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

Abstract

The present invention relates to the technical field of a preparation method of a kit for in vitro determination of adiponectin in the human body, and specifically discloses a preparation method of a kit for in vitro determination of adiponectin in the human body, comprising the following steps: a coupling process; a sealing process: an S-shaped bend is installed on the side interface and the upper interface is kept open, and an injector adds 0.3 - 0.4 volume of a sealing buffer solution into a glass bottle, and 0.1 - 0.2 volume of a sealing buffer solution is poured in through the pouring interface; a preservation process: the pouring interface is closed, the lid is opened, a lower extending tube is installed, the lid is covered, the pouring interface is opened, resuspended by ultrasonic waves with a preservation buffer solution, centrifuged and washed three times, and finally an adiponectin latex antibody is obtained. The present invention is simple in operation and structure, does not rely on the manual control level, has a high preparation efficiency, is not prone to the operation of materials remaining in a small-volume glass bottle, resulting in difficult control of the material ratio, requires a smaller amount of a sealing solution, and the obtained adiponectin latex antibody has good accuracy.

Description

Technical Field

[0001] The present invention relates to the field of preparation methods of assay kits for in vitro determination of adiponectin in human body, and particularly relates to a preparation method of an assay kit for in vitro determination of adiponectin in human body. Background Art

[0002] Current research has found that the level of adiponectin in blood can predict the risk of diabetes. In the existing preparation methods of assay kits for in vitro determination of adiponectin in human body, in the coupling process and the blocking process, 1 mL of material needs to be transferred multiple times through a pipette. The operation is cumbersome, dependent on the manual control level, the preparation efficiency is low, and it is easy to have the operation of the material remaining in a small-volume glass bottle, resulting in difficulty in controlling the material ratio, requiring more blocking solution, and the prepared adiponectin latex antibody has the defect of poor accuracy. Summary of the Invention

[0003] In view of the above problems, the present invention provides a preparation method of an assay kit for in vitro determination of adiponectin in human body, which solves the problems that in the existing coupling process and blocking process, 1 mL of material needs to be transferred multiple times through a pipette, the operation is cumbersome, dependent on the manual control level, the preparation efficiency is low, it is easy to have the operation of the material remaining in a small-volume glass bottle, resulting in difficulty in controlling the material ratio, requiring more blocking solution, and the prepared adiponectin latex antibody has the defect of poor accuracy.

[0004] The technical solution adopted by the present invention is as follows:

[0005] A preparation method of an assay kit for in vitro determination of adiponectin in human body, comprising the following steps:

[0006] Coupling process:

[0007] Open the lid of the coupling and blocking bottle, add 1.0 - 1.2 mL of microspheres into the coupling and blocking bottle, then inject 1.0 - 1.2 mL of adiponectin antibody through the lower insertion tube and the hose and stir to mix evenly, and couple at room temperature for 4 - 5 h;

[0008] Blocking process:

[0009] After the coupling is completed, install an S-shaped bend pipe on the side interface and keep the upper interface open. The syringe adds 0.3 - 0.4 volume of blocking buffer into the glass bottle, closes the upper interface, and fills 0.1 - 0.2 volume of blocking buffer through the filling interface. At this time, the liquid in the straight tube is higher than the expected scale of the bottle body; block overnight at room temperature;

[0010] Storage process:

[0011] Close the filling interface, open the lid, install the lower extension tube, cover the lid, open the filling interface, fill the gas using a syringe, seal at room temperature overnight, transfer the mixture to a centrifuge tube, centrifuge, resuspend with preservation buffer and sonicate, centrifuge and wash three times, and finally obtain adiponectin latex antibody;

[0012] Among them, the coupling and sealing bottle includes a bottle body, the top of the bottle body is equipped with a detachable lid, the lid is equipped with an upper interface, the interface is externally connected to a hose, the bottom of the lid is provided with a lower interface, and the lower interface is detachably equipped with a lower extension tube; one side of the top of the bottle body is equipped with a side interface, the side interface is detachably externally connected to an S-shaped bend pipe, the S-shaped bend pipe at least includes two U-shaped pipes and a straight pipe at the end, the top of the straight pipe is higher than the height of the bottle body, the straight pipe at the end connects the two U-shaped pipes, one of the U-shaped pipes has its bottom facing up and the other U-shaped pipe has its bottom facing down, the end of the straight pipe is provided with a filling interface, the inner diameter of the straight pipe is configured to have a size that can generate capillary action, and a scale is installed on the straight pipe.

[0013] In the improved process of the present invention, by adding 0.3 - 0.4 volume of sealing buffer to the glass bottle through a syringe, closing the filling interface, opening the lid, installing the lower extension tube, covering the lid, opening the filling interface, filling the gas using a syringe, sealing at room temperature overnight, and transferring the mixture to a centrifuge tube, it does not require the coupling process and the sealing process to transfer 1 mL of material multiple times through a pipette. Its operation is simple, the structure is simple, it does not depend on the manual control level, the preparation efficiency is relatively high, it is not easy to have the situation that the material remains in the small-volume glass bottle during the operation, which makes it difficult to control the material ratio, requires a smaller amount of sealing liquid, and the prepared adiponectin latex antibody has good accuracy.

[0014] In the present invention, by increasing the adiponectin index during fasting blood glucose, it can not only reflect the pancreatic islet secretion function of the body, but also reflect the insulin resistance situation. In the atherosclerosis model, the serum adiponectin level is negatively correlated with triglyceride and low-density lipoprotein, and positively correlated with high-density lipoprotein; administering adiponectin treatment can significantly reduce the content of blood triglyceride and low-density lipoprotein, increase the content of high-density lipoprotein, and alleviate the atherosclerotic lesion.

[0015] Optionally, an activated microsphere process is also carried out before the coupling process; the activated microsphere process:

[0016] 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 thereto, and stir for 30 - 40 minutes to complete activation;

[0017] After the activation is completed, transfer the mixture to a centrifuge tube and centrifuge;

[0018] Remove the supernatant, add 1.0 - 1.2 mL of EDC buffer, mix well, then sonicate and centrifuge;

[0019] Add 1 - 1.2 mL of EDC buffer and repeat the rinsing 2 - 3 times. Finally, resuspend with HEPES buffer to obtain microspheres with a predetermined concentration.

[0020] Optionally, a microsphere rinsing process is also carried out before the microsphere activation process; the microsphere rinsing process:

[0021] Add 900 - 1000 μL of MES buffer into a centrifuge tube, then take out 100 - 110 μL of microspheres and add them into it and mix well to obtain a mixture;

[0022] Sonicate the mixture, and then centrifuge the sonicated and mixed mixture;

[0023] Remove the supernatant, add 1.0 - 1.2 mL of MES buffer, mix well, and sonicate; Examine under a microscope whether the microspheres are in a monodisperse state.

[0024] Optionally, the centrifugation conditions in the microsphere activation process and the microsphere rinsing process are: the rotation speed is 13000 - 1400 rmp, the temperature is 4 - 4.5 °C, and the centrifugation time is 20 - 22 min.

[0025] Optionally, in the microsphere activation process and the microsphere rinsing process, each sonication operation is sonicated for 2 - 4 s, stopped for 4 - 6 s, and the above operations are repeated, with a total duration of 2 - 4 min.

[0026] Optionally, the blocking buffer contains 2 - 3% of BSA.

[0027] Optionally, the blocking buffer contains 0.1 - 0.2% of Tween - 20.

[0028] Optionally, the blocking buffer further includes phosphate buffer, which is used to adjust the pH to 7.8 - 8.0.

[0029] Optionally, the amount of HEPES buffer added is 1 - 2 ml.

[0030] Optionally, the filling interface, the U-shaped tube and the straight tube are all connected through a hopper-shaped structure, and a sealing cover is installed on the filling interface. In the present invention, the S-shaped bent tube at least includes two U-shaped tubes and a straight tube at the end. The straight tube at the end connects the two U-shaped tubes, with one U-shaped tube having its bottom facing up and the other U-shaped tube having its bottom facing down. The inner diameter of the straight tube is configured to be a size that can generate capillary action, which can make the liquid level in the straight tube higher than that of the bottle body during closing through capillary action, resulting in a good closing effect and enabling closing with less liquid. The filling interface, the U-shaped tube and the straight tube of the present invention are all connected through a hopper-shaped structure, which can make the outer diameter of the straight tube itself much larger than its inner diameter, thus making the overall structural strength of the straight tube higher and not easily damaged.

[0031] Beneficial effects

[0032] In the improved process of the present invention, 0.3 - 0.4 volume of a closing buffer solution is added to the glass bottle through a syringe. The filling interface is closed, the lid is opened, the lower insertion tube is installed, the lid is covered, the filling interface is opened, and gas is injected using the syringe. After closing at room temperature overnight, the mixture is transferred to a centrifuge tube. It does not require coupling and closing processes that need to transfer 1 mL of material multiple times through a pipette. Its operation is simple, the structure is simple, it does not depend on the manual control level, the preparation efficiency is relatively high, and it is not easy to have the situation where material remains in a small-volume glass bottle during the operation, which is difficult to control the material ratio. A smaller amount of closing solution is required, and the prepared adiponectin latex antibody has good accuracy.

[0033] In the present invention, the S-shaped bent tube at least includes two U-shaped tubes and a straight tube at the end. The straight tube at the end connects the two U-shaped tubes, with one U-shaped tube having its bottom facing up and the other U-shaped tube having its bottom facing down. The inner diameter of the straight tube is configured to be a size that can generate capillary action, which can make the liquid level in the straight tube higher than that of the bottle body during closing through capillary action, resulting in a good closing effect and enabling closing with less liquid.

[0034] The filling interface, the U-shaped tube and the straight tube of the present invention are all connected through a hopper-shaped structure, which can make the outer diameter of the straight tube itself much larger than its inner diameter, thus making the overall structural strength of the straight tube higher and not easily damaged.

[0035] By adding the adiponectin index during fasting blood glucose, the present invention can not only reflect the pancreatic islet secretion function of the body but also reflect the insulin resistance situation. In an atherosclerotic model, the serum adiponectin level is negatively correlated with triglyceride and low-density lipoprotein, and positively correlated with high-density lipoprotein; administering adiponectin treatment can significantly reduce the content of blood triglyceride and low-density lipoprotein, increase the content of high-density lipoprotein, and alleviate atherosclerotic lesions.

[0036] The adiponectin latex antibody prepared by the present invention has a good overall linear relationship, good stability, high accuracy, and good dispersibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 It is an electron microscope result diagram after JSR microsphere coupling in the preparation method of the assay kit for in vitro determination of adiponectin in human body in Example 1 of the present invention.

[0038] Figure 2 It is an electron microscope result diagram after JSR microsphere coupling in the preparation method of the assay kit for in vitro determination of adiponectin in human body in Example 2 of the present invention.

[0039] Figure 3 It is a functional result diagram after coupling of JSR microspheres and antibodies in the preparation method of the assay kit for in vitro determination of adiponectin in human body in Example 1 of the present invention.

[0040] Figure 4 It is a functional result after coupling of Bangs microspheres and antibodies in the preparation method of the assay kit for in vitro determination of adiponectin in human body in Example 2 of the present invention.

[0041] Figure 5 It is a three-dimensional view of the coupling closure bottle in the preparation method of the assay kit for in vitro determination of adiponectin in human body in Example 3 of the present invention.

[0042] Each reference numeral in the figure is as follows:

[0043] 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 OF THE INVENTION

[0044] The following combines the drawings and examples to further describe in detail the specific embodiments of the present invention. The following examples are used to illustrate the present invention, but are not used to limit the scope of the present invention.

[0045] In the description of the present invention, unless otherwise specified, "a plurality of" means two or more; the orientation or positional relationship indicated by terms such as "upper", "lower", "left", "right", "inner", "outer", "front end", "back end", "head", "tail", etc. is based on the orientation or positional relationship shown in the drawings, and is 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 construed as a limitation on the present invention. In the description of the present invention, it should be noted that, unless otherwise clearly specified and defined, the terms "connected" and "connected" should be understood in a broad sense. For example, it may be a fixed connection, a detachable connection, or an integral connection; it may 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.

[0046] Adiponectin of the present invention, also known as Acrp30, AdipoQ, GBP28, apM1, is an endogenous bioactive polypeptide or protein secreted by adipocytes. There are mainly three complex forms of adiponectin in serum, namely the trimer (LMW) in the low molecular form, the hexamer (MMW) in the medium molecular form, and the 12-18 mer (HMW) in the high molecular form. Among them, HMW is the main active form of adiponectin. As an insulin–sensitizing hormone, it can increase the promotion of fatty acid oxidation and glucose uptake in skeletal muscle cells, significantly enhance the inhibitory effect of insulin on gluconeogenesis, inhibit hepatic glucose production, and is an important regulatory factor in the regulatory network of lipid metabolism and blood glucose homeostasis in the body. In an experimental atherosclerosis model, the serum adiponectin level is negatively correlated with triglyceride and low-density lipoprotein, and positively correlated with high-density lipoprotein. Administering adiponectin treatment significantly reduces the content of blood triglyceride and low-density lipoprotein, increases the content of high-density lipoprotein, and alleviates atherosclerotic lesions.

[0047] Currently, the methods used for diabetes include fasting blood glucose (observation index), glucose tolerance test (diagnostic index), glycated hemoglobin (evaluation index), skin advanced glycation end products (not clear), diabetes risk assessment scale (auxiliary evaluation index), adiponectin (early screening index, no fasting required, stable results, convenient operation). Combining the characteristics of each index in clinical practice and the compliance of the subjects, adding the adiponectin index during the routine detection of fasting blood glucose can not only reflect the pancreatic islet secretion function of the body, but also reflect the insulin resistance situation.

[0048] In the present invention, BSA is bovine serum albumin.

[0049] In the present invention, Tween-20, also known as Tween 20 or polyoxyethylene sorbitan monolaurate, is a non-ionic surfactant.

[0050] In the present invention, the MES buffer: 0.1 mol / L, pH = 6.0.

[0051] HEPES buffer: 40 mmol / L HEPES, 50 mM NaCl, pH = 7.2.

[0052] PBS (pH = 8.0) --- Phosphate buffer

[0053] Blocking buffer (2% BSA, 0.1% Tween-20, PBS (pH = 8.0))

[0054] Storage buffer (1% BSA, 0.1% Tween-20, PBS (pH = 8.0)).

[0055] The adiponectin antibody in the present invention can adopt the antibody coupling reagent of 1901 on the market.

[0056] In the present invention, the microspheres adopt JSR microspheres and Bangs microspheres. Among them, JSR microspheres are from Beijing Bermo Biotechnology Co., Ltd., and Bangs microspheres are prepared by Bangs Laboratories.

[0057] Example 1

[0058] The technical solution adopted by the present invention is as follows:

[0059] The present invention discloses a preparation method of a determination kit for in vitro determination of adiponectin in human body, including:

[0060] 1) Rinsing microspheres process:

[0061] Add 900 μL of MES buffer into a 1.5 ml centrifuge tube, then take out 100 μL of microspheres (10% (solid content)) and add them into it and mix well to obtain a mixture;

[0062] Ultrasonic the mixture, insert the probe 1 cm below the liquid surface, and then mix and centrifuge the ultrasonically mixed mixture;

[0063] Remove the supernatant, add 1.0 mL of MES buffer and mix well, and ultrasonic, where repeat 2 - 3 times; Examine under a microscope whether the microspheres are in a monodisperse state. JSR microspheres are used in this example.

[0064] 2) Activating microspheres process:

[0065] Prepare 200 mM EDC solution: Add 19.2 mg of EDC and 500 μL of purified water to a clean centrifuge tube at room temperature.

[0066] Transfer 1.0 mL of washed microspheres (10% solid content) to a container, quickly add 48 μL of 200 mM EDC and 480 μL of 200 mM Sulfo-NHS thereto, and stir for 30 minutes to complete activation.

[0067] Prepare 200 mM Sulfo-NHS: Add 21.7 mg of Sulfo-NHS and 500 μL of activation / coupling buffer (made of purified water) to a clean centrifuge tube.

[0068] After the activation is completed, transfer the mixture to a centrifuge tube and centrifuge.

[0069] Discard the supernatant, add 1.0 mL of EDC buffer thereto, mix well, sonicate and centrifuge.

[0070] Add 1 mL of EDC buffer and repeat the rinsing twice, and finally resuspend with HEPES buffer to obtain microspheres with a predetermined concentration (1%).

[0071] 3) Coupling process:

[0072] Open the lid of the coupling vial, add 1.0 mL of microspheres to the coupling vial, then add 1.0 - 1.2 mL of adiponectin antibody and stir well. Couple at room temperature for 4 h; the antibody coupling reagent in this example is the 7B5 antibody coupling reagent. The total volume of microspheres and antibody in this example is 2 ml.

[0073] 4) Blocking process:

[0074] After the coupling is completed, install an S-shaped bend on the side interface and keep the upper interface open. The injector adds 0.3 volume of blocking buffer to the glass bottle, closes the upper interface, and fills 0.1 volume of blocking buffer through the filling interface. At this time, the liquid in the straight tube is at a scale higher than the bottle body; block overnight at room temperature. Here, 0.3 volume is 0.6 ml and 0.1 volume is 0.2 ml in this example.

[0075] 5) Preservation process:

[0076] Close the filling interface, open the lid, install the lower extension tube, cover the lid, open the filling interface, use the injector to fill with gas, transfer the mixture that has been blocked overnight at room temperature to a centrifuge tube, centrifuge, resuspend and sonicate with the preservation buffer, and centrifuge and wash three times to finally obtain adiponectin latex antibody.

[0077] Among them, the coupling closure bottle includes a bottle body, a detachable cover is installed on the top of the bottle body, an upper interface is installed on the cover, the interface is externally connected to a hose, a lower interface is provided at the bottom of the cover, and a lower extension tube is detachably installed on the lower interface; a side interface is installed on one side of the top of the bottle body, the side interface is detachably externally connected to an S-shaped bend pipe, the S-shaped bend pipe includes at least two U-shaped pipes and a straight pipe at the end, the top of the straight pipe is higher than the height of the bottle body, the straight pipe at the end connects the two U-shaped pipes, one of the U-shaped pipes has its bottom facing up and the other U-shaped pipe has its bottom facing down, an infusion interface is provided at the end of the straight pipe, the inner diameter of the straight pipe is configured to have a size that can produce a capillary effect, and a scale is installed on the straight pipe.

[0078] In this embodiment, the centrifugation conditions in the activated microsphere process and the rinsed microsphere process are as follows: the rotation speed is 13000 rmp, the temperature is 4 - °C, and the centrifugation time is 20 min.

[0079] In this embodiment, in the activated microsphere process and the rinsed microsphere process, each ultrasonic operation is for 2 s, stop for 4 s, repeat the above operation, and the total duration is 2 min.

[0080] In this embodiment, the blocking buffer contains 2% BSA, 0.1% Tween - 20 and phosphate buffer solution. The phosphate buffer solution is used to adjust the pH of the blocking buffer to 7.8. The addition amount of the HEPES buffer solution is 1 ml.

[0081] Example 2

[0082] The technical solution adopted by the present invention is as follows:

[0083] The present invention discloses a preparation method of a determination kit for in vitro determination of adiponectin in human body, including:

[0084] 1) Rinsed microsphere process:

[0085] Add 1000 μL of MES buffer solution into a centrifuge tube, then take out 110 μL of microspheres and add them into it and mix evenly to obtain a mixture;

[0086] Ultrasonic the mixture, and then centrifuge the mixture after ultrasonic mixing and homogenization;

[0087] Remove the supernatant, add 1.2 mL of MES buffer solution and mix evenly, and ultrasonic; check under a microscope whether the microspheres are in a monodisperse state.

[0088] Bangs microspheres are used in this embodiment.

[0089] 2) Activated microsphere process:

[0090] Transfer 1.2 mL of the washed microspheres into a container, quickly add 50 μL of 200 mM EDC and 500 μL of 200 mM Sulfo-NHS thereto, and stir for 40 minutes to complete activation;

[0091] After the activation is completed, transfer the mixture into a centrifuge tube and centrifuge;

[0092] Discard the supernatant, add 1.2 mL of EDC buffer thereto, mix well, then ultrasonicate and centrifuge;

[0093] Add 1.2 mL of EDC buffer and repeat the rinsing 3 times, and finally resuspend with HEPES buffer to obtain microspheres with a predetermined concentration (1% w / v).

[0094] 3) Coupling process:

[0095] Open the lid of the coupling closure bottle, add 1.2 mL of the microspheres into the coupling closure bottle, then add 1.2 mL of adiponectin antibody and stir to mix evenly, and couple at room temperature for 5 h; the antibody coupling reagent in this example uses a 7B5 antibody coupling reagent. In this example, the total volume of the microspheres and the antibody is 2.4 mL.

[0096] 4) Blocking process:

[0097] After the coupling is completed, install an S-shaped bend on the side interface and keep the upper interface open. The syringe adds 0.4 volume of blocking buffer into the glass bottle, closes the upper interface, and fills 0.2 volume of blocking buffer through the filling interface. At this time, the liquid in the straight tube is at a scale higher than the bottle body; block overnight at room temperature; here, 0.4 volume is 0.96 mL and 0.2 volume is 0.48 mL in this example. 5) Storage process:

[0098] Close the filling interface, open the lid, install the lower extension tube, cover the lid, open the filling interface, use the syringe to fill with gas, transfer the mixture after blocking overnight at room temperature into a centrifuge tube, centrifuge, resuspend and ultrasonicate with the storage buffer, centrifuge and wash three times, and finally obtain adiponectin latex antibody.

[0099] Wherein, the coupling closure bottle includes a bottle body, the top of the bottle body is equipped with a detachable lid, the lid is equipped with an upper interface, the interface is externally connected to a hose, the bottom of the lid is provided with a lower interface, and the lower interface is detachably equipped with a lower extension tube; one side of the top of the bottle body is equipped with a side interface, the side interface is detachably externally connected to an S-shaped bend, the S-shaped bend includes 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 end straight tube connects the two U-shaped tubes, one U-shaped tube has its bottom facing up and the other U-shaped tube has its bottom facing down, the end of the straight tube is provided with a filling interface, the inner diameter of the straight tube is configured to have a size that can produce a capillary effect, and a scale is installed on the straight tube.

[0100] In this embodiment, the centrifugation conditions in the activated microsphere process and the rinsed microsphere process are as follows: the rotation speed is 1400 rmp, the temperature is 4.5 °C, and the centrifugation time is 22 min.

[0101] In this embodiment, in the activated microsphere process and the rinsed microsphere process, each ultrasonic operation is for 4 s and then stops for 6 s. The above operations are repeated, and the total duration is 4 min.

[0102] In this embodiment, the blocking buffer contains 3% BSA, 0.2% Tween-20, and phosphate buffer solution. The phosphate buffer solution is used to adjust the pH of the blocking buffer to 8.0. The addition amount of the HEPES buffer solution is 2 ml.

[0103] Example 3

[0104] As Figure 5 shown, the present invention also discloses a coupling closure bottle, which includes a bottle body 1. A detachable cover body 2 is installed at 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. A lower extending 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. The side interface is detachably externally connected to an S-shaped bent tube. The S-shaped bent tube 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. The straight tube at the end connects the two U-shaped tubes. One U-shaped tube has its bottom facing upward, and the other U-shaped tube has its bottom facing downward. The inner diameter of the straight tube is configured to have a size that can generate a capillary effect. An infusion interface 9 is provided at the end of the straight tube. The infusion interface, the U-shaped tubes, and the straight tube are all connected through a hopper-shaped structure. A sealing cover is installed on the infusion interface, and a scale is installed on the straight tube. Its upper interface and lower interface communicate with each other. The bottle body is made of glass material.

[0105] When this embodiment is implemented, the S-shaped bent tube includes at least two U-shaped tubes and a straight tube at the end. The straight tube at the end connects the two U-shaped tubes. One U-shaped tube has its bottom facing upward, and the other U-shaped tube has its bottom facing downward. The inner diameter of the straight tube is configured to have a size that can generate a capillary effect. It can make the liquid level in the straight tube higher than that of the bottle body during sealing through the capillary effect, and the sealing effect is good. Less liquid can be used to complete the sealing. The infusion interface, the U-shaped tubes, and the straight tube are all connected through a hopper-shaped structure. It 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 easily damaged.

[0106] The adiponectin latex antibody detection methods prepared in Embodiment 1 and Embodiment 2 of this example are as follows:

[0107] Configure adiponectin antigen: 0, 2.5, 5.0, 10.0, 20.0 (concentration mg / L or ng / mL):

[0108] Reagent I: 15 mL (specific preparation process is as follows)

[0109] Reagent II: 10 mL (containing 0.5% adiponectin latex antibody) (specific preparation process is as follows) should meet the technical requirements.

[0110] The following are the preparation processes and formulas of Reagent I and Reagent II:

[0111] Reagent I: Calculated in liters: 0.83 g of sodium dihydrogen phosphate dihydrate, 33.92 g of disodium hydrogen phosphate dodecahydrate, 10 g of polyethylene glycol - 8000 (used for impurity removal to exclude serum interference), 1 g of Triton - 100, 1.8 mL of PC300 (preservative);

[0112] a) Add purified water (about 70% of the total preparation volume) to a clean and dry preparation container.

[0113] b) Add sodium dihydrogen phosphate dihydrate, stir and mix well, stir for 10 min until completely dissolved.

[0114] c) Add disodium hydrogen phosphate dodecahydrate, stir and mix well, stir for 10 min until completely dissolved.

[0115] d) Measure the pH and adjust the pH to 8.00 ± 0.5 (adjust the pH with HCl and NaOH).

[0116] e) Add polyethylene glycol - 8000, stir and mix well, stir for 15 min until completely dissolved.

[0117] f) Add Triton - 100, stir and mix well, stir for 15 min until completely dissolved.

[0118] g) Add PC300, stir and mix well, stir for 15 min until completely dissolved.

[0119] h) Make up the volume to 1000 mL with purified water and mix well.

[0120] Reagent II: Calculated in liters: 0.83 g of sodium dihydrogen phosphate dihydrate, 33.92 g of disodium hydrogen phosphate dodecahydrate, 1 g of Triton - 100, 0.5 g of Tween - 20, 1 g of composite enzyme stabilizer AES, 10 g of bovine serum albumin, 250 mL of adiponectin latex antibody, 1.8 mL of PC300;

[0121] a) Add purified water (about 30% of the total preparation volume) to a clean and dry preparation container.

[0122] b) Add sodium dihydrogen phosphate dihydrate, stir and mix evenly, stir for 10 min until completely dissolved.

[0123] c) Add disodium hydrogen phosphate dodecahydrate, stir and mix evenly, stir for 10 min until completely dissolved.

[0124] d) Measure the pH and adjust the pH to 8.00 ± 0.5.

[0125] e) Add Triton X-100, stir and mix evenly, stir for 15 min until completely dissolved.

[0126] f) Add Tween-20, stir and mix evenly, stir for 15 min until completely dissolved.

[0127] g) Add the composite enzyme stabilizer AES, stir and mix evenly, stir for 15 min until completely dissolved.

[0128] h) Add bovine serum albumin, stir and mix evenly, stir for 15 min until completely dissolved.

[0129] i) Add adiponectin latex antibody, stir and mix evenly, stir for 15 min until completely mixed.

[0130] j) Add PC300, stir and mix evenly, stir for 15 min until completely dissolved.

[0131] k) Make up the volume to 1000 mL with purified water and mix well.

[0132] After preparation and passing the inspection, aliquot into the specified packaging.

[0133] The electron microscopy results of the JSR microspheres after conjugation in Example 1 are as Figure 1 shown. The electron microscopy results of the JSR microspheres after conjugation in Example 2 are as Figure 2 shown. By comparing the above-mentioned figures, it can be seen that the dispersion of the two groups of microspheres is good and the particle size is about 200 nm.

[0134] Select adiponectin antigens with concentrations of 0, 1.25, 2.5, 5, 10, 20 μg / mL, and conduct functional tests on the product after conjugation of Example 1 with 7B5 antibody and the product after conjugation of the microspheres of Example 2 with 7B5 antibody.

[0135] The changes in absorbance before and after the binding of the JSR microsphere and 7B5 antibody conjugate reagent to the adiponectin antigen are shown in Table 1 below.

[0136] Table 1 Changes in absorbance before and after the binding of the JSR microsphere and 7B5 antibody conjugate reagent to the adiponectin antigen

[0137]

[0138] The absorbance changes of Bangs microspheres conjugated with 7B5 antibody reagent before and after binding to adiponectin antigen are shown in Table 2 below.

[0139] Table 2 Absorbance changes of Bangs microspheres conjugated with 7B5 antibody reagent before and after binding to adiponectin antigen

[0140]

[0141] The functional results of the JSR microspheres in Example 1 after antibody conjugation are as Figure 3 shown, Figure 3 The ordinate of which corresponds to the absorbance in Table 1 above.

[0142] The functional results of the Bangs microspheres in Example 2 after antibody conjugation are as Figure 4 shown, Figure 4 The ordinate of which corresponds to the absorbance in Table 2 above.

[0143] It can be found from the above experimental results that both JSR and Bangs microspheres have successfully conjugated with antibodies, and the overall linear relationship is good.

[0144] Microsphere particle size, PDI and potential detection results of Example 1 and Example 2:

[0145] The particle size and PDI data of the JSR microspheres in Example 1 before conjugation are shown in Table 3 below.

[0146] The Zeta-potential of the JSR microspheres in Example 1 before conjugation is shown in Table 4 below.

[0147] Table 3 Particle size and PDI data of JSR microspheres before conjugation

[0148]

[0149] Table 4 Zeta-potential of JSR microspheres before conjugation

[0150]

[0151] The particle size and PDI data of the JSR microspheres in Example 1 after conjugation are shown in Table 5 below.

[0152] The Zeta-potential of the JSR microspheres in Example 1 after conjugation is shown in Table 6 below.

[0153] Table 5 Particle size and PDI data of JSR microspheres after conjugation

[0154]

[0155] Table 6 Zeta-potential before conjugation of JSR microspheres

[0156]

[0157] In Example 1, JSR microspheres were used. The measured average particle size was 400.4 nm, which was more than twice larger than that before conjugation. The average PDI was 0.199, and the change was not obvious.

[0158] The zeta potentials of JSR microspheres measured in three parallel determinations were -28.5, -29.5, and -28.7 mV respectively. Generally

[0159] speaking, when the absolute value of the polymer potential is greater than or equal to 30 mV, it can be considered that the stability is good. Therefore, it is considered that the stability of JSR micro

[0160] spheres is acceptable.

[0161] The particle size and PDI data of Bangs microspheres before conjugation in Example 2 are shown in Table 7 below.

[0162] The Zeta-potential of Bangs microspheres before conjugation in Example 2 is shown in Table 8 below.

[0163] Table 7 Particle size and PDI data of Bangs microspheres before conjugation

[0164]

[0165] Table 8 Zeta-potential of Bangs microspheres before conjugation

[0166]

[0167] The particle size and PDI data of Bangs microspheres after conjugation in Example 2 are shown in Table 9 below.

[0168] The Zeta-potential of Bangs microspheres after conjugation in Example 2 is shown in Table 10 below.

[0169] Table 9 Particle size and PDI data of Bangs microspheres after conjugation

[0170]

[0171] Table 10 Zeta-potential of Bangs microspheres after conjugation

[0172]

[0173] 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 it had good dispersibility.

[0174] The zeta potentials of the Bangs microspheres in Example 2 obtained from 3 parallel measurements were -30.3, -30.7, and -32 mV respectively, and the absolute values were all higher than 30, indicating that the Bangs microspheres had good stability.

[0175] For the self-made reagent conjugated with the selected bangs microspheres in Example 2, the precision of the self-made reagent was detected by using adiponectin antigens diluted to 5 μg / mL and 20 μg / mL. The adiponectin antigen at 5 μg / mL was measured in parallel 8 times, and the adiponectin antigen at 20 μg / mL was measured in parallel 7 times. The average value was calculated according to the formula , standard deviation s, and coefficient of variation CV.

[0176] When the concentration of adiponectin antigen was 5 μg / mL, the results of the precision test of Bangs microspheres under the condition of adiponectin antigen concentration of 5 μg / mL are shown in Table 11 below:

[0177] Table 11 Results of the precision test of Bangs microspheres in Example 2 under the condition of adiponectin antigen concentration of 5 μg / mL:

[0178]

[0179] The calculated average value was 0.1739, the standard deviation s was 0.0059, and the coefficient of variation CV was 0.0345, that is

[0180] 3.45%, less than 8%, and the precision can be considered good.

[0181] When the concentration of adiponectin antigen was 20 μg / mL, the results of the precision test of Bangs microspheres under the condition of adiponectin antigen concentration of 20 μg / mL are shown in Table 12 below.

[0182] Table 12 Results of the precision test of Bangs microspheres in Example 2 under the condition of adiponectin antigen concentration of 20 μg / mL:

[0183]

[0184] The calculated average value was 0.2366, the standard deviation s was 0.0312, and the coefficient of variation CV was

[0185] 0.1323, that is, 13.23%, with good precision.

[0186] Accuracy detection of microspheres: Still use adiponectin antigens diluted to 5 μg / mL and 20 μg / mL to detect the accuracy of the self-made reagent of Bangs. Calculated from 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. Calculate the recovery rate by the formula. Among them, the recovery rate is 95.1% when the adiponectin concentration is 5 μg / mL, and the recovery rate is 93.7% when it is 20 μg / mL. Both are close to 100%, and it can be considered that the accuracy is good.

[0187] The above are only the preferred embodiments of the present invention, and do not limit the patent protection scope of the present invention accordingly. Any equivalent structural transformation made by using the content of the specification and drawings of the present invention, directly or indirectly applied in other related technical fields, shall be included in the protection scope of the present invention by the same token.

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, install an S-shaped elbow on the side interface and keep the upper interface open. Add 0.6-0.96 mL of blocking buffer into the glass bottle through the injection device. Close the upper interface and inject 0.2-0.48 mL of blocking buffer through the injection interface. At this time, the liquid in the linear tube is higher than the expected scale of the bottle body. Seal overnight at room temperature. 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 overnight at room temperature, 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 activation, the mixture was 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 repeatedly for 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 measuring adiponectin in vitro 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 pouring interface, the U-shaped tube and the straight tube are all connected through a bucket-shaped structure, and a sealing cover is arranged on the pouring interface.

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