CN114295842A - A kind of adiponectin detection kit and preparation method thereof - Google Patents

Abstract

The invention discloses an adiponectin detection kit and a preparation method thereof. The adiponectin detection kit includes R1 reagent and R2 reagent; the R1 reagent includes the following components: buffer solution, electrolyte, fusion enhancer, surfactant and Preservative; R2 reagent includes the following components: anti-adiponectin monoclonal antibody-coated latex particles, buffer, protective agent, stabilizer, preservative and surfactant; wherein, the surfactant is selected from Tetronic 1307, castor oil polyoxygen One or both of vinyl ethers. The kit of the present invention not only enhances the anti-interference ability of the reagent for special samples such as chyle and lipid turbidity samples, but also reduces the interference of non-specific adsorption by using a new combination of surfactants to cooperate with other components in the kit. Improve the accuracy of sample testing, but also enhance the repeatability and stability of latex reagents.

Description

A kind of adiponectin detection kit and preparation method thereof

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 technique

Adiponectin (ADPN), an endogenous biologically active polypeptide and protein secreted by adipocytes, can exist stably in plasma, accounting for about 0.01% of plasma protein. The earliest adiponectin was found in human subcutaneous adipose tissue, plasma and murine adipocytes.

With the study of adiponectin, it was found that adiponectin is an insulin-sensitizing hormone (An Insulin-sensitizing Hormone), which can improve insulin resistance (Insulin resistance) and arteriosclerosis; Adiponectin level can predict type II diabetes and the development of coronary heart disease, and has been shown in clinical trials to be associated with obesity, diabetes, insulin resistance, atherosclerosis and inflammation. Therefore, the detection of blood adiponectin levels is of great significance for the diagnosis and prognosis of these diseases.

At present, the methods for the determination of 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 equipment and is expensive; the ELISA method is extremely cumbersome and complicated, with long detection time and poor repeatability. The latex-enhanced immune turbidimetric method is to use the surface cross-linked monoclonal or polyclonal antibody of a certain size of latex particles to combine with the antigen to detect the concentration of the antigen; the existing latex-enhanced immune turbidimetric method has high sensitivity, but anti-interference The performance is poor, the repeatability in the low value area is poor, and the preparation process is complicated, which consumes manpower and material resources.

Therefore, the existing technology needs to be further improved.

SUMMARY OF THE INVENTION

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

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

In a first aspect, the present invention provides an adiponectin detection kit, which includes R1 reagent and R2 reagent; wherein:

The R1 reagent includes the following components: buffers, electrolytes, thawing agents, surfactants and preservatives;

The R2 reagent includes the following components: anti-adiponectin monoclonal antibody-coated latex particles, buffers, protective agents, stabilizers, preservatives, and surfactants;

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

Preferably, the R1 reagent includes the following concentrations of each component:

Buffer 0.02-0.1M/L;

Electrolyte 20-40g/L;

Melting agent 30-80g/L;

Surfactant 0.2-10g/L;

Preservative 0.8-1.2g/L.

The R2 reagent includes the following concentrations of each component:

Anti-adiponectin monoclonal antibody coated latex particles 60-100mg/L;

Buffer 0.01-0.05M/L;

Protective agent 5-30g/L;

Stabilizer 5-20g/L;

Preservative 0.8-1.2g/L;

Surfactant 0.2-10g/L.

Preferably, in the adiponectin detection kit, the surfactants include Tetronic 1307 and castor oil polyoxyethylene ether.

The addition of the above surfactants to R1 enhances the anti-interference ability of the reagents to special samples such as lipid turbidity and hemolytic samples, reduces the interference of non-specific adsorption, and avoids false negatives and false positives during sample testing. appears, thereby improving the accuracy of sample testing. Both Tetronic 1307 and castor oil polyoxyethylene ether are good solubilizers, which can disperse and dissolve oils, reduce matrix interference and non-specific adsorption, and neither destroy the protein structure; especially castor oil polyoxyethylene ether, its The HLB value is between 13-14, which 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.

The addition of two surfactants, Tetronic 1307 and castor oil polyoxyethylene ether, into R2 has multiple effects: on the one hand, the latex reagent can be dispersed more uniformly and the repeatability of the latex reagent can be enhanced; on the other hand, castor oil polymerized Oxyethylene ether has excellent emulsifying ability. It can not only form a hydration film on the surface of the protein to protect the protein and increase the stability of the protein, but also form surface tension and enhance the stability of the liquid.

Preferably, in the adiponectin detection kit, the protective agent is selected from one or both 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 is one or more of TRIS buffer, phosphate buffer, HEPES buffer, and citrate buffer.

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

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

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

Preferably, in the adiponectin detection kit, the R1 reagent includes each component in the following concentrations: citrate buffer 0.02-0.1M/L; sodium chloride 20-40g/L; PEG6000 30-80g/L; Tetronic 13070.1-5g/L; castor oil polyoxyethylene ether 0.1-5g/L; preservative 0.8-1.2g/L;

The R2 reagent includes each component at 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- 20g/L; Tetronic1307 0.1-5g/L; Castor oil polyoxyethylene ether 0.1-5g/L; Preservative 0.8-1.2g/L.

Optionally, 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 this basis, preferably, 0.8-1.2 g/L sodium azide is used as the preservative of the R1 reagent, and 0.8-1.2 g/L Proclin300 is used as the preservative in the R2 reagent.

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

Preparation of Reagent R1: According to the component content of Reagent R1, dissolve buffer, electrolyte, melting agent, surfactant, preservative, etc. in pure water in turn, stir evenly, adjust pH to 6.0-7.4, and stir evenly After incubation at 35-40℃ for one night, the R1 reagent was obtained;

Preparation of reagent R2: Add buffer, stabilizer and surfactant to anti-adiponectin monoclonal antibody-coated latex particles while stirring, and finally add preservative and protective agent, and stabilize at 35-40°C on a shaker overnight. That is, the R2 reagent is obtained.

Preferably, the preparation method of the anti-adiponectin monoclonal antibody-coated latex particles is as follows: take latex microspheres with a particle size of 100-300nm in MES buffer, shake at 35-40°C, 100-300r/min, Shake for 3-10min; then add the freshly prepared EDC and NHS solutions, place on a shaker at 35-40℃, 100-300r/min, activate the latex for 10-20min; under stirring, add buffer and antibody, set Shaking at 35-40, 100-300r/min, coupling for 1-5h; the antibody is anti-adiponectin monoclonal antibody.

Preferably, when adding buffers, stabilizers and surfactants, the latter substances should be added after adding the former substances and incubating at 35-40° C. for 20-40 min on a shaker.

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

Further preferred conditions are as follows: take latex microspheres with a particle size of 100-300 nm in HEPES buffer, place on a shaker at 37 °C, 200 r/min, and activate the latex for 15 min; under stirring, add buffer and antibody, set to Shaking at 37°C, 200r/min, coupling for 3h.

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

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

Preparation of Reagent R1: According to the component content of Reagent R1, dissolve buffer, electrolyte, melt enhancer, surfactant, preservative, etc. in pure water in sequence, stir evenly, adjust pH to 6.0-7.4, and stir evenly After incubation at 37°C for one night, reagent R1 was obtained.

Preparation of reagent R2: Take latex microspheres with a particle size of 100-300nm in 0.01M-0.1M MES buffer, place on a shaker at 37°C, 200r/min, and shake for 5min; then add the freshly prepared EDC and NHS The solution was placed on a shaker at 37°C, 200 r/min, and the latex was activated for 15 minutes; under stirring, add HEPES buffer and antibody, placed on a shaker at 37°C, 200 r/min, and removed from the shaker after coupling for 3 hours. , placed on a stirrer, and added HEPES buffer, BSA and surfactant respectively. After adding one substance and incubating it on a shaker for 30 min at 37°C, add another substance, and finally add preservatives and protective agents. , and the reagent R2 was obtained after it was stabilized on a shaker at 37°C overnight.

In a third aspect, the present invention also provides a surfactant for an adiponectin detection kit, which comprises Tetronic 1307 and castor oil polyoxyethylene ether.

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

The present invention has the following beneficial effects:

1. The present invention adopts a new combination of surfactants to cooperate with other components in the kit, which not only enhances the anti-interference ability of R1 reagent for special samples such as lipid turbidity and hemolytic samples, reduces the interference of non-specific adsorption, and avoids The occurrence of false negatives and false positives in the sample testing process improves the accuracy of sample testing; it also enhances the repeatability and stability of latex reagents.

2. The kit completely avoids the shortcomings of enzyme-linked immunosorbent assay and chemiluminescence method, and can not only detect large batches quickly, but also detect a single sample. Compared with the existing latex-enhanced immune turbidimetric method, the preparation process is complex and consumes manpower and material resources. The preparation process of the adiponectin detection kit provided by the present invention is simple, and steps such as centrifugation and ultrasonic dispersion are not required, which saves time and effectively reduces the Lot-to-lot variation of reagents.

Description of drawings

Fig. 1 calibration curve of embodiment 1 of the present invention;

Figure 2 Linear range of Example 1 of the present invention.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, but not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative efforts shall fall within the protection scope of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or commonly used in the field. The methods in the following examples, unless otherwise specified, are conventional methods in the art.

Example 1

The present embodiment provides an adiponectin detection kit with strong anti-interference and good repeatability, the preparation method of which includes the following steps:

(1) Preparation of reagent R1:

Weigh 100 mL of citric acid buffer, place it on a stirrer, and add 3.0 g of NaCl, 5 g of PEG6000, 0.2 g of Tetronic 1307, 0.1 g of castor oil polyoxyethylene ether, and 0.1 g of NaN3 in sequence. After one substance is completely dissolved, another substance is added. After complete dissolution, the pH is adjusted to 7.0, and the reagent R1 is obtained after incubating at 37°C for one night.

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.

(2) Preparation of reagent R2:

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

Take 80 ul of latex microspheres with a particle size of 200 nm in 130 ul of 0.02M MES buffer at pH 6.0, place on a shaker at 37°C, 200 r/min, and shake for 2 min. After that, it was added to the prepared EDC and NHS solution, placed on a shaker at 37°C, 200 r/min, and shaken for 15 minutes to activate the latex; the concentration of EDC and NHS was 0.10M, and the addition ratio was 1:1; In this case, add 3 ml of HEPES buffer (0.02M pH7.4) and antibody solution (mix of mAb 1 and mAb 2, where the mAb is mouse anti-human ADP antibody), and the total amount of antibody added is 30 mg/L. Place on a shaker at 37°C, 200r/min, and couple for 3h.

② Take 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, respectively. The above substances were all added after one substance was added and incubated at 37°C for 30 minutes before adding another substance. Finally, 5ul of proclin300 and 0.1g trehalose were added, and the reagent R2 was obtained after overnight incubation at 37°C.

After the preparation is completed, the concentrations of the components of reagent R2 are: anti-adiponectin monoclonal antibody-coated latex particles 80mg/L, HEPES buffer 25mM/L, BSA 10g/L, Tetronic 1307 1g/L, castor oil polyoxyethylene Ether 1g/L, proclin 300 1g/L, trehalose 20g/L.

Example 2

The present embodiment provides an adiponectin detection kit with strong anti-interference and good repeatability, including R1 reagent and R2 reagent, and the preparation method thereof includes the following steps:

(1) Preparation of reagent R1:

100 ml of citric acid buffer was weighed, placed on a stirrer, and 3.0 g of NaCl, 5 g of PEG6000, 0.2 g of Tetronic 1307, and 0.1 g of NaN ₃ were added in sequence. After one substance is completely dissolved, another substance is added, and after complete dissolution, the pH is adjusted to 7.0, and the reagent R1 is obtained after incubating at 37°C in a shaker for one night.

After the configuration is completed, the concentrations of the components of reagent R1 are: citrate buffer 50mM/L; sodium chloride 30g/L; PEG6000 50g/L; Tetronic 1307 2g/L; NaN ₃ 1g/L.

(2) Preparation of reagent R2:

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

Take 80 ul of latex microspheres with a particle size of 200 nm in 130 ul of 0.02M MES buffer at pH 6.0, place on a shaker at 37°C, 200 r/min, and shake for 2 min. Then, it was added to the prepared EDC and NHS solution, placed on a shaker at 37 °C, 200 r/min, and shaken for 15 min to activate the latex; the concentration of EDC and NHS was 0.10 M, and the addition ratio was 1:1. Then, under stirring, 3 ml of HEPES buffer (0.02M pH7.4) and antibody solution (mixing of monoclonal antibodies 1 and 2) were added, and the total amount of antibody added was 30 mg/L. Place on a shaker at 37°C, 200r/min, and couple for 3h.

② Take 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, respectively. The above substances were all added after one substance was added and incubated at 37°C for 30 minutes before adding another substance. Finally, 5ul of proclin300 and 0.1g trehalose were added, and the reagent R2 was obtained after overnight incubation at 37°C.

After the preparation is completed, the concentrations of the components of reagent R2 are: anti-adiponectin monoclonal antibody-coated latex particles 80mg/L, HEPES buffer 25mM/L, BSA 10g/L, Tetronic 1307 1g/L, castor oil polyoxyethylene Ether 1g/L, proclin 300 1g/L, trehalose 20g/L.

Example 3

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

(1) Preparation of reagent R1:

100 mL of citric acid buffer was weighed, placed on a stirrer, and 3.0 g of NaCl, 5 g of PEG6000, 0.1 g of castor oil polyoxyethylene ether, and 0.1 g of NaN ₃ were added in sequence. After one substance is completely dissolved, another substance is added, and after complete dissolution, the pH is adjusted to 7.0, and the reagent R1 is obtained after incubating at 37°C in a shaker for one night.

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; NaN ₃ 1g/L.

(2) Preparation of reagent R2:

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

Take 80 ul of latex microspheres with a particle size of 200 nm in 130 ul of 0.02M MES buffer at pH 6.0, place on a shaker at 37°C, 200 r/min, and shake for 2 min. Then, it was added to the prepared EDC and NHS solution, placed on a shaker at 37 °C, 200 r/min, and shaken for 15 min to activate the latex; the concentration of EDC and NHS was 0.10 M, and the addition ratio was 1:1. Then, under stirring, 3 ml of HEPES buffer (0.02M pH7.4) and antibody solution (mixing of monoclonal antibodies 1 and 2) were added, and the total amount of antibody added was 30 mg/L. Place on a shaker at 37°C, 200r/min, and couple for 3h.

② Take 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, respectively. The above substances were all added after one substance was added and incubated at 37°C for 30 minutes before adding another substance. Finally, 5ul of proclin300 and 0.1g trehalose were added, and the reagent R2 was obtained after overnight incubation at 37°C.

After the preparation is completed, the concentrations of the components of reagent R2 are: anti-adiponectin monoclonal antibody-coated latex particles 80mg/L, HEPES buffer 25mM/L, BSA 10g/L, Tetronic 1307 1g/L, castor oil polyoxyethylene Ether 1g/L, proclin 300 1g/L, trehalose 20g/L.

Example 4

The present embodiment provides an adiponectin detection kit with strong anti-interference and good repeatability, including R1 reagent and R2 reagent, and the preparation method thereof includes the following steps:

(1) Preparation of reagent R1:

100 mL of citric acid buffer was weighed, placed on a stirrer, and 3.0 g of NaCl, 5 g of PEG6000, 0.5 g of Tween-20, and 0.1 g of NaN ₃ were added in sequence. After one substance is completely dissolved, another substance is added, and after complete dissolution, the pH is adjusted to 7.0, and the reagent R1 is obtained after incubating at 37°C in a shaker for one night.

After the configuration is completed, the concentrations of the components of the reagent R1 are: citric acid buffer 50mM/L; sodium chloride 30g/L; PEG6000 50g/L; Tween-20 5g/L; NaN ₃ 1g/L.

(2) Preparation of reagent R2:

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

Take 80 ul of latex microspheres with a particle size of 200 nm in 130 ul of 0.02M MES buffer at pH 6.0, place on a shaker at 37°C, 200 r/min, and shake for 2 min. Then, it was added to the prepared EDC and NHS solution, placed on a shaker at 37 °C, 200 r/min, and shaken for 15 min to activate the latex; the concentration of EDC and NHS was 0.10 M, and the addition ratio was 1:1. Then, under stirring, 3 ml of HEPES buffer (0.02M pH7.4) and antibody solution (mixing of monoclonal antibodies 1 and 2) were added, and the total amount of antibody added was 30 mg/L. Place on a shaker at 37°C, 200r/min, and couple for 3h.

② Take 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, respectively. The above substances were all added after one substance was added and incubated at 37°C for 30 minutes before adding another substance. Finally, 5ul of proclin300 and 0.1g trehalose were added, and the reagent R2 was obtained after overnight incubation at 37°C.

After the preparation is completed, the concentrations of the components of reagent R2 are: anti-adiponectin monoclonal antibody-coated latex particles 80mg/L, HEPES buffer 25mM/L, BSA 10g/L, Tetronic 1307 1g/L, castor oil polyoxyethylene Ether 1g/L, proclin 300 1g/L, trehalose 20g/L.

Example 5

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

(1) Preparation of reagent R1:

100 mL of citric acid buffer was weighed, placed on a stirrer, and 3.0 g of NaCl, 5 g of PEG6000, 0.2 g of Tetronic 1307, 0.1 g of castor oil polyoxyethylene ether, and 0.1 g of NaN ₃ were added in sequence. After one substance is completely dissolved, another substance is added, and after complete dissolution, the pH is adjusted to 7.0, and the reagent R1 is obtained after incubating at 37°C in a shaker for one night.

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; NaN _31g /L.

(2) Preparation of reagent R2:

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

Take 80 ul of latex microspheres with a particle size of 200 nm in 130 ul of 0.02M MES buffer at pH 6.0, place on a shaker at 37°C, 200 r/min, and shake for 2 min. Then, it was added to the prepared EDC and NHS solution, placed on a shaker at 37 °C, 200 r/min, and shaken for 15 min to activate the latex; the concentration of EDC and NHS was 0.10 M, and the addition ratio was 1:1. Then, under stirring, 3 ml of HEPES buffer (0.02M pH7.4) and antibody solution (mixing of monoclonal antibodies 1 and 2) were added, and the total amount of antibody added was 30 mg/L. Place on a shaker at 37°C, 200r/min, and couple for 3h.

② Take the mixture from the previous step out of the shaker, place it on a stirrer, and add 1 ml of HEPES buffer, 500 ul of 10% BSA, and 10 ul of Triton-100, respectively. The above substances were all added after one substance was added and incubated at 37°C for 30 minutes before adding another substance. Finally, 5ul of proclin300 and 0.1g trehalose were added, and the reagent R2 was obtained after overnight incubation at 37°C.

After the preparation is completed, the concentrations of the components of reagent R2 are: anti-adiponectin monoclonal antibody-coated latex particles 80mg/L, HEPES buffer 25mM/L, BSA 10g/L, Triton-100 2g/L, proclin3001g/L L, trehalose 20g/L.

Example 6 Application of adiponectin detection kit

A detection method of the above-mentioned adiponectin detection kit of the present embodiment:

1. Biochemical analyzer detection

Take the operation of Hitachi 7180 as an example: the measurement wavelength is 700nm, respectively take calibrator solutions (3uL) of different concentrations, add adiponectin R1 reagent (160uL), mix well, incubate at 37°C for 5 minutes, add adiponectin R2 reagent (40uL), mix well, incubate at 37°C for 30 seconds, read the absorbance value A1 of each tube, read the absorbance value A2 of each tube after 4.5 minutes of reaction, calculate the absorbance difference △A=A2-A1, repeat the measurement for each tube 2 times, draw the "concentration-absorbance difference" calibration curve with the average value of the absorbance difference ΔA measured twice in each calibration tube as the ordinate, and the corresponding calibrator concentration as the abscissa. Take the serum or plasma sample to be tested, measure the absorbance difference of the sample in the same way, and substitute it into the calibration curve to calculate the content of adiponectin ADPN in the sample to be tested. If the concentration of adiponectin in serum or plasma exceeds the range of the calibration curve, the sample should 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.

Example 7 Quality evaluation of the kit

(1) Establishment of standard curve

A. Experimental method

Dilute the high-value calibrator (42mg/L) with distilled water, the concentrations are: 0mg/L, 5.25mg/L, 10.5mg/L, 21mg/L, 42mg/L. The standard curve of the adiponectin calibrator of the present invention was determined according to the method in Example 6. The experimental object is the adiponectin detection kit prepared in Example 1.

B. Experimental results and analysis

Figure 1 shows the calibration curve of the kit of Example 1 of the present invention.

(2) Linear range

A. Experimental method

Mix at least 5 dilution concentrations (x) with a high concentration calibrator (42 mg/L) near the upper limit of the linear range and a low concentration calibrator near the lower limit of the linear range or distilled water. The kits were tested separately, and each dilution concentration was tested twice, and the mean value (y) of the test results was obtained respectively. Taking the dilution concentration (x) as the independent variable and the mean value of the detection results (y) as the dependent variable, a linear regression equation was obtained. The experimental object is the adiponectin detection kit prepared in Example 1.

B. Experimental results and analysis

As shown in FIG. 2 , the kit of the present invention has good linearity, and the regression equation is: y=0.9721x-0.0523, R ² =0.9997.

(3) Analytical Sensitivity

A. Experimental method

Use the kit prepared in Example 1 to test the calibrator with a known concentration of 10.5 mg/L, record the absolute value of the absorbance change value generated under the specified parameters of the kit, repeat the test 3 times, take the average value and convert it to 10 mg/L The absorbance change value of L.

B. Experimental results and analysis

The test results were 0.2504, 0.2481, and 0.2492, respectively, and the absorbance change value converted to 10 mg/L was 0.2373, which indicated that the kit had high sensitivity.

(4) Repeated detection

A. Experimental method

The adiponectin sample with the measured value of 5.91 mg/L from Epinocon Company was used as the low-value sample, and the measurement was carried out according to the detection method of the biochemical analyzer. The measurement of each concentration was repeated 10 times, and the measurement mean and standard deviation were calculated respectively ( S), the reproducibility of the kits of Examples 1 and 5 was investigated by calculating the coefficient of variation.

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

B. Experimental results and analysis

Table 1 Repeatability test results of adiponectin detection kit

As shown in the results in Table 1, after Triton-100 was used as the surfactant in Example 5, the reproducibility of the kit for low-value samples was not good, reaching 7.13%, while the new surfactant was added in Example 1. Group (Tetronic 1307 and castor oil ethoxylate), the reproducibility of the kit for low-value samples was significantly improved, with a coefficient of variation as low as 2.02%.

(5) Anti-interference experiment

A. Experimental method

Take the adiponectin sample with a concentration of 5.91mg/L, and add the same volume of different concentrations of interfering substances respectively, so that the concentrations of the added triglyceride interfering substances are 2.5g/L, 5.0g/L, 8.0g/L, respectively. The final concentration of L and bilirubin was 400 mg/L and the final concentration of hemoglobin was 10 g/L. The test kits prepared in Examples 1-5 were used to measure each prepared sample 3 times, and the average value was taken, which was the same as adding Comparing the samples of volume distilled water, observe the relative deviation of ADPN determination value after adding interfering substances and adding distilled water.

B. Experimental results and analysis

Table 2 Anti-interference experimental results

Note: The relative deviation does not exceed ±10% for the basic compliance.

It can be seen from the experimental results in Table 2 that the anti-interference ability is ranked from strong to weak as follows: Example 1> Example 2> Example 3> Example 5> Example 4; wherein, for triglycerides, Examples 1-3 The anti-interference ability basically reaches the standard; for the interference of bilirubin and hemoglobin, 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 of the measured value after adding different concentrations of interfering substances and adding the same volume of distilled water does not exceed 4%. Therefore, when a certain concentration of interfering substances such as triglyceride, bilirubin and hemoglobin exists in the detection sample, it has no effect on the measurement result of the kit of the present invention, and the present invention has strong anti-interference ability.

(5) Stability

A. Experimental method

The kit of the present invention was placed in a 37°C water bath, and the calibrators were calibrated before storage (ie, when not destroyed), after storage for 3 days, 5 days, and 7 days, and tested the subpackaged samples for analysis.

B. Experimental results and analysis

It can be seen from the results in the above table that after being stored at 37°C for 7 days, the kit of Example 1 still maintains a reactivity of more than 99%, which shows that adding Tetronic 1307 and castor oil polyoxyethylene ether to the reagent can significantly improve the reaction activity. The stability of the adiponectin detection kit under normal temperature conditions increases the practicability of the kit of the present invention for clinical diagnosis.

It can be understood that for those of ordinary skill in the art, equivalent replacements or changes can be made according to the technical solutions of the present invention and the inventive concept, and all these changes or replacements should belong to the protection scope of the appended claims of the present invention.

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.

Images