Preparation method of determination kit for determining adiponectin in human body in vitro

Abstract

The invention relates to the technical field of a preparation method of a determination kit for in-vitro determination of adiponectin in a human body, and particularly discloses a preparation method of a determination kit for in-vitro determination of adiponectin in a human body, which comprises the following steps: a coupling process; an S-shaped bent pipe is installed on the side connector, an upper connector is kept in an open state, a perfusion device adds 0.3-0.4 volume of a sealing buffer solution into the glass bottle, and 0.1-0.2 volume of the sealing buffer solution is injected into the glass bottle through an injection connector; and a storage process: closing the filling interface, opening the cover body, installing the up-down extending pipe, covering the cover body, opening the filling interface, carrying out resuspension ultrasonic treatment by using the storage buffer solution, and carrying out centrifugal washing for three times, thereby finally obtaining the adiponectin latex antibody. The device is easy and convenient to operate, simple in structure, independent of the manual control level and high in preparation efficiency, the situation that materials are left in a small-size glass bottle, consequently, the material ratio is difficult to control, and a small amount of confining liquid is needed is avoided, and the prepared adiponectin latex antibody is good in accuracy.

Description

Technical Field

[0001] The present invention relates to the field of a method for preparing a kit for measuring adiponectin in human body in vitro, and in particular to a method for preparing a kit for measuring adiponectin in human body in vitro. Background Art

[0002] Current research has found that the level of adiponectin in the blood can predict the risk of diabetes. The existing preparation method of the in vitro determination kit for adiponectin in the human body requires multiple transfers of 1 mL of material through a pipette in the coupling process and the blocking process. The operation is cumbersome and relies on manual control level. The preparation efficiency is low, and it is easy for materials to remain in the operation of a small volume glass bottle, which makes it difficult to control the material ratio, requires more blocking liquid, and the adiponectin latex antibody obtained has the defect of poor accuracy. Summary of the invention

[0003] In view of the above problems, the present invention proposes a method for preparing an in vitro assay kit for measuring adiponectin in human body, which solves the problem that the existing coupling process and the blocking process need to transfer 1mL of material through a pipette multiple times, the operation is cumbersome, and the level of manual control is relied on. The preparation efficiency is low, and the material is easily left in a small volume glass bottle, which makes it difficult to control the material ratio, requires more blocking liquid, and the adiponectin latex antibody obtained has the defect of poor accuracy.

[0004] The technical solution adopted by the present invention is as follows: A method for preparing a kit for in vitro determination of adiponectin in human body comprises the following steps: Coupling process: Open the cap of the coupling bottle, add 1.0-1.2 mL of microspheres into the coupling bottle, and then inject 1.0-1.2 mL of adiponectin antibody using the lower extension tube and hose, stir and mix, and couple at room temperature for 4-5 hours; Closing process: After the coupling is completed, an S-shaped elbow is installed on the side interface and the upper interface is kept open. The perfusion device adds 0.3-0.4 volume of blocking buffer to the glass bottle, closes the upper interface, and injects 0.1-0.2 volume of blocking buffer through the injection interface. At this time, the liquid in the straight tube is higher than the expected scale of the bottle body; the bottle is sealed at room temperature overnight; Save process: Close the injection interface, open the cover, install the upper and lower insertion tubes, cover the cover, open the injection interface, inject gas using a syringe, seal at room temperature overnight, transfer the mixed solution to a centrifuge tube, centrifuge, resuspend with storage buffer, and wash three times by centrifugation to obtain adiponectin latex antibody; The coupled closed bottle comprises a bottle body, a detachable cover body is mounted on the top of the bottle body, an upper interface is mounted on the cover body, the interface is externally connected to a hose, a lower interface is arranged at the bottom of the cover body, and a lower insertion tube is detachably mounted on the lower interface; a side interface is mounted on one side of the top of the bottle body, the side interface is detachably externally connected to an S-shaped bend, the S-shaped bend comprises at least two U-shaped tubes and a straight tube at the end, the top of the straight tube is higher than the height of the bottle body, the straight tube at the end connects two U-shaped tubes, one of the U-shaped tubes has its bottom facing upwards, and the other has its bottom facing downwards, an injection interface is arranged at the end of the straight tube, the inner diameter of the straight tube is configured to be a size that can produce a capillary effect, and a scale is mounted on the straight tube.

[0005] The improved process of the present invention comprises the following steps: adding 0.3-0.4 volume of blocking buffer solution into a glass bottle through a syringe, closing a filling interface, opening a cover body, installing upper and lower insertion tubes, closing the cover body, opening the filling interface, and filling gas through a syringe. After sealing at room temperature overnight, the mixed solution is transferred into a centrifuge tube. The process does not require a coupling process and a sealing process, which require multiple transfers of 1 mL of material through a pipette. The process is easy to operate, has a simple structure, does not rely on manual control level, has high preparation efficiency, is not prone to the operation of material residue in a small-volume glass bottle, which makes it difficult to control the material ratio, requires a smaller blocking solution, and obtains adiponectin latex antibody with good accuracy.

[0006] The present invention can reflect both the pancreatic islet secretion function and the insulin resistance by increasing the index of adiponectin during fasting blood sugar. In the atherosclerosis model, the serum adiponectin level is negatively correlated with triglycerides and low-density lipoprotein, and positively correlated with high-density lipoprotein. Adiponectin treatment can significantly reduce the levels of blood triglycerides and low-density lipoprotein, increase the level of high-density lipoprotein, and alleviate atherosclerotic lesions.

[0007] Optionally, a microsphere activation step is performed before the coupling step; the microsphere activation step is as follows: Transfer 1.0-1.2 mL of washed microspheres to a container, quickly add 48-50 μL of EDC and 480-500 μL of Sulfo-NHS, and stir for 30-40 minutes to complete activation; After the reaction is complete, the mixture is transferred to a centrifuge tube and centrifuged; Remove the supernatant, add 1.0-1.2 mL of EDC buffer, mix well, sonicate and centrifuge; 1-1.2 mL of EDC buffer was added and washed 2-3 times, and finally, the microspheres were resuspended with HEPES buffer to obtain microspheres of a predetermined concentration.

[0008] Optionally, a microsphere rinsing step is performed before the microsphere activating step; the microsphere rinsing step includes: Add 900-1000 μL of MES buffer to a centrifuge tube, then take out 100-110 μL of microspheres and add them thereto and mix well to obtain a mixture; The mixture is ultrasonically mixed, and then the ultrasonically mixed mixture is mixed and then centrifuged; The supernatant was removed, 1.0-1.2 mL of MES buffer was added, mixed, and sonicated; and the microspheres were checked under a microscope to see whether they were monodispersed.

[0009] Optionally, the centrifugation conditions in the microsphere activation step and the microsphere rinsing step are: a rotation speed of 13000-1400 rpm, a temperature of 4-4.5° C., and a centrifugation time of 20-22 min.

[0010] Optionally, in the microsphere activation step and the microsphere rinsing step, the ultrasonic operation is performed for 2-4 seconds each time, and then stopped for 4-6 seconds, and the above operation is repeated, and the total duration is 2-4 minutes.

[0011] Optionally, the blocking buffer contains 2-3% BSA.

[0012] Optionally, the blocking buffer contains 0.1-0.2% Tween-20.

[0013] Optionally, the blocking buffer further comprises a phosphate buffer, which is used to adjust the pH to 7.8-8.0.

[0014] Optionally, the amount of HEPES buffer added is 1-2 ml.

[0015] Optionally, the filling interface, the U-shaped tube and the straight tube are all connected by a bucket structure, and a sealing cover is installed on the filling interface. The present invention includes at least two U-shaped tubes and a straight tube at the end through an S-shaped elbow, and the straight tube at the end connects the two U-shaped tubes, one of which has a bottom facing upward and the other has a bottom facing downward. The inner diameter of the straight tube is configured to be a size that can produce a capillary effect, which can make the liquid height of the straight tube higher than the bottle body when closed through the capillary effect, and the sealing effect is good, and less liquid can be used to complete the sealing. The filling interface, the U-shaped tube and the straight tube of the present invention are all connected by a bucket structure, which can make the outer diameter of the straight tube itself much larger than the inner diameter of the straight tube, so that the overall structural strength of the straight tube is higher and it is not easy to be damaged.

[0016] Beneficial Effects The improved process of the present invention comprises the following steps: adding 0.3-0.4 volume of blocking buffer solution into a glass bottle through a syringe, closing the injection interface, opening the cover body, installing upper and lower insertion tubes, covering the cover body, opening the injection interface, injecting gas through the syringe, sealing the mixture overnight at room temperature and transferring the mixture into a centrifuge tube; the coupling process and the sealing process need not be repeated through a transfer tube to transfer 1 mL of material; the operation is simple, the structure is simple, the manual control level is not relied on, the preparation efficiency is high, the material is not likely to remain in a small volume glass bottle, which makes it difficult to control the material ratio, and a smaller blocking solution is required, so that the adiponectin latex antibody is prepared with good accuracy.

[0017] The present invention comprises at least two U-shaped tubes and a straight tube at the end through an S-shaped bent tube, wherein the straight tube at the end connects the two U-shaped tubes, wherein the bottom of one U-shaped tube faces upwards, and the bottom of the other U-shaped tube faces downwards, and the inner diameter of the straight tube is configured to have a size that can produce a capillary effect, and the capillary effect can be used to make the liquid height in the straight tube higher than the bottle body when closed, so that the sealing effect is good, and the sealing can be completed with less liquid.

[0018] The filling interface, U-shaped tube and straight tube of the present invention are all connected through a bucket structure, which can make the outer diameter of the straight tube itself much larger than the inner diameter of the straight tube, so that the overall structural strength of the straight tube is higher and not easy to be damaged.

[0019] The present invention can reflect both the pancreatic islet secretion function and the insulin resistance by increasing the index of adiponectin during fasting blood sugar. In the atherosclerosis model, the serum adiponectin level is negatively correlated with triglycerides and low-density lipoprotein, and positively correlated with high-density lipoprotein. Adiponectin treatment can significantly reduce the levels of blood triglycerides and low-density lipoprotein, increase the level of high-density lipoprotein, and alleviate atherosclerotic lesions.

[0020] The adiponectin latex antibody prepared by the invention has good overall linear relationship, good stability, high accuracy and relatively good dispersibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is an electron microscopic result diagram of the JSR microspheres coupled to the method for preparing the assay kit for in vitro assay of adiponectin in human body according to Example 1 of the present invention.

[0022] Figure 2 This is an electron microscopic result diagram of the JSR microspheres coupled to the method for preparing the assay kit for in vitro assay of adiponectin in human body according to Example 2 of the present invention.

[0023] Figure 3This is a functional result diagram of the JSR microspheres and antibodies coupled to the method for preparing the assay kit for in vitro assay of adiponectin in human body according to Example 1 of the present invention.

[0024] Figure 4 This is the functional result of the Bangs microspheres coupled with antibodies in the preparation method of the assay kit for in vitro assay of adiponectin in human body according to Example 2 of the present invention.

[0025] Figure 5 It is a stereoscopic view of a coupled sealed bottle in the method for preparing an in vitro assay kit for measuring adiponectin in human body according to Example 3 of the present invention.

[0026] The reference numerals in the figures are: 1. Bottle body, 2. Cover body, 3. Upper interface, 4. Hose, 5. Lower extension tube, 6. Side interface, 7. U-shaped tube, 8. Straight tube, 9. Filling interface, 10. Lower interface. DETAILED DESCRIPTION

[0027] The specific implementation of the present invention is further described in detail below in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0028] In the description of the present invention, unless otherwise specified, "plurality" means two or more than two; the orientations or positional relationships indicated by the terms "upper", "lower", "left", "right", "inner", "outer", "front end", "rear end", "head", "tail", etc. are based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present invention. In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "connected" and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection, a direct connection, or an indirect connection through an intermediate medium. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0029] Adiponectin of the present invention, also known as Acrp30, AdipoQ, GBP28, apM1, is an endogenous biologically active polypeptide or protein secreted by adipocytes. Adiponectin in serum mainly has three complex forms, namely, low molecular weight trimer (LMW), medium molecular weight hexamer (MMW), and high molecular weight 12-18 polymer (HMW), among which HMW is the main active form of adiponectin. As an insulin-sensitizing hormone, it can increase and promote fatty acid oxidation and sugar absorption in skeletal muscle cells, significantly enhance the inhibitory effect of insulin on gluconeogenesis, and inhibit the production of sugar in the liver. It is an important regulatory factor in the regulatory network of lipid metabolism and blood sugar homeostasis of the body. In the experimental atherosclerosis model, the serum adiponectin level is negatively correlated with triglycerides and low-density lipoprotein, and positively correlated with high-density lipoprotein. Adiponectin treatment significantly reduced blood triglyceride and low-density lipoprotein levels, increased high-density lipoprotein levels, and alleviated atherosclerotic lesions.

[0030] The current methods used for diabetes include fasting blood sugar (observation indicator), glucose tolerance test (diagnosis indicator), glycosylated hemoglobin (assessment indicator), skin glycosylation end products (unclear), diabetes risk assessment scale (auxiliary assessment indicator), adiponectin (early screening indicator, no fasting required, stable results, easy to operate). Combining the clinical characteristics of each indicator and the compliance of the subjects, adding adiponectin to the routine fasting blood sugar test can reflect both the body's pancreatic secretion function and insulin resistance.

[0031] In the present invention, BSA is bovine serum albumin.

[0032] Tween-20 in the present invention, also known as Tween 20 or polyoxyethylene sorbitan monolaurate, is a nonionic surfactant.

[0033] The MES buffer in the present invention is: 0.1 mol / L, pH=6.0.

[0034] HEPES buffer: 40 m mol / L HEPES, 50 mM NaCl, pH = 7.2.

[0035] PBS (pH=8.0) --- Phosphate buffered saline Blocking buffer (2% BSA, 0.1% Tween-20, PBS (pH = 8.0)) Storage buffer (1% BSA, 0.1% Tween-20, PBS (pH = 8.0)).

[0036] The adiponectin antibody in the present invention can be prepared using the commercially available 1901 antibody coupling reagent.

[0037] The microspheres used in the present invention are JSR microspheres and Bangs microspheres, wherein JSR microspheres are produced by Beijing Boerma Biotechnology Co., Ltd., and Bangs microspheres are produced by Bangs Laboratories.

[0038] Example 1 The technical solution adopted by the present invention is as follows: The present invention discloses a method for preparing a kit for in vitro determination of adiponectin in human body, comprising: 1) Rinsing microspheres process: 900 μL of MES buffer was added to a 1.5 ml centrifuge tube, and then 100 μL of microspheres (10% (solid content)) were taken out and added thereto and mixed to obtain a mixture; The mixture was sonicated, with the probe inserted 1 cm below the liquid surface, and the sonicated mixture was mixed and then centrifuged; The supernatant was removed, 1.0 mL of MES buffer was added and mixed, and ultrasonication was repeated 2-3 times; and the microspheres were examined under a microscope to see whether they were in a monodisperse state. In this example, JSR microspheres were used.

[0039] 2) Activation of microspheres: Prepare 200 mM EDC solution: Add 19.2 mg EDC and 500 μL purified water to a clean centrifuge tube at room temperature.

[0040] 1.0 mL of washed microspheres (10% solid content) was transferred to a container, 48 μL of 200 mM EDC and 480 μL of 200 mM Sulfo-NHS were quickly added thereto, and the mixture was stirred for 30 minutes to complete activation; To prepare 200 mM Sulfo-NHS, add 21.7 mg of Sulfo-NHS and 500 μL of activation / coupling buffer (made with purified water) to a clean microcentrifuge tube.

[0041] After the reaction is complete, the mixture is transferred to a centrifuge tube and centrifuged; After removing the supernatant, 1.0 mL of EDC buffer was added and mixed, followed by sonication and centrifugation; The microspheres were washed twice with 1 mL of EDC buffer and resuspended with HEPES buffer to obtain microspheres with a predetermined concentration (1%).

[0042] 3) Coupling process: Open the cap of the coupling bottle, add 1.0 mL of microspheres to the coupling bottle, then add 1.0-1.2 mL of adiponectin antibody, stir and mix, and couple at room temperature for 4 hours; the antibody coupling reagent of this embodiment uses 7B5 antibody coupling reagent. The total volume of microspheres and antibodies in this embodiment is 2 ml.

[0043] 4) Closing process: After the coupling is completed, an S-shaped elbow is installed on the side interface and the upper interface is kept open. The injection device adds 0.3 volume of blocking buffer to the glass bottle, closes the upper interface, and injects 0.1 volume of blocking buffer through the injection interface. At this time, the liquid in the straight tube is higher than the expected scale of the bottle body; the bottle is sealed overnight at room temperature. In this embodiment, the 0.3 volume here is 0.6ml, and the 0.1 volume is 0.2ml.

[0044] 5) Saving process: Close the injection interface, open the cover, install the upper and lower insertion tubes, cover the cover, open the injection interface, use the syringe to inject gas, seal the room temperature overnight, transfer the mixed solution to a centrifuge tube, centrifuge, resuspend with storage buffer, and wash three times by centrifugation to obtain adiponectin latex antibody.

[0045] The coupled closed bottle comprises a bottle body, a detachable cover body is mounted on the top of the bottle body, an upper interface is mounted on the cover body, the interface is externally connected to a hose, a lower interface is arranged at the bottom of the cover body, and a lower insertion tube is detachably mounted on the lower interface; a side interface is mounted on one side of the top of the bottle body, the side interface is detachably externally connected to an S-shaped bend, the S-shaped bend comprises at least two U-shaped tubes and a straight tube at the end, the top of the straight tube is higher than the height of the bottle body, the straight tube at the end connects two U-shaped tubes, one of the U-shaped tubes has its bottom facing upwards, and the other has its bottom facing downwards, an injection interface is arranged at the end of the straight tube, the inner diameter of the straight tube is configured to be a size that can produce a capillary effect, and a scale is mounted on the straight tube.

[0046] In this embodiment, the centrifugal conditions in the microsphere activation process and the microsphere rinsing process are: a rotation speed of 13000 rpm, a temperature of 4° C., and a centrifugal time of 20 min.

[0047] In this embodiment, the ultrasonic operation in the microsphere activation step and the microsphere rinsing step is performed for 2 seconds each time, and then stopped for 4 seconds. The above operation is repeated for a total time of 2 minutes.

[0048] In this embodiment, the blocking buffer contains 2% BSA, 0.1% Tween-20 and phosphate buffer. The phosphate buffer is used to adjust the pH of the blocking buffer to 7.8. The amount of HEPES buffer added is 1 ml.

[0049] Example 2 The technical solution adopted by the present invention is as follows: The present invention discloses a method for preparing a kit for in vitro determination of adiponectin in human body, comprising: 1) Rinsing microspheres process: 1000 μL of MES buffer was added to a centrifuge tube, and then 110 μL of microspheres were taken out and added thereto and mixed to obtain a mixture; The mixture is ultrasonically mixed, and then the ultrasonically mixed mixture is mixed and then centrifuged; The supernatant was removed, 1.2 mL of MES buffer was added, mixed, and sonicated; the microspheres were checked under a microscope to see whether they were monodispersed.

[0050] In this embodiment, Bangs microspheres are used.

[0051] 2) Activation of microspheres: 1.2 mL of washed microspheres were transferred to a container, 50 μL of 200 mM EDC and 500 μL of 200 mM Sulfo-NHS were quickly added thereto, and the mixture was stirred for 40 minutes to complete activation; After the reaction is complete, the mixture is transferred to a centrifuge tube and centrifuged; After removing the supernatant, 1.2 mL of EDC buffer was added and mixed, followed by sonication and centrifugation; The microspheres were washed three times with 1.2 mL of EDC buffer and resuspended in HEPES buffer to obtain microspheres with a predetermined concentration (1% w / v).

[0052] 3) Coupling process: Open the lid of the coupling bottle, add 1.2 mL of microspheres to the coupling bottle, then add 1.2 mL of adiponectin antibody, stir and mix, and couple at room temperature for 5 hours; the antibody coupling reagent of this example uses 7B5 antibody coupling reagent. The total volume of microspheres and antibodies in this example is 2.4 ml.

[0053] 4) Closing process: After the coupling is completed, an S-shaped elbow is installed on the side interface and the upper interface is kept open. The injection device adds 0.4 volume of blocking buffer to the glass bottle, closes the upper interface, and injects 0.2 volume of blocking buffer through the injection interface. At this time, the liquid in the straight tube is higher than the expected scale of the bottle body; it is sealed overnight at room temperature; in this embodiment, the 0.4 volume here is 0.96ml, and the 0.2 volume is 0.48ml. 5) Storage process: Close the injection interface, open the cover, install the upper and lower insertion tubes, cover the cover, open the injection interface, use the syringe to inject gas, seal the room temperature overnight, transfer the mixed solution to a centrifuge tube, centrifuge, resuspend with storage buffer, and wash three times by centrifugation to obtain adiponectin latex antibody.

[0054] The coupled closed bottle comprises a bottle body, a detachable cover body is mounted on the top of the bottle body, an upper interface is mounted on the cover body, the interface is externally connected to a hose, a lower interface is arranged at the bottom of the cover body, and a lower insertion tube is detachably mounted on the lower interface; a side interface is mounted on one side of the top of the bottle body, the side interface is detachably externally connected to an S-shaped bend, the S-shaped bend comprises at least two U-shaped tubes and a straight tube at the end, the top of the straight tube is higher than the height of the bottle body, the straight tube at the end connects two U-shaped tubes, one of the U-shaped tubes has its bottom facing upwards, and the other has its bottom facing downwards, an injection interface is arranged at the end of the straight tube, the inner diameter of the straight tube is configured to be a size that can produce a capillary effect, and a scale is mounted on the straight tube.

[0055] In this embodiment, the centrifugal conditions in the microsphere activation process and the microsphere rinsing process are: a rotation speed of 1400 rpm, a temperature of 4.5° C., and a centrifugal time of 22 min.

[0056] In this embodiment, the ultrasonic operation in the microsphere activation step and the microsphere rinsing step is performed for 4 seconds each time, and then stopped for 6 seconds. The above operation is repeated for a total time of 4 minutes.

[0057] In this embodiment, the blocking buffer contains 3% BSA, 0.2% Tween-20 and phosphate buffer. The phosphate buffer is used to adjust the pH of the blocking buffer to 8.0. The amount of HEPES buffer added is 2 ml.

[0058] Example 3 like Figure 5 As shown, the present invention also discloses a coupled closed bottle, which includes a bottle body 1, a detachable cover body 2 is installed on the top of the bottle body, an upper interface 3 is installed on the cover body, the interface is externally connected to a hose 4, a lower interface 10 is provided at the bottom of the cover body, and a lower insertion tube 5 is detachably installed on the lower interface; a side interface 6 is installed on one side of the top of the bottle body, and the side interface is detachably externally connected to an S-shaped elbow, and the S-shaped elbow includes at least two U-shaped tubes 7 and a straight tube 8 at the end, the top of the straight tube is higher than the height of the bottle body, and the straight tube at the end connects two U-shaped tubes, one of which has a bottom facing upward and the other has a bottom facing downward, and the inner diameter of the straight tube is configured to be a size that can produce a capillary effect, and a pouring interface 9 is provided at the end of the straight tube, and the pouring interface, the U-shaped tube and the straight tube are all connected through a bucket structure, and a sealing cover is installed on the pouring interface, and a scale is installed on the straight tube. The upper interface and the lower interface are mutually connected. The bottle body is made of glass.

[0059] When this embodiment is implemented, the S-shaped elbow includes at least two U-shaped tubes and a straight tube at the end, wherein the straight tube at the end connects the two U-shaped tubes, wherein the bottom of one U-shaped tube faces upwards, and the bottom of the other U-shaped tube faces downwards, and the inner diameter of the straight tube is configured to be a size that can produce a capillary effect, which can make the liquid height of the straight tube higher than the bottle body when closed through the capillary effect, and the sealing effect is good, and less liquid can be used to complete the sealing. The filling interface, the U-shaped tube and the straight tube are all connected through a bucket structure, which can make the outer diameter of the straight tube itself much larger than the inner diameter of the straight tube, so that the overall structural strength of the straight tube is higher and it is not easy to be damaged.

[0060] The adiponectin latex antibody detection method prepared in Example 1 and Example 2 is as follows: Configuration of adiponectin antigen: 0, 2.5, 5.0, 10.0, 20.0 (concentration mg / L or ng / mL): Reagent I: 15mL (specific preparation process is as follows) Reagent II: 10mL (containing 0.5% adiponectin latex antibody) (the specific preparation process is as follows) should meet the technical requirements.

[0061] The following is the preparation process and formula of Reagent I and Reagent II: Reagent I: calculated by liter: sodium dihydrogen phosphate dihydrate 0.83 g, disodium hydrogen phosphate dodecahydrate 33.92 g, polyethylene glycol-8000 10 g (for removing impurities and eliminating serum interference), Triton-100 1 g, PC300 1.8 mL (preservative); a) Add purified water (approximately 70% of the total preparation volume) into a clean and dry preparation container.

[0062] b) Add sodium dihydrogen phosphate dihydrate and stir to mix, stirring for 10 minutes until completely dissolved.

[0063] c) Add disodium hydrogen phosphate dodecahydrate and stir to mix, stirring for 10 minutes until completely dissolved.

[0064] d) Measure pH and adjust pH to 8.00±0.5 (use HCl or NaOH to adjust pH).

[0065] e) Add polyethylene glycol-8000 and stir to mix well for 15 minutes until completely dissolved.

[0066] f) Add Triton-100 and stir to mix well for 15 minutes until it is completely dissolved.

[0067] g) Add PC300, stir to mix, and stir for 15 minutes until it is completely dissolved.

[0068] h) Add purified water to make up to 1000 mL and mix thoroughly.

[0069] Reagent II: calculated by liter: sodium dihydrogen phosphate dihydrate 0.83 g, sodium dihydrogen phosphate dodecahydrate 33.92 g, Triton-100 1 g, Tween-20 0.5 g, complex enzyme stabilizer AES 1 g, bovine serum albumin 10 g, adiponectin latex antibody 250 mL, PC300 1.8 mL; a) Add purified water (approximately 30% of the total preparation volume) into a clean and dry preparation container.

[0070] b) Add sodium dihydrogen phosphate dihydrate and stir to mix, stirring for 10 minutes until completely dissolved.

[0071] c) Add disodium hydrogen phosphate dodecahydrate and stir to mix, stirring for 10 minutes until completely dissolved.

[0072] d) Measure pH and adjust the pH to 8.00±0.5.

[0073] e) Add Triton-100 and stir to mix well for 15 minutes until completely dissolved.

[0074] f) Add Tween-20, stir to mix, and stir for 15 minutes until completely dissolved.

[0075] g) Add the complex enzyme stabilizer AES, stir to mix, and stir for 15 minutes until it is completely dissolved.

[0076] h) Add bovine serum albumin and stir to mix, stirring for 15 minutes until completely dissolved.

[0077] i) Add adiponectin latex antibody and stir to mix thoroughly for 15 minutes until completely mixed.

[0078] j) Add PC300, stir to mix, and stir for 15 minutes until it is completely dissolved.

[0079] k) Add purified water to make up to 1000 mL and mix thoroughly.

[0080] After configuration and passing the test, it is packaged into designated packaging.

[0081] The electron microscopic results of the JSR microspheres after coupling in Example 1 are as follows Figure 1 The electron microscopic results of the JSR microspheres after coupling in Example 2 are shown in Figure 2 By comparing the above figures, it can be seen that the dispersion of the two groups of microspheres is good, and the particle size is about 200 nm.

[0082] Adiponectin antigens at concentrations of 0, 1.25, 2.5, 5, 10, and 20 μg / mL were selected to perform functional tests on the product of Example 1 coupled with the 7B5 antibody and the product of Example 2 coupled with the microspheres with the 7B5 antibody.

[0083] The absorbance changes of JSR microspheres and 7B5 antibody coupling reagents before and after binding to adiponectin antigen are shown in Table 1 below.

[0084] Table 1 Absorbance changes before and after JSR microspheres and 7B5 antibody coupling reagents bind to adiponectin antigen The absorbance changes of Bangs microspheres and 7B5 antibody coupling reagent before and after binding to adiponectin antigen are shown in Table 2 below.

[0085] Table 2 Absorbance changes before and after the Bangs microspheres and 7B5 antibody conjugate bind to adiponectin antigen The functional results of the JSR microspheres in Example 1 after coupling with antibodies are as follows: Figure 3 As shown, Figure 3 The ordinate corresponds to the absorbance in Table 1 above.

[0086] The functional results of the Bangs microspheres in Example 2 after coupling with antibodies are as follows Figure 4 As shown, Figure 4 The ordinate corresponds to the absorbance in Table 2 above.

[0087] From the above experimental results, it can be found that both JSR and Bangs microspheres were successfully coupled with antibodies, and the overall linear relationship was good.

[0088] The microsphere particle size, PDI and potential test results of Example 1 and Example 2 are as follows: The particle size and PDI data of the JSR microspheres before coupling in Example 1 are shown in Table 3 below.

[0089] The zeta potential of the JSR microspheres of Example 1 before coupling is shown in Table 4 below.

[0090] Table 3 Particle size and PDI data of JSR microspheres before coupling Table 4 Zeta potential of JSR microspheres before coupling The particle size and PDI data of the JSR microspheres after coupling in Example 1 are shown in Table 5 below.

[0091] The zeta potential of the JSR microspheres after coupling in Example 1 is shown in Table 6 below.

[0092] Table 5 Particle size and PDI data of JSR microspheres after coupling Table 6 Zeta potential of JSR microspheres before coupling In this Example 1, JSR microspheres were used, and the average particle size was measured to be 400.4 nm, which is more than twice as large as that of the uncoupled microspheres. The average PDI was 0.199, with no significant change.

[0093] The zeta potentials of JSR microspheres measured three times were -28.5, -29.5, and -28.7 mV, respectively. In general, when the absolute value of the polymer potential is greater than or equal to 30mV, it can be considered that the stability is good, so it is believed that JSR micro The stability of the ball is acceptable.

[0094] The particle size and PDI data of the Bangs microspheres before coupling in Example 2 are shown in Table 7 below.

[0095] The Zeta potential of the Bangs microspheres of Example 2 before coupling is shown in Table 8 below.

[0096] Table 7 Particle size and PDI data of Bangs microspheres before coupling Table 8 Zeta potential of Bangs microspheres before coupling The particle size and PDI data of the Bangs microspheres after coupling in Example 2 are shown in Table 9 below.

[0097] The Zeta potential of the Bangs microspheres after coupling in this Example 2 is shown in Table 10 below.

[0098] Table 9 Particle size and PDI data of Bangs microspheres after coupling Table 10 Zeta potential after Bangs microsphere coupling The average particle size of the Bangs microspheres in Example 2 was measured to be 254.7 nm, and the average PDI was 0.105, indicating that the aggregation phenomenon was not obvious and the microspheres had good dispersibility.

[0099] The zeta potentials of the Bangs microspheres in Example 2 obtained by three parallel measurements were -30.3, -30.7, and -32 mV, respectively, and their absolute values ​​were all higher than 30, indicating that the Bangs microspheres had good stability.

[0100] For the homemade reagent coupled with bangs microspheres in Example 2, the precision of the homemade reagent was tested by diluting the adiponectin antigen of 5 μg / mL and 20 μg / mL. The adiponectin antigen of 5 μg / mL was measured 8 times in parallel, and the adiponectin antigen of 20 μg / mL was measured 7 times in parallel. The average value was calculated according to the formula , standard deviation s, coefficient of variation CV.

[0101] When the adiponectin antigen concentration was 5 μg / mL, the precision test results of Bangs microspheres at the adiponectin antigen concentration of 5 μg / mL are shown in Table 11 below: Table 11 Results of precision testing of Bangs microspheres in Example 2 at an adiponectin antigen concentration of 5 μg / mL: Calculated average is 0.1739, the standard deviation s is 0.0059, and the coefficient of variation CV is 0.0345, that is, 3.45%, which is less than 8%, can be considered as good precision.

[0102] When the adiponectin antigen concentration was 20 μg / mL, the precision test results of Bangs microspheres at the adiponectin antigen concentration of 20 μg / mL are shown in Table 12 below.

[0103] Table 12 Precision test results of Bangs microspheres in Example 2 at an adiponectin antigen concentration of 20 μg / mL: Calculated average value is 0.2366, the standard deviation s is 0.0312, and the coefficient of variation CV is 0.1323, or 13.23%, has good precision.

[0104] Microsphere accuracy test: The accuracy of Bangs homemade reagent was still tested using adiponectin antigen diluted to 5 μg / mL and 20 μg / mL. According to the fitting curve, the absorbance difference should be 0.183 when the adiponectin concentration is 5 μg / mL, and the absorbance difference should be 0.253 when it is 20 μg / mL. The recovery rate was calculated by the formula, and the recovery rate was 95.1% when the adiponectin concentration was 5μg / mL, and the recovery rate was 93.7% when it was 20 μg / mL, both close to 100%, which can be considered to have good accuracy.

[0105] The above description is only a preferred embodiment of the present invention, and does not limit the patent protection scope of the present invention. Any equivalent structural transformation made by using the contents of the present invention and the drawings, directly or indirectly used in other related technical fields, is also included in the protection scope of the present invention.

Claims

1. A method for preparing a kit for in vitro determination of adiponectin in human body, characterized in that: The following steps are involved: Coupling process: Open the cap of the coupling bottle, add 1.0-1.2 mL of microspheres into the coupling bottle, and then inject 1.0-1.2 mL of adiponectin antibody using the lower extension tube and hose, stir and mix, and couple at room temperature for 4-5 hours; Closing process: After the coupling is completed, an S-shaped elbow is installed on the side interface and the upper interface is kept open. The perfusion device adds 0.3-0.4 volume of blocking buffer to the glass bottle, closes the upper interface, and injects 0.1-0.2 volume of blocking buffer through the injection interface. At this time, the liquid in the straight tube is higher than the expected scale of the bottle body; the bottle is sealed at room temperature overnight; Save process: Close the injection interface, open the cover, install the upper and lower insertion tubes, cover the cover, open the injection interface, inject gas using a syringe, seal at room temperature overnight, transfer the mixed solution to a centrifuge tube, centrifuge, resuspend with storage buffer, and wash three times by centrifugation to obtain adiponectin latex antibody; The coupled closed bottle comprises a bottle body, a detachable cover body is mounted on the top of the bottle body, an upper interface is mounted on the cover body, the interface is externally connected to a hose, a lower interface is arranged at the bottom of the cover body, and a lower insertion tube is detachably mounted on the lower interface; a side interface is mounted on one side of the top of the bottle body, the side interface is detachably externally connected to an S-shaped bend, the S-shaped bend comprises at least two U-shaped tubes and a straight tube at the end, the top of the straight tube is higher than the height of the bottle body, the straight tube at the end connects two U-shaped tubes, one of the U-shaped tubes has its bottom facing upwards, and the other has its bottom facing downwards, an injection interface is arranged at the end of the straight tube, the inner diameter of the straight tube is configured to be a size that can produce a capillary effect, and a scale is mounted on the straight tube.

2. The method for preparing a kit for determining adiponectin in human body in vitro according to claim 1, characterized in that: Before the coupling step, a microsphere activation step is also performed; the microsphere activation step is: Transfer 1.0-1.2 mL of washed microspheres to a container, quickly add 48-50 μL of EDC and 480-500 μL of Sulfo-NHS, and stir for 30-40 minutes to complete activation; After the reaction is complete, the mixture is transferred to a centrifuge tube and centrifuged; Remove the supernatant, add 1.0-1.2 mL of EDC buffer, mix well, sonicate and centrifuge; 1-1.2 mL of EDC buffer was added and washed 2-3 times, and finally, the microspheres were resuspended with HEPES buffer to obtain microspheres of a predetermined concentration.

3. The method for preparing a kit for determining adiponectin in human body in vitro according to claim 2, characterized in that: Before the microsphere activation process, a microsphere rinsing process is also performed; the microsphere rinsing process: Add 900-1000 μL of MES buffer to a centrifuge tube, then take out 100-110 μL of microspheres and add them thereto and mix well to obtain a mixture; The mixture is ultrasonically mixed, and then the ultrasonically mixed mixture is mixed and then centrifuged; The supernatant was removed, 1.0-1.2 mL of MES buffer was added, mixed, and sonicated; and the microspheres were checked under a microscope to see whether they were monodispersed.

4. The method for preparing a kit for in vitro determination of adiponectin in human body according to claim 3, characterized in that: The centrifugal conditions in the microsphere activation process and the microsphere rinsing process are: a rotation speed of 13000-1400 rpm, a temperature of 4-4.5° C., and a centrifugal time of 20-22 min.

5. The method for preparing a kit for measuring adiponectin in vitro in human body according to claim 3, characterized in that: In the microsphere activation step and the microsphere rinsing step, the ultrasonic operation is performed for 2-4 seconds each time, and then stopped for 4-6 seconds. The above operation is repeated for a total time of 2-4 minutes.

6. A method for preparing a kit for in vitro determination of adiponectin in human body according to claim 1, 2, 3, 4 or 5, characterized in that: The blocking buffer contained 2-3% BSA.

7. The method for preparing a kit for in vitro determination of adiponectin in human body according to claim 6, characterized in that: The blocking buffer contains 0.1-0.2% Tween-20.

8. The method for preparing a kit for in vitro determination of adiponectin in human body according to claim 7, characterized in that: The blocking buffer also includes a phosphate buffer, which is used to adjust the pH to 7.8-8.

0.

9. The method for preparing a kit for measuring adiponectin in vitro in human body according to claim 2, 3, 4 or 5, characterized in that: The amount of HEPES buffer added is 1-2 ml.

10. The method for preparing a kit for measuring adiponectin in human body in vitro according to claim 1, 2, 3, 4 or 5, characterized in that: The filling interface, the U-shaped tube and the straight tube are all connected through a bucket structure, and a sealing cover is installed on the filling interface.

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