Kit for determining adiponectin by latex enhanced immunoturbidimetry

Abstract

A kit for determining adiponectin by latex enhanced immunoturbidimetry comprises a first reagent, a second reagent, a calibrator and a quality control product, the first reagent comprises the following components by mass concentration: 10 mmol / L to 100 mmol / L of a buffer agent, 20 g / L to 70 g / L of inorganic salt, 1 g / L to 20 g / L of a stabilizer, 3 g / L to 20 g / L of a coagulant, 3 g / L to 20 g / L of a surfactant and 0.05 g / L to 5 g / L of a preservative; the second reagent is obtained by diluting a latex microsphere particle suspension coated with an adiponectin antibody with a preservation solution to a certain weight, and the content of latex microsphere particles coated with the adiponectin antibody in the second reagent is 0.5 g / L-3 g / L (based on the mass of latex microspheres); the preserving fluid comprises the following components: 10-50mmol / L of a buffering agent, 5g / L-100g / L of a stabilizing agent and 0.05 g / L-2g / L of a preservative.

Description

Technical Field

[0001] The invention relates to the field of immunoassay, and in particular to a kit for measuring adiponectin (ADP) by nano-enhanced immunoturbidimetry. Background Art

[0002] Adiponectin is an insulin-sensitizing hormone. As a metabolic disease marker and an indicator for monitoring prognosis, it plays an important role in regulating insulin sensitivity and glucose metabolism. It can dynamically reflect the body's insulin resistance level and is a risk prediction indicator for diabetes. Adiponectin can be used to screen out high-risk people with diabetes whose blood sugar is still normal but whose physiological metabolism is already abnormal. In addition, plasma ADP levels are significantly negatively correlated with the incidence of myocardial infarction. Therefore, plasma ADP levels can better predict the degree of early atherosclerosis. In addition, adiponectin also has anti-inflammatory, anti-atherosclerotic, tumor cell growth inhibition and apoptosis promotion effects. It is a predictive indicator of risk factors for multiple diseases such as diabetes, metabolic syndrome, coronary heart disease, and tumors.

[0003] Adiponectin determination method: The detection technologies that have been patented in China include a human serum adiponectin detection kit based on microparticle chemiluminescence immunoassay (publication number CN105759061A), a competitive inhibition enzyme-linked immunosorbent assay kit and method for adiponectin (publication number CN102721819A), an adiponectin content detection kit and its preparation method (publication number CN104237522A), and an adiponectin content detection kit based on latex-enhanced immunoturbidimetric analysis method and its preparation method (authorization number CN104237522B). Among them, the latex-enhanced immunoturbidimetric method is to coat the antibody of adiponectin on latex particles with a diameter of 90-200nm, so that the volume of the adiponectin antigen and adiponectin antibody conjugate is increased. After the light passes through, the intensity changes of the transmitted light and the scattered light are more significant, thereby improving the sensitivity of the test. The common material of latex particles is polystyrene. The advantages of latex-enhanced immunoturbidimetric method are reflected in short detection time, simple operation steps, and wide clinical application.

[0004] According to the "Expert Consensus on Screening and Intervention of High-Risk Populations for Diabetes" (2022, and verified by data from 400 healthy individuals), the reference range of ADP is women: >3.4 mg / L, men: >3 mg / L, so the accuracy, linearity, and repeatability of the reagent in detecting low-value samples is particularly important.

[0005] However, the existing latex-enhanced immunoturbidimetry method for determining adiponectin uses chemical adsorption to couple latex and adiponectin antibodies, and the detection accuracy and repeatability are poor in the range close to the actual detection limit. The operation steps are complicated and the coupling process is cumbersome.

[0006] Therefore, providing an improved latex-enhanced immunoturbidimetric method for determining adiponectin, which can improve the detection accuracy and repeatability within the actual detection limit while simplifying the process steps, has become an urgent problem to be solved in the prior art. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides an improved latex enhanced immunoturbidimetric kit for detecting adiponectin. At the same time, by changing the antibody and microsphere coupling process and optimizing the preparation process, the accuracy, linearity, repeatability and other performances of the reagent in detecting low-value samples are greatly improved, and the efficiency of reagent preparation is also improved, the stability of reagent performance is ensured, the batch difference of the reagent is reduced, and the reagent cost is reduced. The accuracy, linearity, repeatability and other performances of the reagent in detecting low-value samples are improved.

[0008] The technical solution of the present invention is as follows:

[0009] A kit for determining adiponectin by latex enhanced immunoturbidimetry, comprising a first reagent, a second reagent, a calibrator and a quality control product, wherein the first reagent is composed of the following components in mass concentration: a buffer of 10 mmol / L to 100 mmol / L, an inorganic salt of 20 g / L to 70 g / L, a stabilizer of 1 g / L to 20 g / L, a coagulant of 3 g / L to 20 g / L, a surfactant of 3 g / L to 20 g / L, and a preservative of 0.05 g / L to 5 g / L;

[0010] The second reagent is obtained by diluting a suspension of latex microsphere particles coated with adiponectin antibodies with a preservation solution to determine the weight, and the content of latex microsphere particles coated with adiponectin antibodies in the second reagent is 0.5g / L-3g / L (based on the mass of latex microspheres); the composition and content of the preservation solution are: buffer 10-50mmol / L, stabilizer 5g / L-100g / L, preservative 0.05g / L-2g / L;

[0011] The calibration product and the quality control product are obtained by dissolving the adiponectin antigen in a dispersion, and the components and mass concentrations of the dispersion are: buffer 5mmol / L-100mmol / L, inorganic salt 5g / L-40g / L, stabilizer 5g / L-80g / L, preservative 0.05g / L-2g / L; the adiponectin antigen content of the calibration product is 1mg / L-100mg / L; the adiponectin antigen content of the quality control product is 1mg / L-100mg / L.

[0012] The kit for determining adiponectin by latex-enhanced immunoturbidimetry further comprises:

[0013] The buffer in the first reagent is selected from any one or more of a phosphate buffer, a 3-[NN-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid buffer, a barbiturate buffer, a N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid, a 2-(N-morpholine)ethanesulfonic acid, and a 3-morpholinepropanesulfonic acid buffer;

[0014] The surfactant in the first reagent is selected from any one or more of ethylphenyl polyethylene glycol and Triton X-100.

[0015] The stabilizer in the first reagent is selected from any one or more of bovine serum albumin (BSA), sucrose, trehalose, and ovalbumin,

[0016] The preservative in the first reagent is selected from any one or more of Proclin 300, sodium azide, phenol, gentamicin, and thimerosal.

[0017] The inorganic salt in the first reagent is selected from any one or more of magnesium chloride, sodium chloride, potassium chloride, calcium chloride, and aluminum chloride.

[0018] The coagulant in the first reagent is selected from any one or more of polyethylene glycol 8000, polyethylene glycol 6000, and polyethylene glycol 2000.

[0019] The kit for determining adiponectin by latex-enhanced immunoturbidimetry is further preferably configured such that the contents of the components in the first reagent are:

[0020] The buffer is a phosphate buffer with a concentration of 10mmol / L to 50mmol / L.

[0021] The inorganic salt is sodium chloride, with a concentration of 30g / L to 60g / L.

[0022] The stabilizer is bovine serum albumin, with a concentration of 1g / L to 10g / L.

[0023] The coagulant is polyethylene glycol 6000, with a concentration of 3g / L to 10g / L.

[0024] The surfactant is Triton X-100 with a concentration of 3g / L to 10g / L.

[0025] The preservative is sodium azide with a concentration of 0.05g / L to 2g / L.

[0026] The kit for determining adiponectin by latex-enhanced immunoturbidimetry is further preferably wherein the components and contents of the first reagent are:

[0027] The buffer is a phosphate buffer with a pH of 7.3 to 7.5 and a concentration of 10 mmol / L to 15 mmol / L.

[0028] The inorganic salt is sodium chloride, with a concentration of 40g / L to 60g / L.

[0029] The stabilizer is bovine serum albumin, with a concentration of 1.5g / L to 2.5g / L.

[0030] The coagulant is polyethylene glycol 6000, with a concentration of 4g / L to 6g / L.

[0031] The surfactant is Triton X-100 with a concentration of 4g / L to 6g / L.

[0032] The preservative is sodium azide with a concentration of 0.5g / L to 2g / L.

[0033] The kit for determining adiponectin by latex-enhanced immunoturbidimetry further comprises:

[0034] The stabilizer of the preservation solution in the second reagent is selected from any one or more of sucrose, trehalose, and glucose, preferably 70 to 90 g / L of sucrose;

[0035] The preservative in the second reagent is selected from any one or more of Proclin 300, sodium azide, phenol, gentamicin, and thimerosal, preferably 0.5 to 2 g / L of sodium azide;

[0036] The buffer in the storage solution in the second reagent is selected from any one or more of phosphate buffer, 3-[NN-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid buffer, barbiturate buffer, N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid, and 3-morpholinepropanesulfonic acid buffer; preferably, N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid buffer with a pH of 7.9 to 8.1 and a content of 8 to 12 mol / L;

[0037] The method for preparing the latex microsphere particle suspension coated with adiponectin antibody in the second reagent comprises the following steps:

[0038] 1) diluting the latex microsphere suspension with a weakly acidic buffer to obtain a buffered microsphere suspension,

[0039] 2) Prepare a chelating agent solution by diluting with a weakly acidic buffer,

[0040] 3) The chelating agent solution is then added to the buffered microsphere suspension and shaken to mix, thereby activating the microspheres to obtain an activated microsphere suspension.

[0041] 4) Diluting the antibody solution with a buffer to obtain a diluted antibody solution

[0042] 5) Add the diluted antibody solution to the activated microsphere suspension and shake to mix for coating reaction to obtain a coated microsphere suspension.

[0043] 6) adding a blocking agent to the coated microsphere suspension to perform a blocking reaction to obtain a suspension of latex microsphere particles coated with adiponectin antibodies;

[0044] The blocking agent is selected from: any one or more of bovine serum albumin (BSA), sucrose, trehalose, and ovalbumin;

[0045] The weak acid buffer is 2-(N-morpholine)ethanesulfonic acid, and the buffers in the remaining steps are selected from: any one or more of a phosphate buffer, a 3-[NN-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid buffer, a barbiturate buffer, a N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid, and a 3-morpholinepropanesulfonic acid buffer;

[0046] The chelating agent in the second reagent is selected from any one or more of sodium ethylenediaminetetraacetate, 1-ethyl-3[3-dimethylaminopropyl]carbodiimide hydrochloride, and diethylenetriaminepentaacetic acid.

[0047] The kit for determining adiponectin by latex-enhanced immunoturbidimetry is further characterized in that: in the method for preparing the suspension of latex microsphere particles coated with adiponectin antibodies in the second reagent, preferably:

[0048] In step 1), the mass percentage content of microspheres in the latex microsphere suspension is 8% to 12%, and the particle size of the microspheres is 90-200 nm: the acidic buffer is 2-(N-morpholine)ethanesulfonic acid buffer, with a concentration of 8 to 12 mmol / L and a pH value of 5.5 to 5.7, and the volume of the latex microsphere suspension is 50% to 70% of the volume of the weakly acidic buffer.

[0049] In step 2), the chelating agent is 1-ethyl-3[3-dimethylaminopropyl]carbodiimide hydrochloride, and the concentration of the chelating agent solution is 1 mg / mL-6 mg / mL.

[0050] Step 3) specifically comprises adding the chelating agent solution obtained in step 2) to the buffered microsphere suspension obtained in step 1), and shaking on a shaker for 15 min to 30 min to activate the microspheres, wherein the volume of the chelating agent solution is 60% to 80% of the volume of the buffered microsphere suspension;

[0051] In step 4), the antibody solution is diluted with 3-morpholinepropanesulfonic acid buffer having a concentration of 40 mmol / L to 60 mmol / L and a pH of 7.7 to 7.9, wherein the volume of the antibody solution is 10% to 30% of the volume of the buffer solution and the antibody concentration is 5 to 20 mg / L.

[0052] In step 5), the diluted antibody solution obtained in step 4) is added to the activated microsphere suspension solution for coating reaction to obtain a coated microsphere suspension. The reaction time is 1-18 hours. The reaction container needs to be shaken on a shaker at a speed of 50-280 rpm.

[0053] In step 6), the blocking agent is bovine serum albumin, and the blocking reaction is completed by shaking on a shaker for 1-2 hours. The mass ratio of bovine serum albumin as a blocking agent to latex microspheres is 1.5-2 mg / g.

[0054] In the kit for determining adiponectin by latex-enhanced immunoturbidimetry, the method for preparing the suspension of latex microsphere particles coated with adiponectin antibodies in the second reagent is further preferably:

[0055] The concentration of the chelating agent solution in step 2) is 2.5-3.5 mg / ml;

[0056] The antibody in step 3) is a rabbit polyclonal antibody, and the antibody concentration is 8-12 mol / L;

[0057] In step 5), the reaction time is 1 to 2 hours, and the shaking speed is 150-250 rpm.

[0058] The kit for determining adiponectin by latex-enhanced immunoturbidimetry, further, the buffer in the dispersion is selected from any one or more of phosphate buffer, 3-[NN-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid buffer, barbiturate buffer, N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid, 2-(N-morpholine)ethanesulfonic acid, 3-morpholinepropanesulfonic acid buffer, preferably a phosphate buffer with a pH of 7.3 to 7.5;

[0059] The stabilizer in the dispersion is selected from any one or more of bovine serum albumin, sucrose, trehalose, and ovalbumin; preferably 8 to 12 g / L of bovine serum albumin

[0060] The preservative in the dispersion is selected from any one or more of Proclin 300, sodium azide, phenol, gentamicin, and thimerosal, preferably 0.5 to 2 g / L of sodium azide.

[0061] The inorganic salts in the dispersion are independently selected from any one or more of magnesium chloride, sodium chloride, potassium chloride, calcium chloride and aluminum chloride, preferably 8 to 10 g / L of sodium chloride.

[0062] The kit for determining adiponectin by latex-enhanced immunoturbidimetry, further, the volume ratio of the first reagent to the second reagent is 4:1.

[0063] The kit provided by the present invention is based on latex immunoturbidimetry (PETIA), the principle of which is to use a method combining physical adsorption and chemical coupling to bind specific antibodies to the surface of latex microspheres of a certain particle size. When the microspheres cross-linked with antibodies bind to antigens, they quickly aggregate together in a short period of time, thereby changing the absorbance of the reaction solution. According to the increase in absorbance, the turbidity of the immune complex can be measured at a wavelength of 600nm, and the content of adiponectin (ADP) in serum can be quantitatively detected. Compared with the prior art, the beneficial effects of the present invention are:

[0064] 1) Different from other latex reagents, the present invention adopts a method combining physical adsorption and chemical coupling in the process of coupling antibodies and microspheres, wherein physical adsorption is mainly used, and a high-concentration antibody solution is added to a high-concentration microsphere solution, and violent stirring is performed at a high speed, so that the antibody solution can be dispersed in the entire system at the moment of contact with the microsphere solution, and the physical adsorption process is completed within 10 minutes. A small amount of chemical coupling is performed in the next 50 minutes, thereby achieving a process that takes 4-18 hours for traditional chemical coupling in a short time.

[0065] 2) To ensure the stability of the system, the present invention has made detailed adjustments to the concentration of each substance in the reaction system, the reaction time, the stirring speed, etc. On the basis of ensuring the detection performance of the reagent, there is no need to centrifuge and repeatedly wash the latex microsphere particle suspension coated with the adiponectin antibody. The latex microsphere particle suspension coated with the adiponectin antibody is directly diluted to the required concentration with a preservation solution and then ultrasonically dispersed to obtain the second reagent. This further saves operating steps, reduces material consumption and operating difficulty, and is more suitable for large-scale production; the overall preparation environment of the test kit does not require special requirements, and room temperature is sufficient; the type of chelating agent is simplified from two to one; in addition, the high concentration of antibodies and latex microspheres during coupling reduces the total volume of the solution in the coupling step, which also makes it possible to prepare a large amount in a smaller container, ensuring the stability of the reagent performance, reducing the difference between reagent batches, and reducing the cost of the reagent.

[0066] 3) The present invention provides a reagent kit, the linear minimum value of the reagent detection sample can be 0.2 mg / L, the reagent can detect 0.5 mg / L sample within the batch coefficient of variation can reach within 4%, the inter-batch coefficient of variation can reach within 5%, and the day-to-day coefficient of variation can reach within 7%. The detection performance can meet actual needs. DETAILED DESCRIPTION

[0067] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the technical solutions of the present invention are described in detail below in conjunction with specific embodiments.

[0068] Example 1: Preparation of adiponectin (ADP) assay kit

[0069] 1. The first reagent (R1) is:

[0070]

[0071] 2. The raw materials and specifications of the second reagent (R2) are as follows:

[0072] N-[Tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid buffer (pH 8.0)

[0073] 2-(N-morpholine)ethanesulfonic acid buffer (pH 5.5)

[0074] 3-Morpholinepropanesulfonic acid buffer (pH 7.8) 50mmol / L

[0075] 1-Ethyl-3[3-dimethylaminopropyl]carbodiimide hydrochloride(EDC)

[0076] BSA

[0077] sucrose

[0078] Sodium azide 1g / L

[0079] Latex microsphere particles coated with adiponectin antibody 0.5g / L~3g / L (based on the mass of latex microspheres)

[0080] The second reagent preparation method is as follows:

[0081] (1) Buffered latex microsphere suspension: 30 ml of latex microsphere suspension (microsphere particle size: 150 nm, microsphere content: 10% wt) was diluted with 43 mL of 10 mmol / L 2-(N-morpholine)ethanesulfonic acid buffer (pH: 5.5) to obtain a buffered microsphere suspension.

[0082] (2) Preparation of chelating agent solution: Weigh the chelating agent, 1-ethyl-3-methyl-(3-dimethylaminopropyl)carbamide (EDC), and dissolve it in 10 mmol / L 2-(N-morpholine)ethanesulfonic acid buffer (pH 5.5) to obtain a chelating agent solution with a chelating agent concentration of 3 mg / ml.

[0083] (3) Activating microspheres: adding a chelating agent solution to the buffered microsphere suspension obtained in step (1) to activate the microspheres for 20 min to obtain an activated microsphere suspension. The volume of the chelating agent solution is 80% of the volume of the microsphere suspension. The process needs to be carried out on a shaker.

[0084] (4) Diluting the adiponectin antibody solution: dilute 12 mL of the adiponectin antibody solution (antibody concentration: 10 mg / mL) with 60 mL of 50 mmol / L 3-morpholinepropanesulfonic acid buffer (pH: 7.8) to obtain a diluted antibody solution.

[0085] (5) Add the diluted antibody solution to the activated microsphere suspension for coating reaction to obtain a coated microsphere suspension. The reaction time is 1 hour. The process needs to be carried out on a shaker at a speed of 200 rpm.

[0086] (6) Add 50 μL BSA (100 g / L) to the coated microsphere suspension and seal for 1 h to obtain a suspension of latex microsphere particles coated with adiponectin antibodies. This process needs to be carried out on a shaker.

[0087] (6) Preparation of preservation solution: Add bovine serum albumin (10 g / L), sucrose (80 g / L) and preservative (sodium azide 1 g / L) to 10 mmol / L N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid buffer (pH = 8.0).

[0088] (7) The latex microsphere particle suspension coated with adiponectin (ADP) antibody in step 6 was weighed to 500 mL with preservation solution and then directly dispersed by ultrasonication. After the end of ultrasonication, preservation solution was added to weigh to 1 L and then dispersed by ultrasonication to obtain the second reagent (3 g / L based on the mass of latex microspheres).

[0089] 3. Calibrators and quality control products are obtained by dissolving adiponectin (ADP) antigen in dispersion.

[0090] The dispersion consists of the following components in mass concentrations:

[0091]

[0092] The ADP antigen concentration of the standard is 64 mg / L, and the ADP antigen concentration of the quality control is 5 mg / L

[0093] The standard product is diluted with purified water to multiple levels. When calibrated, the standard values ​​of antigen content are 0mg / L (purified water), 4mg / L, 8mg / L, 32mg / L, 64mg / L.

[0094] Example 2: Usage of the detection kit

[0095] Take Hitachi 7180 biochemical analyzer as an example: first add 160μL of the first reagent, then add 3μL of the calibrator, mix well, incubate for 5 minutes, add 40μL of the second reagent, mix well and incubate for 30 seconds, detect the absorbance value A1 of each calibration point at a wavelength of 700nm, 5min later, detect the absorbance value A2 of each calibration point at a wavelength of 700nm, calculate the absorbance difference ΔA=A2-A1, repeat the measurement twice for each tube, use the absorbance value ΔA measured twice for each calibrator tube as the ordinate, and the corresponding concentration as the abscissa, make a "concentration-absorbance difference" calibration curve, take the sample to be tested, determine the absorbance difference of the sample by the same method, substitute it into the calibration curve, and you can calculate the content of adiponectin (ADP) in the sample to be tested.

[0096] Example 3: Taking the kit prepared in Example 1 as an example, the detection performance of the kit of the present invention was tested using a Hitachi 7180 fully automatic biochemical analyzer.

[0097] 1. Intra-batch reproducibility

[0098] The ADP samples with detection concentrations of 0.5 mg / L, 1 mg / L and 2 mg / L were tested 10 times using the kit prepared in Example 1, and the average value, standard deviation (SD) and coefficient of variation (CV) were calculated. The results are shown in Table 3 below, and it can be seen that the repeatability CV is ≤4.0%, and the intra-batch repeatability is good.

[0099] Table 1: Intra-batch repeatability test results of the kit prepared in Example 1

[0100] 2. Batch-to-batch reproducibility

[0101] The ADP samples with concentrations of 0.5 mg / L, 1 mg / L and 2 mg / L were tested with three batches of reagents and repeated 10 times using the kit prepared in Example 1, and the average value, standard deviation (SD) and coefficient of variation (CV) were calculated. The results are shown in Table 3 below, and it can be seen that the repeatability CV is ≤5.0%, and the batch-to-batch repeatability is good.

[0102] Table 2: Results of the inter-batch repeatability test of the kit prepared in Example 1

[0103]

[0104] 3. Daily repeatability

[0105] The ADP samples with concentrations of 0.5 mg / L, 1 mg / L and 2 mg / L were tested with three batches of reagents respectively, and the kit prepared in Example 1 was used to repeat the test 10 times, and the average value, standard deviation (SD) and coefficient of variation (CV) were calculated. The results are shown in Table 3 below, and it can be seen that the repeatability CV is ≤7.0%, and the repeatability between days is good.

[0106] Table 3: Test results of the repeatability of the test kit prepared in Example 1

[0107]

[0108]

[0109] 4. Linear

[0110] The 42 mg / L ADP sample was diluted with diluent in equal proportions, and the diluted samples were measured with the kit prepared in Example 1. Each sample was measured 3 times, and the average value was calculated. The relative deviation was calculated by comparing with the theoretical concentration. The results are shown in Table 2 below. It can be seen that the linear range of the test results of the kit can reach 0.2-42.0 mg / L, and the linear correlation coefficient R=0.9997, indicating that the linear range is wide and the linear relationship is good.

[0111] Table 4: Detection linearity test results of the kit prepared in Example 1

[0112]

[0113] 5. Accuracy

[0114] The recovery rate should be in the range of 85.0% to 115.0%.

[0115] A high concentration ADP sample (A) with a concentration of about 42 mg / L was added to a serum or other corresponding matrix sample B, with a volume ratio of A to B of 1:9. Each sample was tested three times and the average value was calculated. The recovery rate R was calculated back. The results are shown in Table 3 below, and it can be seen that the recovery rate is 107.33%, which is a good recovery rate.

[0116] Table 5: Test results of the recovery rate of the kit prepared in Example 1

[0117]

[0118] The above specific examples are only used to illustrate the invention to help understand the invention, and are not intended to limit the invention. Those skilled in the art may make several modifications and improvements based on the concept of the invention, which all belong to the protection scope of the invention.

[0119] As can be seen from the above, the advantages and beneficial effects of the present invention are:

[0120] The adiponectin assay kit provided by the present invention adopts a reaction mode combining physical adsorption and chemical coupling to improve the binding efficiency of antibodies and microspheres, thereby greatly improving the accuracy, repeatability and linearity of the reagent in detecting low-value samples and reducing the batch-to-batch differences of the reagent. The steps of centrifugation and repeated washing are omitted while ensuring that the performance of the reagent remains basically unchanged; the overall preparation environment does not require special requirements and can be room temperature; the type of chelating agent is simplified from two to one; in addition, the high concentration of antibodies and latex microspheres during coupling reduces the total volume of the solution in the coupling step, which also makes it possible to prepare a large amount in a smaller container.

[0121] Those skilled in the art should understand that the discussion of any of the above embodiments is only exemplary and is not intended to imply that the scope of the present disclosure (including the claims) is limited to these examples; under the concept of the present invention, the technical features in the above embodiments or different embodiments can also be combined, and there are many other changes in different aspects of the present invention as described above, which are not provided in detail for the sake of simplicity. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A kit for determining adiponectin by latex-enhanced immunoturbidimetry, comprising a first reagent, a second reagent, a calibrator and a quality control product, It is characterized in that The first reagent is composed of the following components in mass concentration: buffer 10mmol / L to 100mmol / L, inorganic salt 20g / L to 70g / L, stabilizer 1g / L to 20g / L, coagulant 3g / L to 20g / L, surfactant 3g / L to 20g / L, preservative 0.05g / L to 5g / L; The second reagent is obtained by diluting a suspension of latex microsphere particles coated with adiponectin antibodies with a preservation solution to determine the weight, and the content of latex microsphere particles coated with adiponectin antibodies in the second reagent is 0.5g / L-3g / L (based on the mass of latex microspheres); the composition and content of the preservation solution are: buffer 10-50mmol / L, stabilizer 5g / L-100g / L, preservative 0.05g / L-2g / L; The calibration product is obtained by dissolving adiponectin antigen in a dispersion, and the components and mass concentrations of the dispersion are: buffer 5mmol / L-100mmol / L, inorganic salt 5g / L-40g / L, stabilizer 5g / L-80g / L, preservative 0.05g / L-2g / L; the adiponectin antigen content of the calibration product is 1mg / L-100mg / L; the adiponectin antigen content of the quality control product is 1mg / L-100mg / L.

2. A kit for determining adiponectin by latex-enhanced immunoturbidimetry as claimed in claim 1, Features: The buffer in the first reagent is selected from any one or more of phosphate buffer, 3-[NN-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid buffer, barbiturate buffer, N-[tri(hydroxymethyl)methyl]-3-aminopropanesulfonic acid, 2-(N-morpholine)ethanesulfonic acid, and 3-morpholinepropanesulfonic acid buffer; the surfactant in the first reagent is selected from any one or more of ethylphenyl polyethylene glycol and Triton X-100; the stabilizer in the first reagent is selected from any one or more of bovine serum albumin, sucrose, trehalose, and ovalbumin; the preservative in the first reagent is selected from any one or more of Proclin 300, sodium azide, phenol, gentamicin, and thimerosal; the inorganic salt in the first reagent is selected from any one or more of magnesium chloride, sodium chloride, potassium chloride, calcium chloride, and aluminum chloride; and the coagulant in the first reagent is selected from any one or more of polyethylene glycol 8000, polyethylene glycol 6000, and polyethylene glycol 2000.

3. A kit for determining adiponectin by latex-enhanced immunoturbidimetry as claimed in claim 2, It is characterized in that The components and contents of the first reagent are as follows: the buffer is phosphate buffer with a concentration of 10mmol / L to 50mmol / L, the inorganic salt is sodium chloride with a concentration of 30g / L to 60g / L, the stabilizer is bovine serum albumin with a concentration of 1g / L to 10g / L, the coagulant is polyethylene glycol 6000 with a concentration of 3g / L to 10g / L, the surfactant is Triton X-100 with a concentration of 3g / L to 10g / L, and the preservative is sodium azide with a concentration of 0.05g / L to 2g / L.

4. A kit for determining adiponectin by latex-enhanced immunoturbidimetry as claimed in claim 3, It is characterized in that The components and contents of the first reagent are as follows: the buffer is a phosphate buffer with a pH of 7.3 to 7.5 and a concentration of 10 mmol / L to 15 mmol / L; the inorganic salt is sodium chloride and a concentration of 40 g / L to 60 g / L; the stabilizer is bovine serum albumin and a concentration of 1.5 g / L to 2.5 g / L; the coagulant is polyethylene glycol 6000 and a concentration of 4 g / L to 6 g / L; the surfactant is Triton X-100 and a concentration of 4 g / L to 6 g / L; the preservative is sodium azide and a concentration of 0.5 g / L to 2 g / L.

5. A kit for determining adiponectin by latex-enhanced immunoturbidimetry according to any one of claims 1 to 4, Features The stabilizer of the storage solution in the second reagent is selected from any one or more of sucrose, trehalose, and glucose, the preservative of the storage solution in the second reagent is selected from any one or more of Proclin300, sodium azide, phenol, gentamicin, and thimerosal, and the buffer in the storage solution in the second reagent is selected from any one or more of phosphate buffer, 3-[NN-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid buffer, barbiturate buffer, N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid, and 3-morpholinepropanesulfonic acid buffer; The method for preparing the latex microsphere particle suspension coated with adiponectin antibody in the second reagent comprises the following steps: 1) diluting the latex microsphere suspension with a weakly acidic buffer to obtain a buffered microsphere suspension, 2) Prepare a chelating agent solution by diluting with a weakly acidic buffer, 3) adding the chelating agent solution to the buffered microsphere suspension and shaking to mix, thereby activating the microspheres to obtain an activated microsphere suspension, 4) Diluting the antibody solution with a buffer to obtain a diluted antibody solution 5) adding the diluted antibody solution to the activated microsphere suspension and shaking to mix for coating reaction to obtain a coated microsphere suspension. 6) adding a blocking agent to the coated microsphere suspension to perform a blocking reaction to obtain a suspension of latex microsphere particles coated with adiponectin antibodies; The blocking agent is selected from any one or more of bovine serum albumin (BSA), sucrose, trehalose, and ovalbumin; The weak acid buffer is 2-(N-morpholine)ethanesulfonic acid, and the buffers in the remaining steps are selected from any one or more of a phosphate buffer, a 3-[NN-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid buffer, a barbiturate buffer, a N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid, and a 3-morpholinepropanesulfonic acid buffer; The chelating agent in the second reagent is selected from any one or more of sodium ethylenediaminetetraacetate, 1-ethyl-3[3-dimethylaminopropyl]carbodiimide hydrochloride, and diethylenetriaminepentaacetic acid.

6. A kit for determining adiponectin by latex-enhanced immunoturbidimetry as claimed in claim 5, Features: The stabilizer of the storage solution in the second reagent is 70-90 g / L sucrose, the preservative of the storage solution in the second reagent is 0.5-2 g / L sodium azide, and the buffer in the storage solution in the second reagent is N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid buffer with a pH of 7.9-8.1 and a content of 8-12 mol / L; In the preparation method of the latex microsphere particle suspension coated with adiponectin antibody in the second reagent: In step 1), the mass percentage content of microspheres in the latex microsphere suspension is 8% to 12%, and the particle size of the microspheres is 90-200 nm; the acidic buffer is 2-(N-morpholine)ethanesulfonic acid buffer, with a concentration of 8 to 12 mmol / L and a pH value of 5.5 to 5.7, and the volume of the latex microsphere suspension is 50% to 70% of the volume of the weakly acidic buffer; In step 2), the chelating agent is 1-ethyl-3[3-dimethylaminopropyl]carbodiimide hydrochloride, and the concentration of the chelating agent solution is 1 mg / mL-6 mg / mL; Step 3) specifically comprises adding the chelating agent solution obtained in step 2) to the buffered microsphere suspension obtained in step 1), and shaking on a shaker for 15 min to 30 min to activate the microspheres, wherein the volume of the chelating agent solution is 60% to 80% of the volume of the buffered microsphere suspension; In step 4), the antibody solution is diluted with a 3-morpholinepropanesulfonic acid buffer having a concentration of 40 mmol / L to 60 mmol / L and a pH of 7.7 to 7.9, wherein the volume of the antibody solution is 10% to 30% of the volume of the buffer solution and the antibody concentration is 5 to 20 mg / L; In step 5), the diluted antibody solution obtained in step 4) is added to the activated microsphere suspension solution for coating reaction to obtain a coated microsphere suspension. The reaction time is 1-18 hours. The reaction container needs to be shaken on a shaker at a speed of 50-280 rpm. In step 6), the blocking agent is bovine serum albumin, and the blocking reaction is completed by shaking on a shaker for 1-2 hours. The mass ratio of bovine serum albumin as a blocking agent to latex microspheres is 1.5-2 mg / g.

7. A kit for determining adiponectin by latex-enhanced immunoturbidimetry as claimed in claim 6, Features In the preparation method of the latex microsphere particle suspension coated with adiponectin antibody in the second reagent: The concentration of the chelating agent solution in step 2) is 2.5-3.5 mg / ml; The antibody in step 3) is a rabbit polyclonal antibody, and the antibody concentration is 8-12 mol / L; In step 5), the reaction time is 1 to 2 hours, and the shaking speed is 150-250 rpm.

8. A kit for determining adiponectin by latex-enhanced immunoturbidimetry as claimed in claim 7, Features The buffer in the calibration standard is selected from any one or more of phosphate buffer, 3-[NN-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid buffer, barbiturate buffer, N-[tri(hydroxymethyl)methyl]-3-aminopropanesulfonic acid, 2-(N-morpholine)ethanesulfonic acid, and 3-morpholinepropanesulfonic acid buffer; the stabilizer in the calibration standard is selected from any one or more of bovine serum albumin, sucrose, trehalose, and ovalbumin; the preservative in the calibration standard is selected from any one or more of Proclin300, sodium azide, phenol, gentamicin, and thimerosal; the inorganic salts in the calibration standard are independently selected from any one or more of magnesium chloride, sodium chloride, potassium chloride, calcium chloride, and aluminum chloride.

9. A kit for determining adiponectin by latex-enhanced immunoturbidimetry as claimed in claim 8, Features: The buffer in the calibration material is a phosphate buffer with a pH of 7.3 to 7.5; the stabilizer in the calibration material is 8 to 12 g / L bovine serum albumin; the preservative in the calibration material is 0.5 to 2 g / L sodium azide; and the inorganic salt in the calibration material is 8 to 10 g / L sodium chloride.

10. A kit for determining adiponectin by latex-enhanced immunoturbidimetry according to any one of claims 6 to 9, It is characterized in that The volume ratio of the first reagent to the second reagent is 4:1.

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