: 20~200 mg / L; The mass ratio of sodium deoxycholate in reagent 1 is 0.05%~0.4%; The solvent is purified water, and the pH of reagent 1 is 4.5~9.0; (2) The components and concentrations of reagent 2 are as follows: (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) buffer: 50 mM; Rabbit anti-human SAA antibody coated latex particles: 1%; Polyoxyethylene sorbitan fatty acid ester: 1~5g / L; Trehalose: 2g / L; Glycerin: 1 g / L; The solvent is purified water, and the pH of reagent 2 is 4.5~9.0; The upper limit of the detection linearity of the latex-enhanced turbidimetric assay kit can reach 500 mg / L.

2. The method for improving the linear range of the latex-enhanced turbidimetric assay kit according to claim 1, characterized in that, Reagent 2 in the latex-enhanced turbidimetric assay kit is a dispersion of antibody-coated latex particles.

3. The method for improving the linear range of the latex-enhanced turbidimetric assay kit according to claim 2, characterized in that, The antibody coated in reagent 2 corresponds to the detection substance.

4. The method for improving the linear range of the latex-enhanced turbidimetric assay kit according to claim 1, characterized in that, The latex particles coated with the rabbit anti-human SAA antibody are microspheres of a single particle size.

5. The method for improving the linear range of the latex-enhanced turbidimetric assay kit according to claim 4, characterized in that, The latex particles have a particle size of 30~200nm.

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