CN114295842A - Adiponectin detection kit and preparation method thereof - Google Patents
Adiponectin detection kit and preparation method thereof Download PDFInfo
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- CN114295842A CN114295842A CN202111655950.4A CN202111655950A CN114295842A CN 114295842 A CN114295842 A CN 114295842A CN 202111655950 A CN202111655950 A CN 202111655950A CN 114295842 A CN114295842 A CN 114295842A
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Abstract
The 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 includes the following components: buffer solution, electrolyte, melting promoter, surfactant and preservative; the R2 reagent includes the following components: the anti-adiponectin monoclonal antibody comprises latex particles coated with the anti-adiponectin monoclonal antibody, buffer solution, protective agent, stabilizing agent, preservative and surfactant; wherein the surfactant is selected from one or two of Tetronic1307 and castor oil polyoxyethylene ether. The kit disclosed by the invention adopts the novel surfactant combination to be matched with other components in the kit, so that the anti-interference capability of the reagent on special samples such as chyle and fat turbidity samples is enhanced, the interference of non-specific adsorption is reduced, the accuracy of sample test is improved, and the repeatability and stability of the latex reagent are enhanced.
Description
Technical Field
The invention relates to the field of in-vitro diagnostic reagents, in particular to an adiponectin detection kit and a preparation method thereof.
Background
Adiponectin (ADPN), an endogenous biologically active polypeptide and protein secreted by adipocytes, is stable in plasma and accounts for approximately 0.01% of plasma proteins. The earliest adiponectin was found in human subcutaneous adipose tissue, plasma, and murine adipocytes.
With the research on adiponectin, it is found that adiponectin is An Insulin-sensitizing Hormone (An Insulin-sensitizing Hormone) and can improve Insulin resistance (Insulin resistance) and arteriosclerosis; adiponectin levels are predictive of the development of type II diabetes and coronary heart disease and have been shown in clinical trials to be associated with obesity, diabetes, insulin resistance, atherosclerosis and inflammation. Therefore, the measurement of blood adiponectin levels is of great importance for the diagnosis and prognosis of these diseases.
At present, methods for measuring adiponectin mainly use chemiluminescence immunoassay (CLIA), enzyme-linked immunosorbent assay (ELISA), and latex-enhanced immunoturbidimetry. Among them, the chemiluminescence method has high sensitivity, but needs special instruments and is expensive; the enzyme linked immunosorbent assay has the disadvantages of extremely complicated and complex operation process, long detection time and poor repeatability. The latex enhanced immunoturbidimetry is to detect the concentration of antigen by combining surface cross-linked monoclonal or polyclonal antibody of latex particles with certain particle size with the antigen; the existing latex enhanced immunoturbidimetry has high sensitivity, but has poor anti-interference performance, poor repeatability in a low-value area, complex preparation process and high manpower and material resource consumption.
Therefore, the prior art is in need of further improvement.
Disclosure of Invention
Aiming at the problems, the invention provides a novel adiponectin detection kit with strong anti-interference capability and good repeatability and a preparation method thereof.
In order to solve the problems, the invention provides the following technical scheme:
in a first aspect, the present invention provides an adiponectin detection kit comprising an R1 reagent and an R2 reagent; wherein:
the R1 reagent includes the following components: buffer solution, electrolyte, melting promoter, surfactant and preservative;
the R2 reagent includes the following components: the anti-adiponectin monoclonal antibody comprises latex particles coated with the anti-adiponectin monoclonal antibody, buffer solution, protective agent, stabilizing agent, preservative and surfactant;
wherein the surfactant is selected from one or more of Tetronic1307, castor oil polyoxyethylene ether, Tween-20, Triton-100 and Brij-35.
Preferably, the R1 reagent includes the following concentrations of the ingredients:
buffer solution 0.02-0.1M/L;
20-40g/L of electrolyte;
30-80g/L of a melting promoter;
0.2-10g/L of surfactant;
0.8-1.2g/L of preservative.
The R2 reagent included the following ingredients at the following concentrations:
60-100mg/L of anti-adiponectin monoclonal antibody coated latex particles;
buffer solution 0.01-0.05M/L;
5-30g/L of protective agent;
5-20g/L of stabilizer;
0.8-1.2g/L of preservative;
0.2-10g/L of surfactant.
Preferably, in the adiponectin detection kit, the surfactant comprises Tetronic1307 and castor oil polyoxyethylene ether.
After the surfactant is added into the R1, the anti-interference capability of the reagent on special samples such as lipid turbidity samples and hemolysis samples is enhanced, and the interference of non-specific adsorption is reduced; and the occurrence of false negative and false positive in the sample testing process is also avoided, so that the accuracy of sample testing is improved. Both the Tetronic1307 and the castor oil polyoxyethylene ether are good solubilizers, can disperse and dissolve grease, can reduce matrix interference and nonspecific adsorption, and do not damage protein structures; especially castor oil polyoxyethylene ether, its HLB value is between 13-14, have very good degreasing ability; in addition, Tetronic1307 has super-strong antistatic capability of zwitterions, and can avoid false positive caused by electrostatic adsorption.
Two surfactants, namely Tetronic1307 and castor oil polyoxyethylene ether are added into R2, so that the oil-water mixed surfactant has multiple functions: on one hand, the latex reagent can be dispersed more uniformly, and the repeatability of the latex reagent is enhanced; on the other hand, the castor oil polyoxyethylene ether has excellent emulsifying capacity, can form a hydration film on the surface of the protein, plays roles of protecting the protein and increasing the stability of the protein, can form surface tension and enhances the stability of liquid.
Preferably, in the adiponectin detection kit, the protective agent is one or two of trehalose and sucrose.
Preferably, in the adiponectin detection kit, the stabilizer is selected from one or more of casein and bovine serum albumin.
Optionally, in the adiponectin detection kit, the buffer solution is one or more of TRIS buffer solution, phosphate buffer solution, HEPES buffer solution and citric acid buffer solution.
Optionally, the electrolyte is one or more of sodium chloride, potassium chloride and calcium chloride.
Optionally, the melting promoter is one or more of PEG6000, PEG8000 and PEG 12000.
Optionally, the preservative is one or more of sodium azide and Proclin 300.
Preferably, in the adiponectin detection kit, the R1 reagent comprises the following components in concentration: 0.02-0.1M/L of citric acid buffer solution; 20-40g/L of sodium chloride; PEG 600030-80 g/L; tetronic 13070.1-5 g/L; 0.1-5g/L of castor oil polyoxyethylene ether; 0.8-1.2g/L of preservative;
the R2 reagent included the following components at the following concentrations: coating latex particles with anti-adiponectin monoclonal antibody: 60-100 mg/L; HEPES buffer solution 0.01-0.1M/L; 5-30g/L of trehalose; 5-20g/L of bovine serum albumin; tetronic 13070.1-5 g/L; 0.1-5g/L of castor oil polyoxyethylene ether; 0.8-1.2g/L of preservative.
Alternatively, the pH of the R1 reagent is 6.0-7.4 and the pH of the R2 reagent is 7.0-7.8. Preferably, the pH of the R1 reagent is 7.0 and the pH of the R2 reagent is 7.4.
On the basis, preferably, 0.8-1.2g/L of sodium azide is used as the preservative of the reagent R1, and 0.8-1.2g/L of Proclin300 is used as the preservative of the reagent R2.
In a second aspect, the present invention provides a method for preparing the adiponectin detection kit, comprising the steps of:
preparation of R1 reagent: according to the component content of the reagent R1, sequentially dissolving a buffer solution, an electrolyte, a melting promoter, a surfactant, a preservative and the like in pure water respectively, uniformly stirring, adjusting the pH to 6.0-7.4, uniformly stirring, and then placing in an oven at 35-40 ℃ for incubation overnight to obtain a reagent R1;
preparation of reagent R2: and (3) respectively adding a buffer solution, a stabilizer and a surfactant into the adiponectin monoclonal antibody-coated latex particles while stirring, finally adding a preservative and a protective agent, and stabilizing the mixture in a shaker at 35-40 ℃ for one night to obtain the R2 reagent.
Preferably, the preparation method of the anti-adiponectin monoclonal antibody coated latex particle comprises the following steps: taking latex microspheres with the particle size of 100-; then adding the prepared EDC and NHS solution, placing the solution in a shaking table at the temperature of 35-40 ℃, activating the latex for 10-20min at the speed of 100-300 r/min; under the condition of stirring, adding a buffer solution and an antibody, placing the mixture in a 35-40 shaking table, coupling for 1-5h at the speed of 100-; the antibody is anti-adiponectin monoclonal antibody.
Preferably, when adding the buffer, the stabilizer and the surfactant, the former substance is added and incubated at 35-40 deg.C for 20-40min in a shaker, and then the latter substance is added.
Preferably, the preparation method of the anti-adiponectin monoclonal antibody coated latex particle comprises the following steps: the preparation method of the anti-adiponectin monoclonal antibody coated latex particle comprises the following steps: taking latex microspheres with the particle size of 100-; then adding the prepared EDC and NHS solution, placing the solution in a shaking table at the temperature of 35-40 ℃, activating the latex for 10-20min at the speed of 100-300 r/min; under the condition of stirring, adding a buffer solution and an antibody, placing the mixture in a shaker at the temperature of 35-40 ℃, performing coupling for 1-5h at the speed of 100-300 r/min.
Further preferred conditions are: placing latex microspheres with the particle size of 100-300nm in HEPES buffer solution, placing the latex microspheres in a shaking table at 37 ℃, and activating the latex for 15min at 200 r/min; while stirring, buffer and antibody were added, and the mixture was shaken at 37 ℃ for 200r/min and coupled for 3 h.
Preferably, the concentration of the HEPES buffer is 0.01M to 0.1M.
Preferably, the method for preparing the adiponectin detection kit comprises the following steps:
preparation of reagent R1: according to the component content of the reagent R1, sequentially dissolving a buffer solution, an electrolyte, a melting promoter, a surfactant, a preservative and the like in pure water respectively, uniformly stirring, adjusting the pH to 6.0-7.4, uniformly stirring, and placing in an oven at 37 ℃ for incubation for one night to obtain the reagent R1.
Preparation of reagent R2: taking latex microspheres with the particle size of 100-300nm into 0.01-0.1M MES buffer solution, placing the latex microspheres in a shaking table at 37 ℃, shaking at 200r/min, and shaking uniformly for 5 min; adding the prepared EDC and NHS solution, placing in a shaker at 37 ℃, and activating the latex for 15min at 200 r/min; adding HEPES buffer solution and an antibody under the condition of stirring, placing the mixture in a shaking table at 37 ℃, performing 200R/min, coupling for 3h, then taking the mixture out of the shaking table, placing the mixture on a stirrer, adding the HEPES buffer solution, BSA and a surfactant respectively, adding another substance after one substance is added and incubated in the shaking table at 37 ℃ for 30min, finally adding a preservative and a protective agent, and obtaining the reagent R2 after the shaking table is stabilized at 37 ℃ for one night.
In a third aspect, the invention also provides a surfactant for an adiponectin detection kit, which comprises Tetronic1307 and castor oil polyoxyethylene ether.
Alternatively, Tetronic1307 can be used at a concentration of 0.1-5g/L and castor oil polyoxyethylene ether can be used at a concentration of 0.1-5 g/L.
The invention has the following beneficial effects:
1. the novel surfactant combination is adopted to be matched with other components in the kit, so that the anti-interference capability of the R1 reagent on special samples such as turbid lipid samples and hemolytic samples is enhanced, the interference of nonspecific adsorption is reduced, the occurrence of false negative and false positive in the sample testing process is avoided, and the accuracy of sample testing is improved; the reproducibility and stability of the latex reagent is also enhanced.
2. The kit completely avoids the defects of enzyme-linked immunosorbent assay and chemiluminescence assay, can rapidly detect in large batch, and can detect a single sample. Compared with the existing latex enhanced immunoturbidimetry preparation method which is complex in process and consumes manpower and material resources, the adiponectin detection kit provided by the invention is simple in preparation process, does not need the steps of centrifugation, ultrasonic dispersion and the like, saves time and effectively reduces the batch difference of reagents.
Drawings
FIG. 1 calibration curve for inventive example 1;
FIG. 2 Linear Range of inventive example 1.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. In the present invention, the equipment and materials used are commercially available or commonly used in the art, if not specified. The methods in the following examples are conventional in the art unless otherwise specified.
Example 1
The embodiment provides an adiponectin detection kit with strong anti-interference performance and good repeatability, and the preparation method comprises the following steps:
(1) preparation of reagent R1:
100mL of citric acid buffer solution was weighed, placed on a stirrer, and 3.0g of NaCl, 5g of PEG6000, 0.2g of Tetronic1307, 0.1g of castor oil polyoxyethylene ether, and 0.1g of NaN3 were added in this order. Adding another substance after one substance is completely dissolved, adjusting the pH to 7.0 after the other substance is completely dissolved, and placing the mixture in a shaking table at 37 ℃ for incubation for one night to obtain the reagent R1.
After the preparation, the concentrations of the components of the reagent R1 are as follows: 50mM/L of citric acid buffer solution; 30g/L of sodium chloride; PEG 600050 g/L; tetronic 13072 g/L; 1g/L of castor oil polyoxyethylene ether; NaN 31 g/L.
(2) Preparation of reagent R2:
preparing anti-adiponectin monoclonal antibody coated latex particles:
80ul of latex microspheres with the particle size of 200nm are taken and put in 130ul of 0.02M MES buffer solution with the pH value of 6.0, and then put in a shaking table at 37 ℃ for 200r/min and then evenly shaken for 2 min. Then adding the solution into the prepared EDC and NHS solution, placing the solution in a shaker at 37 ℃, shaking at 200r/min, and shaking for 15min to activate the latex; the concentration of EDC and NHS is 0.10M, the addition ratio is 1: 1; thereafter, 3ml of HEPES buffer (0.02M pH7.4) and an antibody solution (mixture of monoclonal antibody 1 and monoclonal antibody 2, wherein the monoclonal antibody is a mouse anti-human ADP antibody) were added with stirring in a total amount of 30 mg/L. The mixture is placed on a shaker at 37 ℃ and coupled for 3h at a speed of 200 r/min.
② taking the mixture in the last step out of the shaking table, placing the mixture on a stirrer, and respectively adding 1ml of HEPES buffer solution, 500ul of 10% BSA, 5ul of Tetronic1307 and 5ul of castor oil polyoxyethylene ether. After one substance is added and incubated for 30min at 37 ℃ in a shaking table, the other substance is added, and finally 5ul proclin300 and 0.1g trehalose are added, and the substances are incubated for one night at 37 ℃ in a shaking table to obtain a reagent R2.
After the preparation is completed, the concentrations of the components of the reagent R2 are respectively as follows: the anti-adiponectin monoclonal antibody coating latex particles are 80mg/L, HEPES buffer solution 25mM/L, BSA 10g/L, Tetronic 13071 g/L, castor oil polyoxyethylene ether 1g/L, proclin3001g/L and trehalose 20 g/L.
Example 2
The embodiment provides an adiponectin detection kit with strong interference resistance and good repeatability, which comprises an R1 reagent and an R2 reagent, and the preparation method comprises the following steps:
(1) preparation of reagent R1:
100ml of citric acid buffer solution is weighed, placed on a stirrer and sequentially added with 3.0g of NaCl、5g PEG6000、0.2g Tetronic 1307、0.1g NaN3. Adding another substance after one substance is completely dissolved, adjusting the pH to 7.0 after the other substance is completely dissolved, and placing the mixture in a shaking table at 37 ℃ for incubation for one night to obtain the reagent R1.
After the preparation, the concentrations of the components of the reagent R1 are as follows: 50mM/L of citric acid buffer solution; 30g/L of sodium chloride; PEG 600050 g/L; tetronic 13072 g/L; NaN3 1g/L。
(2) Preparation of reagent R2:
preparing anti-adiponectin monoclonal antibody coated latex particles:
80ul of latex microspheres with the particle size of 200nm are taken and put in 130ul of 0.02M MES buffer solution with the pH value of 6.0, and then put in a shaking table at 37 ℃ for 200r/min and then evenly shaken for 2 min. Then adding the solution into the prepared EDC and NHS solution, placing the solution in a shaker at 37 ℃, shaking at 200r/min, and shaking for 15min to activate the latex; EDC and NHS were added at a concentration of 0.10M and a ratio of 1: 1. Thereafter, 3ml of HEPES buffer (0.02M pH7.4) and an antibody solution (mixture of mAbs 1 and 2) were added with stirring to give an antibody addition total of 30 mg/L. The mixture is placed on a shaker at 37 ℃ and coupled for 3h at a speed of 200 r/min.
② taking the mixture in the last step out of the shaking table, placing the mixture on a stirrer, and respectively adding 1ml of HEPES buffer solution, 500ul of 10% BSA, 5ul of Tetronic1307 and 5ul of castor oil polyoxyethylene ether. After one substance is added and incubated for 30min at 37 ℃ in a shaking table, the other substance is added, and finally 5ul proclin300 and 0.1g trehalose are added, and the substances are incubated for one night at 37 ℃ in a shaking table to obtain a reagent R2.
After the preparation is completed, the concentrations of the components of the reagent R2 are respectively as follows: the anti-adiponectin monoclonal antibody coating latex particles are 80mg/L, HEPES buffer solution 25mM/L, BSA 10g/L, Tetronic 13071 g/L, castor oil polyoxyethylene ether 1g/L, proclin3001g/L and trehalose 20 g/L.
Example 3
The embodiment provides an adiponectin detection kit, which comprises an R1 reagent and an R2 reagent, and the preparation method comprises the following steps:
(1) preparation of reagent R1:
weighing 100mL of citric acid buffer solution, placing on a stirrer, and sequentially adding3.0g NaCl, 5g PEG6000, 0.1g castor oil polyoxyethylene ether, 0.1g NaN3. Adding another substance after one substance is completely dissolved, adjusting the pH to 7.0 after the other substance is completely dissolved, and placing the mixture in a shaking table at 37 ℃ for incubation for one night to obtain the reagent R1.
After the preparation, the concentrations of the components of the reagent R1 are as follows: 50mM/L of citric acid buffer solution; 30g/L of sodium chloride; PEG 600050 g/L; 1g/L of castor oil polyoxyethylene ether; NaN3 1g/L。
(2) Preparation of reagent R2:
preparing anti-adiponectin monoclonal antibody coated latex particles:
80ul of latex microspheres with the particle size of 200nm are taken and put in 130ul of 0.02M MES buffer solution with the pH value of 6.0, and then put in a shaking table at 37 ℃ for 200r/min and then evenly shaken for 2 min. Then adding the solution into the prepared EDC and NHS solution, placing the solution in a shaker at 37 ℃, shaking at 200r/min, and shaking for 15min to activate the latex; EDC and NHS were added at a concentration of 0.10M and a ratio of 1: 1. Thereafter, 3ml of HEPES buffer (0.02M pH7.4) and an antibody solution (mixture of mAbs 1 and 2) were added with stirring to give an antibody addition total of 30 mg/L. The mixture is placed on a shaker at 37 ℃ and coupled for 3h at a speed of 200 r/min.
② taking the mixture in the last step out of the shaking table, placing the mixture on a stirrer, and respectively adding 1ml of HEPES buffer solution, 500ul of 10% BSA, 5ul of Tetronic1307 and 5ul of castor oil polyoxyethylene ether. After one substance is added and incubated for 30min at 37 ℃ in a shaking table, the other substance is added, and finally 5ul proclin300 and 0.1g trehalose are added, and the substances are incubated for one night at 37 ℃ in a shaking table to obtain a reagent R2.
After the preparation is completed, the concentrations of the components of the reagent R2 are respectively as follows: the anti-adiponectin monoclonal antibody coating latex particles are 80mg/L, HEPES buffer solution 25mM/L, BSA 10g/L, Tetronic 13071 g/L, castor oil polyoxyethylene ether 1g/L, proclin3001g/L and trehalose 20 g/L.
Example 4
The embodiment provides an adiponectin detection kit with strong interference resistance and good repeatability, which comprises an R1 reagent and an R2 reagent, and the preparation method comprises the following steps:
(1) preparation of reagent R1:
weighing 100mL lemonAcid buffer solution, placed on the stirrer, and sequentially added with 3.0g NaCl, 5g PEG6000, 0.5g Tween-20, and 0.1g NaN3. Adding another substance after one substance is completely dissolved, adjusting the pH to 7.0 after the other substance is completely dissolved, and placing the mixture in a shaking table at 37 ℃ for incubation for one night to obtain the reagent R1.
After the preparation, the concentrations of the components of the reagent R1 are as follows: 50mM/L of citric acid buffer solution; 30g/L of sodium chloride; PEG 600050 g/L; tween-205 g/L; NaN3 1g/L。
(2) Preparation of reagent R2:
preparing anti-adiponectin monoclonal antibody coated latex particles:
80ul of latex microspheres with the particle size of 200nm are taken and put in 130ul of 0.02M MES buffer solution with the pH value of 6.0, and then put in a shaking table at 37 ℃ for 200r/min and then evenly shaken for 2 min. Then adding the solution into the prepared EDC and NHS solution, placing the solution in a shaker at 37 ℃, shaking at 200r/min, and shaking for 15min to activate the latex; EDC and NHS were added at a concentration of 0.10M and a ratio of 1: 1. Thereafter, 3ml of HEPES buffer (0.02M pH7.4) and an antibody solution (mixture of mAbs 1 and 2) were added with stirring to give an antibody addition total of 30 mg/L. The mixture is placed on a shaker at 37 ℃ and coupled for 3h at a speed of 200 r/min.
② taking the mixture in the last step out of the shaking table, placing the mixture on a stirrer, and respectively adding 1ml of HEPES buffer solution, 500ul of 10% BSA, 5ul of Tetronic1307 and 5ul of castor oil polyoxyethylene ether. After one substance is added and incubated for 30min at 37 ℃ in a shaking table, the other substance is added, and finally 5ul proclin300 and 0.1g trehalose are added, and the substances are incubated for one night at 37 ℃ in a shaking table to obtain a reagent R2.
After the preparation is completed, the concentrations of the components of the reagent R2 are respectively as follows: the anti-adiponectin monoclonal antibody coating latex particles are 80mg/L, HEPES buffer solution 25mM/L, BSA 10g/L, Tetronic 13071 g/L, castor oil polyoxyethylene ether 1g/L, proclin3001g/L and trehalose 20 g/L.
Example 5
The embodiment provides an adiponectin detection kit, which comprises an R1 reagent and an R2 reagent, and the preparation method comprises the following steps:
(1) preparation of reagent R1:
weighing 100mL lemonAcid buffer solution, placed on a stirrer, and sequentially added with 3.0g of NaCl, 5g of PEG6000, 0.2g of Tetronic1307, 0.1g of castor oil polyoxyethylene ether and 0.1g of NaN3. Adding another substance after one substance is completely dissolved, adjusting the pH to 7.0 after the other substance is completely dissolved, and placing the mixture in a shaking table at 37 ℃ for incubation for one night to obtain the reagent R1.
After the preparation, the concentrations of the components of the reagent R1 are as follows: 50mM/L of citric acid buffer solution; 30g/L of sodium chloride; PEG 600050 g/L; tetronic 13072 g/L; 1g/L of castor oil polyoxyethylene ether; NaN3 1g/L。
(2) Preparation of reagent R2:
preparing anti-adiponectin monoclonal antibody coated latex particles:
80ul of latex microspheres with the particle size of 200nm are taken and put in 130ul of 0.02M MES buffer solution with the pH value of 6.0, and then put in a shaking table at 37 ℃ for 200r/min and then evenly shaken for 2 min. Then adding the solution into the prepared EDC and NHS solution, placing the solution in a shaker at 37 ℃, shaking at 200r/min, and shaking for 15min to activate the latex; EDC and NHS were added at a concentration of 0.10M and a ratio of 1: 1. Thereafter, 3ml of HEPES buffer (0.02M pH7.4) and an antibody solution (mixture of mAbs 1 and 2) were added with stirring to give an antibody addition total of 30 mg/L. The mixture is placed on a shaker at 37 ℃ and coupled for 3h at a speed of 200 r/min.
② taking the mixture of the last step out of the shaker, placing the mixture on a stirrer, and respectively adding 1ml of HEPES buffer solution, 500ul of 10% BSA, and 10ul of triton-100. After one substance is added and incubated for 30min at 37 ℃ in a shaking table, the other substance is added, and finally 5ul proclin300 and 0.1g trehalose are added, and the substances are incubated for one night at 37 ℃ in a shaking table to obtain a reagent R2.
After the preparation is completed, the concentrations of the components of the reagent R2 are respectively as follows: the anti-adiponectin monoclonal antibody coating latex particles are 80mg/L, HEPES buffer solution 25mM/L, BSA 10g/L, triton-1002 g/L, proclin3001g/L and trehalose 20 g/L.
EXAMPLE 6 use of adiponectin detection kit
The detection method of the adiponectin detection kit of the embodiment comprises the following steps:
1. biochemical analyzer detection
Taking Hitachi 7180 operation as an example: measuring the wavelength at 700nm, respectively taking calibrator solutions (3uL) with different concentrations, adding an adiponectin R1 reagent (160uL), uniformly mixing, incubating at 37 ℃ for 5 minutes, adding an adiponectin R2 reagent (40uL), uniformly mixing, incubating at 37 ℃ for 30 seconds, reading the absorbance value A1 of each tube, reacting for 4.5 minutes, reading the absorbance value A2 of each tube, calculating the absorbance difference delta A which is A2-A1, repeatedly measuring for 2 times in each tube, taking the average value of the absorbance difference delta A measured 2 times in each calibrator tube as a vertical coordinate, taking the corresponding calibrator concentration as an abscissa, and drawing a calibration curve of 'concentration-absorbance difference'. And (3) taking a serum or plasma sample to be detected, determining the absorbance difference of the sample by the same method, and substituting the absorbance difference into the calibration curve to calculate the content of adiponectin ADPN in the sample to be detected. If the concentration of the adiponectin in the serum or the plasma exceeds the range of the calibration curve, the sample needs to be diluted and then detected so as to ensure the accuracy of the detection result. The kit is not only suitable for Hitachi 7180, but also suitable for semi-automatic and full-automatic biochemical analyzers of other brands and models, and specific parameters can be adjusted according to the instruments.
Example 7 quality evaluation of the kit
(1) Preparation of standard curve
A. Experimental methods
Diluting the high-value calibrator (42mg/L) by using distilled water in a multiple ratio, wherein the concentrations are as follows: 0mg/L, 5.25mg/L, 10.5mg/L, 21mg/L, 42 mg/L. The calibration curve of the adiponectin calibrator of the present invention was measured according to the method in example 6. The subject was the adiponectin detection kit prepared in example 1.
B. Results and analysis of the experiments
FIG. 1 shows a calibration curve of the kit of example 1 of the present invention.
(2) Linear range
A. Experimental methods
At least 5 dilution concentrations (x) are mixed with a high concentration calibrator (42mg/L) near the upper limit of the linear range and a low concentration calibrator or distilled water near the lower limit of the linear range. The kits were tested separately, 2 times for each dilution concentration, and the mean (y) of the test results was determined separately. And (3) taking the dilution concentration (x) as an independent variable and taking the detection result mean value (y) as a dependent variable to obtain a linear regression equation. The subject was the adiponectin detection kit prepared in example 1.
B. Results and analysis of the experiments
As shown in FIG. 2, the kit of the invention has good linearity, and the regression equation is as follows: y 0.9721x-0.0523, R2＝0.9997。
(3) Sensitivity of analysis
A. Experimental methods
The calibrator having a known concentration of 10.5mg/L was tested using the kit prepared in example 1, the absolute value of the absorbance change value generated under the parameters specified in the kit was recorded, and the test was repeated 3 times, averaged and converted to an absorbance change value of 10 mg/L.
B. Results and analysis of the experiments
The test results are respectively 0.2504, 0.2481 and 0.2492, and the absorbance change value converted into 10mg/L is 0.2373, which indicates that the kit has higher sensitivity.
(4) Detection of repeatability
A. Experimental methods
The low-value sample of adiponectin whose measured value of Nocongong department of the Itanium is 5.91mg/L was used as the sample, the measurement was performed according to the biochemical analyzer detection method, each concentration was repeatedly measured 10 times, the measurement mean and the standard deviation (S) were calculated, and the repetitive investigation was performed on the kits of examples 1 and 5 by calculating the coefficient of variation.
Since the reproducibility of the kit is mainly related to the R2 reagent, the experiment was mainly performed on the kits prepared in examples 1 and 5 for reproducibility.
B. Results and analysis of the experiments
TABLE 1 results of the reproducibility test of adiponectin assay kits
As shown in the results in Table 1, in example 5, the repeatability of the kit on the low-value sample is poor and reaches 7.13% after the Triton-100 is used as the surfactant, while in example 1, the repeatability of the kit on the low-value sample is obviously improved after a new surfactant group (Tetronic 1307 and castor oil polyoxyethylene ether) is added, and the coefficient of variation is as low as 2.02%.
(5) Anti-interference experiment
A. Experimental methods
The adiponectin samples with the concentration of 5.91mg/L were taken, the interfering substances with different concentrations in the same volume were added to make the concentrations of the added triglyceride interfering substances 2.5g/L, 5.0g/L, 8.0g/L, bilirubin 400mg/L and hemoglobin 10g/L, the measurement was repeated 3 times using the kits prepared in examples 1 to 5, respectively, and the average value was taken to observe the relative deviation of the ADPN measurement value after the addition of the interfering substance and the distilled water compared with the samples with the same volume of distilled water.
B. Results and analysis of the experiments
TABLE 2 anti-interference test results
Note: the relative deviation is not more than +/-10 percent, and the standard is basically reached.
From the experimental results in table 2, the interference rejection is ranked from strong to weak: example 1 > example 2 > example 3 > example 5 > example 4; wherein, for triglyceride, the anti-interference ability of the embodiments 1 to 3 basically reaches the standard; the interference rejection of examples 1-5 was substantially met for bilirubin and hemoglobin interference. Wherein the anti-interference ability of the kit of example 1 is optimal, and the relative deviation between the measured values of the interfering substances with different concentrations and the measured values after the distilled water with the same volume is added is not more than 4%. Therefore, when the interference substances such as triglyceride, bilirubin and hemoglobin with certain concentration exist in the detection sample, the interference substances have no influence on the detection result of the kit, and the anti-interference capability of the kit is strong.
(5) Stability of
A. Experimental methods
The kit of the invention is placed in a water bath at 37 ℃, and the calibration product is calibrated and tested for analysis after being stored for 3 days, 5 days and 7 days before (namely, when the kit is not damaged).
B. Results and analysis of the experiments
From the results in the above table, the kit of example 1 still maintained more than 99% of reactivity after being stored at 37 ℃ for 7 days, which shows that the stability of the adiponectin detection kit at normal temperature can be significantly improved by adding Tetronic1307 and castor oil polyoxyethylene ether to the reagent, and the practicability of the kit of the invention for clinical diagnosis is increased.
It should be understood that the technical solutions and concepts of the present invention may be equally replaced or changed by those skilled in the art, and all such changes or substitutions should fall within the protection scope of the appended claims.
Claims (10)
1. An adiponectin detection kit, which is characterized by comprising an R1 reagent and an R2 reagent; wherein:
the R1 reagent includes the following components: buffer solution, electrolyte, melting promoter, surfactant and preservative;
the R2 reagent includes the following components: the anti-adiponectin monoclonal antibody comprises latex particles coated with the anti-adiponectin monoclonal antibody, buffer solution, protective agent, stabilizing agent, preservative and surfactant;
wherein the surfactant is selected from one or two of Tetronic1307 and castor oil polyoxyethylene ether.
2. The adiponectin detection kit of claim 1, wherein the surfactant comprises Tetronic1307 and castor oil ethoxylate.
3. The adiponectin detection kit according to claim 1, wherein the protective agent is one or two selected from trehalose and sucrose.
4. The adiponectin detection kit according to claim 1, wherein the stabilizer is one or more selected from casein and bovine serum albumin.
5. The adiponectin detection kit according to claim 1, wherein the buffer solution is one or more of TRIS buffer solution, phosphate buffer solution, HEPES buffer solution, and citric acid buffer solution; the electrolyte is one or more of sodium chloride, potassium chloride and calcium chloride; the melting promoter is one or more of PEG6000, PEG8000 and PEG 12000; the preservative is one or more of sodium azide and Proclin 300.
6. The adiponectin detection kit according to claim 1, wherein the R1 reagent comprises the following concentrations of the respective components: 0.02-0.1M/L of citric acid buffer solution; 20-40g/L of sodium chloride; PEG 600030-80 g/L; tetronic 13070.1-5 g/L; 0.1-5g/L of castor oil polyoxyethylene ether; 0.8-1.2g/L of preservative;
the R2 reagent included the following components at the following concentrations: coating latex particles with anti-adiponectin monoclonal antibody: 60-100 mg/L; HEPES buffer solution 0.01-0.05M/L; 5-30g/L of trehalose; 5-20g/L of bovine serum albumin; tetronic 13070.1-5 g/L; 0.1-5g/L of castor oil polyoxyethylene ether; 0.8-1.2g/L of preservative.
7. The adiponectin detection kit according to claim 6, wherein the preservative in the reagent R1 is sodium azide of 0.8 to 1.2g/L, and the preservative in the reagent R2 is Proclin300 of 0.8 to 1.2 g/L.
8. The method of preparing the adiponectin detection kit according to claim 1, comprising the steps of:
preparation of R1 reagent: according to the component content of the reagent R1, sequentially dissolving a buffer solution, an electrolyte, a melting promoter, a surfactant, a preservative and the like in pure water respectively, uniformly stirring, adjusting the pH to 6.0-7.4, uniformly stirring, and then placing in an oven at 35-40 ℃ for incubation overnight to obtain a reagent R1;
preparation of reagent R2: and (3) respectively adding a buffer solution, a stabilizer and a surfactant into the adiponectin monoclonal antibody-coated latex particles while stirring, finally adding a preservative and a protective agent, and stabilizing the mixture in a shaker at 35-40 ℃ for one night to obtain the R2 reagent.
9. The method for preparing the adiponectin detection kit according to claim 8, wherein the method for preparing the anti-adiponectin monoclonal antibody-coated latex particle comprises: taking latex microspheres with the particle size of 100-; then adding the prepared EDC and NHS solution, placing the solution in a shaking table at the temperature of 35-40 ℃, activating the latex for 10-20min at the speed of 100-300 r/min; under the condition of stirring, adding a buffer solution and the anti-adiponectin monoclonal antibody, placing the mixture in a shaking table at the temperature of 35-40 ℃, shaking the mixture at the speed of 100-300r/min, and coupling the mixture for 1-5 h.
10. The method of claim 9, wherein the buffer, the stabilizer and the surfactant are added after the former substance is added and incubated at 35-40 ℃ for 20-40min in a shaker.
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