Adiponectin detection kit and preparation method thereof

Abstract

The present invention discloses an adiponectin detection kit and a preparation method thereof, wherein the adiponectin detection kit comprises an R1 reagent and an R2 reagent; the R1 reagent comprises the following components: a buffer solution, an electrolyte, a fusogen, a surfactant and a preservative; the R2 reagent comprises the following components: anti-adiponectin monoclonal antibody coated latex particles, a buffer solution, a protective agent, a stabilizer, a preservative and a surfactant; wherein the surfactant is selected from one or two of Tetronic 1307 and castor oil polyoxyethylene ether. The kit of the present invention not only enhances the anti-interference ability of the reagent to special samples such as chyle and lipid turbidity samples, reduces the interference of nonspecific adsorption, improves the accuracy of sample testing, but also enhances the repeatability and stability of the latex reagent by adopting a new surfactant combination in combination with other components in the kit.

Description

Technical Field

[0001] The present invention relates to the field of in vitro diagnostic reagents, and in particular to an adiponectin detection kit and a preparation method thereof. Background Art

[0002] Adiponectin (ADPN) is an endogenous bioactive polypeptide and protein secreted by adipocytes. It can stably exist in plasma, accounting for about 0.01% of plasma protein. Adiponectin was first discovered in human subcutaneous adipose tissue, plasma, and adipocytes of rodents.

[0003] With the research on adiponectin, it is found that adiponectin is an insulin-sensitizing hormone that can improve insulin resistance and arteriosclerosis; adiponectin levels can predict the development of type II diabetes and coronary heart disease, and clinical trials show that adiponectin has a certain correlation with obesity, diabetes, insulin resistance, atherosclerosis and inflammation. Therefore, the detection of adiponectin levels in the blood is of great significance for the diagnosis and prognosis of these diseases.

[0004] At present, the methods for determining adiponectin mainly use chemiluminescence immunoassay (CLIA), enzyme-linked immunosorbent assay (ELISA) and latex-enhanced immunoturbidimetry. Among them, the chemiluminescence method has high sensitivity, but requires special instruments and is expensive; the operation process of the enzyme-linked immunosorbent assay is extremely cumbersome and complicated, the detection time is long, and the repeatability is poor. The latex-enhanced immunoturbidimetry uses the surface cross-linked monoclonal or polyclonal antibodies of latex particles of a certain particle size to bind to the antigen to detect the concentration of the antigen; the existing latex-enhanced immunoturbidimetry has high sensitivity, but poor anti-interference, poor repeatability in the low value area, and a complex preparation process, which consumes manpower and material resources.

[0005] Therefore, the prior art needs to be further improved. Summary of the invention

[0006] In view of the above problems, the present invention provides a novel adiponectin detection kit with strong anti-interference ability and good repeatability, and provides a preparation method of the kit.

[0007] To solve the above problems, the present invention provides the following technical solutions:

[0008] In a first aspect, the present invention provides a test kit for adiponectin, which comprises a reagent R1 and a reagent R2; wherein:

[0009] R1 reagent includes the following components: buffer, electrolyte, solubility promoter, surfactant and preservative;

[0010] The R2 reagent includes the following components: anti-adiponectin mAb-coated latex particles, buffer, protective agent, stabilizer, preservative, and surfactant;

[0011] Wherein, the surfactant is selected from one or more of Tetronic 1307, castor oil polyoxyethylene ether, Tween-20, Triton-100 and Brij-35.

[0012] Preferably, the R1 reagent includes the following components in the following concentrations:

[0013] Buffer 0.02-0.1M / L;

[0014] Electrolytes 20-40 g / L;

[0015] Fusion promoter 30-80g / L;

[0016] Surfactant 0.2-10g / L;

[0017] Preservative 0.8-1.2g / L.

[0018] The R2 reagent contains the following components at the following concentrations:

[0019] Anti-adiponectin mAb coated latex particles 60-100 mg / L;

[0020] Buffer 0.01-0.05M / L;

[0021] Protective agent 5-30g / L;

[0022] Stabilizer 5-20g / L;

[0023] Preservatives 0.8-1.2g / L;

[0024] Surfactant 0.2-10g / L.

[0025] Preferably, in the adiponectin detection kit, the surfactant includes Tetronic 1307 and castor oil polyoxyethylene ether.

[0026] After adding the above surfactants to R1, the anti-interference ability of the reagent to special samples such as turbid and hemolytic samples is enhanced, and the interference of nonspecific adsorption is reduced; it also avoids the occurrence of false negatives and false positives during sample testing, thereby improving the accuracy of sample testing. Tetronic 1307 and castor oil polyoxyethylene ether are both good solubilizers, which can disperse and dissolve oils, reduce matrix interference and nonspecific adsorption, and neither of them destroys the protein structure; especially castor oil polyoxyethylene ether, its HLB value is between 13-14, and it has a good degreasing ability; in addition, Tetronic 1307 is a zwitterion with super antistatic ability, which can avoid false positives caused by electrostatic adsorption.

[0027] Adding two surfactants, Tetronic 1307 and castor oil polyoxyethylene ether, to R2 has multiple functions: on the one hand, it can make the latex reagent disperse more evenly and enhance the repeatability of the latex reagent; on the other hand, castor oil polyoxyethylene ether has excellent emulsifying ability, which can not only form a hydration film on the protein surface to protect the protein and increase the stability of the protein, but also form surface tension and enhance the stability of the liquid.

[0028] Preferably, in the adiponectin detection kit, the protective agent is selected from one or both of trehalose and sucrose.

[0029] Preferably, in the adiponectin detection kit, the stabilizer is selected from one or more of casein and bovine serum albumin.

[0030] Optionally, in the adiponectin detection kit, the buffer is one or more of TRIS buffer, phosphate buffer, HEPES buffer, and citric acid buffer.

[0031] Optionally, the electrolyte is one or more of sodium chloride, potassium chloride and calcium chloride.

[0032] Optionally, the fusogenic agent is one or more of PEG6000, PEG8000, and PEG12000.

[0033] Optionally, the preservative is one or more of sodium azide and Proclin300.

[0034] Preferably, in the adiponectin detection kit, the R1 reagent comprises the following components at the following concentrations: citric acid buffer 0.02-0.1M / L; sodium chloride 20-40g / L; PEG6000 30-80g / L; Tetronic 1307 0.1-5g / L; castor oil polyoxyethylene ether 0.1-5g / L; preservative 0.8-1.2g / L;

[0035] The R2 reagent includes the following components in the following concentrations: anti-adiponectin monoclonal antibody coated latex particles: 60-100 mg / L; HEPES buffer 0.01-0.1 M / L; trehalose 5-30 g / L; bovine serum albumin 5-20 g / L; Tetronic 1307 0.1-5 g / L; castor oil polyoxyethylene ether 0.1-5 g / L; preservative 0.8-1.2 g / L.

[0036] Optionally, the pH value of the R1 reagent is 6.0-7.4, and the pH value of the R2 reagent is 7.0-7.8. Preferably, the pH value of the R1 reagent is 7.0, and the pH value of the R2 reagent is 7.4.

[0037] On this basis, preferably, the preservative in the R1 reagent is 0.8-1.2 g / L sodium azide, and the preservative in the R2 reagent is 0.8-1.2 g / L Proclin300.

[0038] In a second aspect, the present invention provides a method for preparing the above-mentioned adiponectin detection kit, which comprises the following steps:

[0039] Preparation of R1 reagent: according to the component content of reagent R1, dissolve the buffer, electrolyte, solubility promoter, surfactant, preservative, etc. in pure water in turn, stir evenly, adjust the pH to 6.0-7.4, stir evenly, and incubate in a 35-40℃ oven overnight to obtain R1 reagent;

[0040] Preparation of reagent R2: Add buffer, stabilizer and surfactant to the anti-adiponectin monoclonal antibody coated latex particles while stirring, and finally add preservatives and protective agents. After stabilizing on a shaker at 35-40°C overnight, the R2 reagent is obtained.

[0041] Preferably, the preparation method of the anti-adiponectin monoclonal antibody coated latex particles is: take latex microspheres with a particle size of 100-300nm in MES buffer, shake at 35-40°C, 100-300r / min, and shake for 3-10min; then add freshly prepared EDC and NHS solutions, place in a 35-40°C shaker, 100-300r / min, activate the latex for 10-20min; while stirring, add buffer and antibody, place in a 35-40 shaker, 100-300r / min, and couple for 1-5h; the antibody is an anti-adiponectin monoclonal antibody.

[0042] Preferably, when adding a buffer, a stabilizer and a surfactant, the latter should be added after the former is added and incubated on a shaker at 35-40° C. for 20-40 min.

[0043] Preferably, the preparation method of the anti-adiponectin monoclonal antibody coated latex particles is as follows: latex microspheres with a particle size of 100-300 nm are placed in MES buffer, shaken at 35-40°C, 100-300 r / min, and shaken for 3-10 min; then, freshly prepared EDC and NHS solutions are added, and the latex is activated for 10-20 min at a shaker at 35-40°C, 100-300 r / min; while stirring, buffer and antibody are added, and the mixture is shaken at 35-40°C, 100-300 r / min, and coupled for 1-5 h.

[0044] Further preferred conditions are: take latex microspheres with a particle size of 100-300 nm in HEPES buffer, place it in a 37°C shaker at 200 r / min, and activate the latex for 15 minutes; add buffer and antibody while stirring, place it in a 37°C shaker at 200 r / min, and couple for 3 hours.

[0045] Preferably, the concentration of HEPES buffer is 0.01M-0.1M.

[0046] Preferably, the preparation method of the adiponectin detection kit comprises the following steps:

[0047] Preparation of reagent R1: According to the component content of reagent R1, dissolve the buffer solution, electrolyte, solubility promoter, surfactant, preservative, etc. in pure water in turn, stir evenly, adjust the pH to 6.0-7.4, stir evenly and incubate in a 37°C oven overnight to obtain reagent R1.

[0048] Preparation of reagent R2: take latex microspheres with a particle size of 100-300nm in 0.01M-0.1M MES buffer, place it in a 37℃ shaker at 200r / min, and shake evenly for 5min; then add the freshly prepared EDC and NHS solutions, place it in a 37℃ shaker at 200r / min, and activate the latex for 15min; under stirring, add HEPES buffer and antibody, place it in a 37℃ shaker at 200r / min, and after coupling for 3h, take it out of the shaker, place it on a stirrer, and add HEPES buffer, BSA and surfactant respectively. After adding one substance, incubate it in a 37℃ shaker for 30min before adding another substance. Finally, add preservatives and protective agents, and stabilize it in a 37℃ shaker overnight to obtain reagent R2.

[0049] In a third aspect, the present invention further provides a surfactant for an adiponectin detection kit, comprising Tetronic 1307 and castor oil polyoxyethylene ether.

[0050] Optionally, the use concentration of Tetronic 1307 is 0.1-5 g / L, and the use concentration of castor oil polyoxyethylene ether is 0.1-5 g / L.

[0051] The present invention has the following beneficial effects:

[0052] 1. The present invention adopts a new surfactant combination in combination with other components in the kit, which not only enhances the anti-interference ability of the R1 reagent for special samples such as lipophilic and hemolytic samples, reduces the interference of nonspecific adsorption, avoids the occurrence of false negatives and false positives in the sample testing process, thereby improving the accuracy of the sample testing; but also enhances the repeatability and stability of the latex reagent.

[0053] 2. The kit completely avoids the shortcomings of enzyme-linked immunosorbent assay and chemiluminescence assay, and can detect both large quantities of samples quickly and single samples. Compared with the complex preparation process of the existing latex-enhanced immunoturbidimetric method, which consumes manpower and material resources, the preparation process of the adiponectin detection kit provided by the present invention is simple, and does not require steps such as centrifugation and ultrasonic dispersion, which saves time and effectively reduces the batch difference of the reagent. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 The calibration curve of Example 1 of the present invention;

[0055] Figure 2 The linear range of Example 1 of the present invention. DETAILED DESCRIPTION

[0056] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments are conventional methods in the art unless otherwise specified.

[0057] Example 1

[0058] This embodiment provides a detection kit for adiponectin with strong anti-interference and good repeatability, and the preparation method thereof comprises the following steps:

[0059] (1) Preparation of reagent R1:

[0060] Weigh 100 mL of citric acid buffer, place it on a stirrer, and add 3.0 g NaCl, 5 g PEG6000, 0.2 g Tetronic 1307, 0.1 g castor oil polyoxyethylene ether, and 0.1 g NaN3 in sequence. After one substance is completely dissolved, add the other substance. After they are completely dissolved, adjust the pH to 7.0, and incubate it on a shaker at 37°C overnight to obtain reagent R1.

[0061] After the configuration is completed, the concentrations of the components of reagent R1 are: citric acid buffer 50mM / L; sodium chloride 30g / L; PEG6000 50g / L; Tetronic 1307 2g / L; castor oil polyoxyethylene ether 1g / L; NaN3 1g / L.

[0062] (2) Preparation of reagent R2:

[0063] ① Preparation of anti-adiponectin monoclonal antibody coated latex particles:

[0064] Take 80ul of latex microspheres with a particle size of 200nm and put them in 130ul of 0.02M pH6.0 MES buffer, place them in a 37℃ shaker, 200r / min, and shake them for 2min. Then add the freshly prepared EDC and NHS solution, place them in a 37℃ shaker, 200r / min, and shake them for 15min to activate the latex; the concentration of EDC and NHS is 0.10M, and the addition ratio is 1:1; then, under stirring, add 3ml HEPES buffer (0.02M pH7.4) and antibody solution (monoclonal antibody 1 and monoclonal antibody 2 are mixed, and the monoclonal antibody is mouse anti-human ADP antibody), and the total amount of antibody added is 30mg / L. Place them in a 37℃ shaker, 200r / min, and couple for 3h.

[0065] ②Take the mixture from the previous step out of the shaker, place it on a stirrer, and add 1ml of HEPES buffer, 500ul of 10% BSA, 5ul of Tetronic 1307, and 5ul of castor oil polyoxyethylene ether. After adding one of the above substances, incubate it on a shaker at 37℃ for 30min before adding another substance. Finally, add 5ul of proclin300 and 0.1g of trehalose, and incubate it on a shaker at 37℃ overnight to obtain reagent R2.

[0066] After the preparation was completed, the concentrations of the components of reagent R2 were: anti-adiponectin monoclonal antibody coated latex particles 80 mg / L, HEPES buffer 25 mM / L, BSA 10 g / L, Tetronic 1307 1 g / L, castor oil polyoxyethylene ether 1 g / L, proclin300 1 g / L, and trehalose 20 g / L.

[0067] Example 2

[0068] This embodiment provides an adiponectin detection kit with strong anti-interference and good repeatability, including R1 reagent and R2 reagent, and a preparation method thereof, comprising the following steps:

[0069] (1) Preparation of reagent R1:

[0070] Weigh 100 ml of citric acid buffer, place it on a stirrer, and add 3.0 g NaCl, 5 g PEG6000, 0.2 g Tetronic 1307, and 0.1 g NaN3 in order. After one substance is completely dissolved, add the other substance. After they are completely dissolved, adjust the pH to 7.0, and incubate it in a shaker at 37°C overnight to obtain reagent R1.

[0071] After the configuration is completed, the concentrations of the components of reagent R1 are: citric acid buffer 50mM / L; sodium chloride 30g / L; PEG6000 50g / L; Tetronic 1307 2g / L; NaN3 1g / L.

[0072] (2) Preparation of reagent R2:

[0073] ① Preparation of anti-adiponectin monoclonal antibody coated latex particles:

[0074] Take 80ul of latex microspheres with a particle size of 200nm and put them in 130ul of 0.02M pH6.0 MES buffer, place it in a 37℃ shaker, 200r / min, and shake it for 2min. Then add the freshly prepared EDC and NHS solution, place it in a 37℃ shaker, 200r / min, and shake it for 15min to activate the latex; the concentration of EDC and NHS is 0.10M, and the addition ratio is 1:1. Then, under stirring, add 3ml HEPES buffer (0.02M pH7.4) and antibody solution (monoclonal antibodies 1 and 2 mixed), and the total amount of antibody added is 30mg / L. Place it in a 37℃ shaker, 200r / min, and couple for 3h.

[0075] ②Take the mixture from the previous step out of the shaker, place it on a stirrer, and add 1ml of HEPES buffer, 500ul of 10% BSA, 5ul of Tetronic 1307, and 5ul of castor oil polyoxyethylene ether. After adding one of the above substances, incubate it on a shaker at 37℃ for 30min before adding another substance. Finally, add 5ul of proclin300 and 0.1g of trehalose, and incubate it on a shaker at 37℃ overnight to obtain reagent R2.

[0076] After the preparation was completed, the concentrations of the components of reagent R2 were: anti-adiponectin monoclonal antibody coated latex particles 80 mg / L, HEPES buffer 25 mM / L, BSA 10 g / L, Tetronic 1307 1 g / L, castor oil polyoxyethylene ether 1 g / L, proclin300 1 g / L, and trehalose 20 g / L.

[0077] Example 3

[0078] This embodiment provides an adiponectin detection kit, including R1 reagent and R2 reagent, and a preparation method thereof, comprising the following steps:

[0079] (1) Preparation of reagent R1:

[0080] Weigh 100 mL of citric acid buffer, place it on a stirrer, and add 3.0 g NaCl, 5 g PEG6000, 0.1 g castor oil polyoxyethylene ether, and 0.1 g NaN3 in sequence. After one substance is completely dissolved, add the other substance. After they are completely dissolved, adjust the pH to 7.0, and incubate it on a shaker at 37°C overnight to obtain reagent R1.

[0081] After the configuration is completed, the concentrations of the components of reagent R1 are: citric acid buffer 50mM / L; sodium chloride 30g / L; PEG6000 50g / L; castor oil polyoxyethylene ether 1g / L; NaN3 1g / L.

[0082] (2) Preparation of reagent R2:

[0083] ① Preparation of anti-adiponectin monoclonal antibody coated latex particles:

[0084] Take 80ul of latex microspheres with a particle size of 200nm and put them in 130ul of 0.02M pH6.0 MES buffer, place it in a 37℃ shaker, 200r / min, and shake it for 2min. Then add the freshly prepared EDC and NHS solution, place it in a 37℃ shaker, 200r / min, and shake it for 15min to activate the latex; the concentration of EDC and NHS is 0.10M, and the addition ratio is 1:1. Then, under stirring, add 3ml HEPES buffer (0.02M pH7.4) and antibody solution (monoclonal antibodies 1 and 2 mixed), and the total amount of antibody added is 30mg / L. Place it in a 37℃ shaker, 200r / min, and couple for 3h.

[0085] ②Take the mixture from the previous step out of the shaker, place it on a stirrer, and add 1ml of HEPES buffer, 500ul of 10% BSA, 5ul of Tetronic 1307, and 5ul of castor oil polyoxyethylene ether. After adding one of the above substances, incubate it on a shaker at 37℃ for 30min before adding another substance. Finally, add 5ul of proclin300 and 0.1g of trehalose, and incubate it on a shaker at 37℃ overnight to obtain reagent R2.

[0086] After the preparation was completed, the concentrations of the components of reagent R2 were: anti-adiponectin monoclonal antibody coated latex particles 80 mg / L, HEPES buffer 25 mM / L, BSA 10 g / L, Tetronic 1307 1 g / L, castor oil polyoxyethylene ether 1 g / L, proclin300 1 g / L, and trehalose 20 g / L.

[0087] Example 4

[0088] This embodiment provides an adiponectin detection kit with strong anti-interference and good repeatability, including R1 reagent and R2 reagent, and a preparation method thereof, comprising the following steps:

[0089] (1) Preparation of reagent R1:

[0090] Weigh 100 mL of citric acid buffer, place it on a stirrer, and add 3.0 g NaCl, 5 g PEG6000, 0.5 g Tween-20, and 0.1 g NaN3 in sequence. After one substance is completely dissolved, add the other substance. After it is completely dissolved, adjust the pH to 7.0, and incubate it in a shaker at 37°C overnight to obtain reagent R1.

[0091] After the configuration is completed, the concentrations of the components of reagent R1 are: citric acid buffer 50mM / L; sodium chloride 30g / L; PEG6000 50g / L; Tween-20 5g / L; NaN3 1g / L.

[0092] (2) Preparation of reagent R2:

[0093] ① Preparation of anti-adiponectin monoclonal antibody coated latex particles:

[0094] Take 80ul of latex microspheres with a particle size of 200nm and put them in 130ul of 0.02M pH6.0 MES buffer, place it in a 37℃ shaker, 200r / min, and shake it for 2min. Then add the freshly prepared EDC and NHS solution, place it in a 37℃ shaker, 200r / min, and shake it for 15min to activate the latex; the concentration of EDC and NHS is 0.10M, and the addition ratio is 1:1. Then, under stirring, add 3ml HEPES buffer (0.02M pH7.4) and antibody solution (monoclonal antibodies 1 and 2 mixed), and the total amount of antibody added is 30mg / L. Place it in a 37℃ shaker, 200r / min, and couple for 3h.

[0095] ②Take the mixture from the previous step out of the shaker, place it on a stirrer, and add 1ml of HEPES buffer, 500ul of 10% BSA, 5ul of Tetronic 1307, and 5ul of castor oil polyoxyethylene ether. After adding one of the above substances, incubate it on a shaker at 37℃ for 30min before adding another substance. Finally, add 5ul of proclin300 and 0.1g of trehalose, and incubate it on a shaker at 37℃ overnight to obtain reagent R2.

[0096] After the preparation was completed, the concentrations of the components of reagent R2 were: anti-adiponectin monoclonal antibody coated latex particles 80 mg / L, HEPES buffer 25 mM / L, BSA 10 g / L, Tetronic 1307 1 g / L, castor oil polyoxyethylene ether 1 g / L, proclin300 1 g / L, and trehalose 20 g / L.

[0097] Example 5

[0098] This embodiment provides an adiponectin detection kit, including R1 reagent and R2 reagent, and a preparation method thereof, comprising the following steps:

[0099] (1) Preparation of reagent R1:

[0100] Weigh 100 mL of citric acid buffer, place it on a stirrer, and add 3.0 g NaCl, 5 g PEG6000, 0.2 g Tetronic 1307, 0.1 g castor oil polyoxyethylene ether, and 0.1 g NaN3 in sequence. After one substance is completely dissolved, add the other substance. After they are completely dissolved, adjust the pH to 7.0, and incubate it on a shaker at 37°C overnight to obtain reagent R1.

[0101] After the configuration is completed, the concentrations of the components of reagent R1 are: citric acid buffer 50mM / L; sodium chloride 30g / L; PEG6000 50g / L; Tetronic 1307 2g / L; castor oil polyoxyethylene ether 1g / L; NaN3 1g / L.

[0102] (2) Preparation of reagent R2:

[0103] ① Preparation of anti-adiponectin monoclonal antibody coated latex particles:

[0104] Take 80ul of latex microspheres with a particle size of 200nm and put them in 130ul of 0.02M pH6.0 MES buffer, place it in a 37℃ shaker, 200r / min, and shake it for 2min. Then add the freshly prepared EDC and NHS solution, place it in a 37℃ shaker, 200r / min, and shake it for 15min to activate the latex; the concentration of EDC and NHS is 0.10M, and the addition ratio is 1:1. Then, under stirring, add 3ml HEPES buffer (0.02M pH7.4) and antibody solution (monoclonal antibodies 1 and 2 mixed), and the total amount of antibody added is 30mg / L. Place it in a 37℃ shaker, 200r / min, and couple for 3h.

[0105] ②Take the mixture from the previous step out of the shaker, place it on a stirrer, and add 1ml of HEPES buffer, 500ul of 10% BSA, and 10ul of Triton-100 respectively. After adding one of the above substances, incubate it in a shaker at 37℃ for 30min before adding another substance. Finally, add 5ul of proclin300 and 0.1g of trehalose, and incubate it in a shaker at 37℃ overnight to obtain reagent R2.

[0106] After the preparation, the concentrations of the components of reagent R2 were: anti-adiponectin monoclonal antibody coated latex particles 80 mg / L, HEPES buffer 25 mM / L, BSA 10 g / L, Triton-100 2 g / L, proclin 300 1 g / L, and trehalose 20 g / L.

[0107] Example 6 Application of adiponectin detection kit

[0108] A detection method of the above-mentioned adiponectin detection kit in this embodiment:

[0109] 1. Biochemical analyzer detection

[0110] Take Hitachi 7180 operation as an example: the measurement wavelength is 700nm, take different concentrations of calibration solution (3uL), add adiponectin R1 reagent (160uL), mix, incubate at 37℃ for 5 minutes, add adiponectin R2 reagent (40uL), mix, incubate at 37℃ for 30 seconds, read the absorbance value A1 of each tube, react for 4.5 minutes, read the absorbance value A2 of each tube, calculate the absorbance difference △A=A2-A1, repeat the measurement twice for each tube, take the average value of the absorbance difference △A measured twice for each calibration tube as the ordinate, and the corresponding calibration concentration as the abscissa, draw the "concentration-absorbance difference" calibration curve. Take the serum or plasma sample to be tested, measure the absorbance difference of the sample in the same way, substitute it into the calibration curve, and the content of adiponectin ADPN in the sample to be tested can be calculated. If the concentration of adiponectin in serum or plasma exceeds the range of the calibration curve, the sample needs to be diluted before testing to ensure the accuracy of the test results. This kit is not only suitable for Hitachi 7180, but also for semi-automatic and fully automatic biochemical analyzers of other brands and models. The specific parameters can be adjusted according to the instrument.

[0111] Example 7 Quality Evaluation of the Kit

[0112] (1) Preparation of standard curve

[0113] A. Experimental Methods

[0114] The high value calibrator (42 mg / L) was diluted with distilled water in multiples to concentrations of 0 mg / L, 5.25 mg / L, 10.5 mg / L, 21 mg / L, and 42 mg / L. The standard curve of the adiponectin calibrator of the present invention was determined according to the method in Example 6. The experimental subject was the adiponectin detection kit prepared in Example 1.

[0115] B. Experimental Results and Analysis

[0116] like Figure 1 Shown is the calibration curve of the kit of Example 1 of the present invention.

[0117] (2) Linear range

[0118] A. Experimental Methods

[0119] Use a high concentration calibrator (42 mg / L) close to the upper limit of the linear range and a low concentration calibrator or distilled water close to the lower limit of the linear range to mix into at least 5 dilution concentrations (x). Test the kit separately, test each dilution concentration twice, and calculate the mean (y) of the test results respectively. Use the dilution concentration (x) as the independent variable and the mean (y) of the test results as the dependent variable to calculate the linear regression equation. The experimental object is the adiponectin detection kit prepared in Example 1.

[0120] B. Experimental Results and Analysis

[0121] like Figure 2 As shown, the kit of the present invention has good linearity, and the regression equation is: y = 0.9721x-0.0523, R 2 =0.9997.

[0122] (3) Analytical sensitivity

[0123] A. Experimental Methods

[0124] The kit prepared in Example 1 was used to test a calibrator with a known concentration of 10.5 mg / L, and the absolute value of the absorbance change produced under the parameters specified in the kit was recorded. The test was repeated 3 times, and the average value was taken and converted to an absorbance change value of 10 mg / L.

[0125] B. Experimental Results and Analysis

[0126] The test results were 0.2504, 0.2481, and 0.2492, respectively, which converted to an absorbance change of 0.2373 at 10 mg / L, indicating that the kit has a high sensitivity.

[0127] (4) Repeatability testing

[0128] A. Experimental Methods

[0129] The adiponectin sample with a measured value of 5.91 mg / L produced by Ipnocon was used as a low-value sample and was measured according to the biochemical analyzer detection method. Each concentration was measured 10 times, and the mean and standard deviation (S) were calculated respectively. The coefficient of variation was calculated to investigate the repeatability of the kits of Examples 1 and 5.

[0130] Since the repeatability of the kit is mainly related to the R2 reagent, this experiment mainly examines the repeatability of the kits prepared in Example 1 and Example 5.

[0131] B. Experimental Results and Analysis

[0132] Table 1 Repeatability test results of adiponectin detection kit

[0133]

[0134]

[0135] As shown in the results of Table 1, after using Triton-100 as a surfactant in Example 5, the repeatability of the kit for low-value samples was poor, reaching 7.13%. After adding a new surfactant group (Tetronic 1307 and castor oil polyoxyethylene ether) in Example 1, the repeatability of the kit for low-value samples was significantly improved, and its coefficient of variation was as low as 2.02%.

[0136] (5) Anti-interference experiment

[0137] A. Experimental Methods

[0138] A sample of adiponectin with a concentration of 5.91 mg / L was taken, and the same volumes of interfering substances of different concentrations were added, so that the concentrations of the triglyceride interfering substances after addition were 2.5 g / L, 5.0 g / L, 8.0 g / L, the final concentration of bilirubin was 400 mg / L, and the final concentration of hemoglobin was 10 g / L, respectively. Each prepared sample was measured 3 times using the kit prepared in Examples 1-5, and the average value was taken. The average value was compared with the sample added with the same volume of distilled water to observe the relative deviation of the ADPN measurement value after adding the interfering substance and adding distilled water.

[0139] B. Experimental Results and Analysis

[0140] Table 2 Anti-interference test results

[0141]

[0142] Note: The relative deviation does not exceed ±10% to basically meet the standard.

[0143] From the experimental results in Table 2, it can be seen that the anti-interference ability is ranked from strong to weak as follows: Example 1> Example 2> Example 3> Example 5> Example 4; among them, for triglycerides, the anti-interference ability of Examples 1-3 basically meets the standard; for bilirubin and hemoglobin interference, the anti-interference ability of Examples 1-5 basically meets the standard. Among them, the anti-interference ability of the kit of Example 1 is the best, and the relative deviation between the measured value after adding different concentrations of interfering substances and the measured value after adding the same volume of distilled water does not exceed 4%. Therefore, when a certain concentration of interfering substances such as triglycerides, bilirubin and hemoglobin exists in the test sample, it has no effect on the measurement results of the kit of the present invention, and the anti-interference ability of the present invention is strong.

[0144] (5) Stability

[0145] A. Experimental Methods

[0146] The kit of the present invention was placed in a 37° C. water bath, and the calibration products were calibrated before storage (i.e., before being damaged), after 3 days, 5 days, and 7 days of storage, and the subpackaged samples were tested for analysis.

[0147] B. Experimental Results and Analysis

[0148]

[0149]

[0150] From the results in the above table, it can be seen that after being stored at 37°C for 7 days, the kit of Example 1 still maintains more than 99% of the reaction activity, which indicates that the stability of the adiponectin detection kit at room temperature can be significantly improved by adding Tetronic 1307 and castor oil polyoxyethylene ether to the reagent, thereby increasing the practicality of the kit of the present invention for clinical diagnosis.

[0151] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solution and concept of the present invention, and all these changes or substitutions should fall within the protection scope of the claims attached to the present invention.

Claims

1. A test kit for adiponectin, characterized in that: Comprising R1 reagent and R2 reagent; wherein: R1 reagent includes the following components: buffer, electrolyte, solubility promoter, surfactant and preservative; The R2 reagent includes the following components: anti-adiponectin mAb-coated latex particles, buffer, protective agent, stabilizer, preservative, and surfactant; Wherein, the surfactant is composed of Tetronic 1307 and castor oil polyoxyethylene ether; Specifically, the R1 reagent includes the following components in the following concentrations: citric acid buffer 0.02-0.1M / L; sodium chloride 20-40g / L; PEG6000 30-80g / L; Tetronic 1307 0.1-5g / L; castor oil polyoxyethylene ether 0.1-5g / L; preservative 0.8-1.2g / L; The R2 reagent includes the following components in the following concentrations: anti-adiponectin monoclonal antibody coated latex particles: 60-100 mg / L; HEPES buffer 0.01-0.05 M / L; trehalose 5-30 g / L; bovine serum albumin 5-20 g / L; Tetronic 1307 0.1-5 g / L; castor oil polyoxyethylene ether 0.1-5 g / L; preservative 0.8-1.2 g / L.

2. The adiponectin detection kit according to claim 1, characterized in that The preservative in the R1 reagent is 0.8-1.2 g / L sodium azide, and the preservative in the R2 reagent is 0.8-1.2 g / L Proclin300.

3. The method for preparing the adiponectin detection kit according to claim 1, characterized in that: The following steps are involved: Preparation of R1 reagent: according to the component content of reagent R1, dissolve the buffer, electrolyte, solubility promoter, surfactant, preservative, etc. in pure water in turn, stir evenly, adjust the pH to 6.0-7.4, stir evenly, and incubate in a 35-40℃ oven overnight to obtain R1 reagent; Preparation of reagent R2: Add buffer, stabilizer and surfactant to the anti-adiponectin monoclonal antibody coated latex particles while stirring, and finally add preservatives and protective agents. After stabilizing on a shaker at 35-40°C overnight, the R2 reagent is obtained.

4. The method for preparing the adiponectin detection kit according to claim 3, characterized in that: The preparation method of the anti-adiponectin monoclonal antibody coated latex particles is as follows: taking latex microspheres with a particle size of 100-300 nm in MES buffer, shaking at 35-40° C., 100-300 r / min, and shaking for 3-10 minutes; then adding freshly prepared EDC and NHS solutions, placing in a 35-40° C. shaker, 100-300 r / min, activating the latex for 10-20 minutes; adding buffer and anti-adiponectin monoclonal antibody under stirring, placing in a 35-40° C. shaker, 100-300 r / min, and coupling for 1-5 hours.

5. The method for preparing the adiponectin detection kit according to claim 4, characterized in that: When adding buffer, stabilizer and surfactant, add the former substance and incubate on a shaker at 35-40℃ for 20-40min before adding the latter substance.

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