Adiponectin latex immunoturbidimetric assay kit and preparation method thereof

Abstract

The present invention relates to an adiponectin latex immunoturbidimetric assay kit and a preparation method thereof, comprising reagent R1, reagent R2, a calibrator, and a quality control product. Reagent R1 contains an anti-interference agent comprising polyoxyethylene lauryl ether sulfate sodium salt, with a mass content of 0.1%-0.5%; an emulsifier, with a mass content of 0.2%-1%; and triaton, with a mass content of 0.2%-1%. Compared with the prior art, the kit of the present invention has strong anti-interference ability and is applicable to testing blood samples of patients with different physical conditions, particularly capable of accurately measuring special blood samples containing a large number of interfering factors.

Description

Technical Field

[0001] The invention relates to the field of biochemical technology, and in particular to a method for preparing a latex immunoturbidimetric adiponectin detection kit with high sensitivity and strong specificity. Background Art

[0002] Diabetes is a metabolic disease caused by insufficient insulin secretion or peripheral tissue insensitivity to insulin. It is characterized by a persistent state of hyperglycemia and may cause long-term damage, dysfunction or failure of various tissues and organs (such as the eyes, kidneys, heart, blood vessels, nerves, etc.). In layman's terms, diabetes is a common metabolic disorder, that is, blood sugar rises and then flows out in the urine, so there is sugar in the urine. Specifically, it is related to excessive intake of total energy, fat, carbohydrates, lack of exercise, and overnutrition, so it is jokingly called a "rich disease." If the disease is not controlled well, it will cause complications in the future, such as cardiovascular disease, cerebrovascular disease, retinal vascular disease, renal artery sclerosis, limb arteriosclerosis, etc. These complications are the main causes of diabetic blindness, kidney failure, heart attack, stroke and lower limb amputation.

[0003] Data from the World Health Organization indicates that diabetes ranks third among chronic non-communicable diseases in terms of prevalence, disability, mortality, and overall health risk. With socioeconomic development and changes in lifestyle, the incidence of type 2 diabetes is increasing annually worldwide. Diabetes can be predicted, foreseen, detected early, and prevented. Numerous studies have found that adiponectin is an independent risk factor for type 2 diabetes and can significantly enhance diabetes risk prediction.

[0004] Adiponectin is a protein hormone primarily secreted by fat cells. It circulates abundantly and plays a key role in regulating insulin sensitivity and glucose metabolism. Adiponectin levels in the blood are negatively correlated with body weight, abdominal obesity, and insulin resistance. It is an independent risk factor for type 2 diabetes and can significantly enhance the ability to predict diabetes risk.

[0005] Adiponectin testing doesn't require fasting; simply drawing 2 milliliters of venous blood can detect metabolic abnormalities and thus predict diabetes risk. Adiponectin screening offers significant advantages over standard blood sugar testing: unlike blood sugar and glycated hemoglobin, which are diagnostic indicators for diabetes, adiponectin is a predictor of diabetes risk. Adiponectin can identify individuals at high risk for diabetes who may have normal blood sugar levels but metabolic abnormalities. More importantly, compared to conventional risk factors such as age, gender, BMI, and waist circumference, which suffer from limitations such as crude estimation methods, limited prediction accuracy, and significant racial disparity, adiponectin dynamically reflects the body's insulin resistance level and is closely linked to individual metabolic health. Furthermore, adiponectin levels in the blood are stable, and testing is unaffected by factors such as food intake, mood swings, lack of sleep, and changes in dietary habits. Furthermore, adiponectin testing is highly automated, simple to operate, and highly resistant to interference, making it compatible with all types of biochemical analyzers.

[0006] Adiponectin testing can be used for early screening of diabetes, as well as for evaluating the effectiveness of drug treatment or lifestyle intervention. It can also be used for the classification and efficacy evaluation of type 2 diabetes, and is suitable for surveys and clinical diagnosis.

[0007] Currently, the adiponectin (ADPN) detection methods on the market include enzyme immunoassay (ELISA), radioimmunoassay (RIA), latex-enhanced turbidimetric immunoassay, etc. However, the ELISA and RIA detection methods are cumbersome to operate and take a long time to detect; the ordinary turbidimetric immunoassay is not sensitive enough; and the latex-enhanced turbidimetric immunoassay also has weak anti-interference ability. Some test kits can even measure negative values ​​for special blood samples such as chylous blood samples, which cannot well reflect the renal function of patients with special constitutions.

[0008] Patent application CN202110426188.6 discloses a latex-enhanced immunoturbidimetric kit for adiponectin and its preparation method. It includes R1 reagent, R2 reagent, adiponectin quality control product, and adiponectin calibrator, wherein: the R1 reagent includes a buffer, an electrolyte, a stabilizer, a surfactant, and a preservative; the R2 reagent includes latex microspheres coupled with adiponectin antibodies, a buffer, a stabilizer, an electrolyte, and a preservative, the latex microspheres being a polymer containing polystyrene, with carboxyl groups on the surface and a particle size between 70-200 nm; the adiponectin quality control product includes adiponectin and a quality control product diluent; the adiponectin calibrator includes adiponectin and a calibrator diluent. This technology improves detection sensitivity and a wide linear range to a certain extent, but has poor anti-interference ability, low sensitivity, and low discrimination of samples at low values ​​of 3-4 mg / L. The reference range of the modified reagent is greater than 5 mg / L. At the same time, the stability of the reagents using this system after opening the bottle cannot be sustained.

[0009] Therefore, it is necessary to improve the preparation of the previous ADPN latex immunoturbidimetric assay kit to enhance the anti-interference ability of the ADPN latex immunoturbidimetric assay reagent and expand the applicable population. Summary of the Invention

[0010] The present invention aims to overcome the above-mentioned shortcomings of the prior art by providing a kit for the immunoturbidimetric determination of adiponectin (Adiponectin) with a latex immunoturbidimetric assay and a method for its preparation. The kit has a strong anti-interference capability and is suitable for testing blood samples from patients with different physical conditions, particularly for accurately measuring blood samples containing a high number of interfering factors.

[0011] The purpose of the present invention can be achieved by the following technical scheme: an adiponectin latex immunoturbidimetric assay kit, comprising a reagent R1, a reagent R2, a calibrator and a quality control product, wherein the reagent R1 contains an anti-interference agent, which includes polyoxyethylene lauryl ether sulfate sodium salt with a mass content of 0.1%-0.5%; an emulsifier with a mass content of 0.2%-1%; and a triaton with a mass content of 0.2%-1%.

[0012] Furthermore, the polyoxyethylene lauryl ether sulfate sodium salt includes Kao EMAL 20C;

[0013] The emulsifier includes emulsifier OP-10;

[0014] The triton includes tritonx-405.

[0015] Furthermore, the reagent R1 also includes a buffer, a sensitizer, an electrolyte and a preservative;

[0016] The buffer is a common buffer with a concentration of 25-150 mM and a pH of 6.5-9.0;

[0017] The mass content of the sensitizer is 0.5%-10%;

[0018] The electrolyte mass content is 0.5%-5%;

[0019] The mass content of the preservative is 0.02%-2%.

[0020] Furthermore, the buffer described in reagent R1 includes phosphate buffer, HEPES buffer, TRIS buffer or MOPSO buffer, with a pH of 7.0-8.0;

[0021] The sensitizer includes one or more of polyethylene glycol 6000, polyethylene glycol 8000 or PAA;

[0022] The electrolyte includes common potassium chloride;

[0023] The preservative comprises one or both of sodium azide and Proclin 300.

[0024] Furthermore, the reagent R2 includes latex microspheres linked to ADPN antibodies, a buffer, a stabilizer, and a preservative;

[0025] The diameter of the latex microspheres connected with ADPN antibodies is 100-300 nm, and the mass content of the latex particles connected with ADPN antibodies is 2% to 10%;

[0026] The pH value of the buffer solution is 6.5-9.0;

[0027] The mass content of the stabilizer is 0.5%-5%.

[0028] The mass content of the preservative is 0.02%-2%.

[0029] Furthermore, in reagent R2,

[0030] The buffer is selected from one or more of phosphate buffered saline (PBS), tris (hydroxymethylaminomethane) buffer (Tris), 2-(N-morpholino)ethanesulfonic acid buffer (MES), borate buffer, glycine buffer or hydroxyethylpiperazineethanesulfonic acid buffer; the pH value of the buffer is 7.0-8.0;

[0031] The latex microspheres connected with ADPN antibodies are ADPN monoclonal antibodies or polyclonal antibodies connected to polystyrene carboxyl microspheres;

[0032] The stabilizer is one or more of sorbitol, bovine serum albumin, and trehalose.

[0033] The preservative is one or both of sodium azide and Proclin 300.

[0034] Furthermore, the calibrator and quality control product are recombinant or natural ADPN protein.

[0035] The preparation method of the adiponectin latex immunoturbidimetric assay kit comprises the following steps:

[0036] I. Preparation of reagent R1:

[0037] Take an appropriate amount of purified water, then add buffer, anti-interference agent, sensitizer, electrolyte, and preservative in sequence. Stir each time until the material is completely dissolved. Adjust the pH value to 6.5-9.0 and filter with a 0.22μm filter membrane.

[0038] II. Preparation of reagent R2:

[0039] (1) Activation of polystyrene latex microspheres: Take latex microspheres with a particle size of 120 to 250 nm, add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and activate them at 20°C-25°C for 30 minutes;

[0040] (2) Antibody coupling: Add adiponectin antibody to activated polystyrene latex microspheres and react at 20°C-25°C for 2 h for covalent coupling;

[0041] (3) Washing to remove uncoupled antibodies: The latex microsphere reaction solution obtained in step (2) is centrifuged to remove the supernatant to remove free antibodies and small molecule impurities;

[0042] (4) Blocking the carboxyl sites on the microspheres that are not coupled to the antibody: Add bovine serum albumin to the latex microspheres obtained in step (3) and block for 1 hour;

[0043] (5) Mixing and storage: Using R2 diluent containing a stabilizer, a preservative, and a buffer, dilute the solution obtained in step (4) by 3 to 15 times to obtain reagent R2, which is stored at 4°C;

[0044] III. Preparation of calibrators and quality control products: Dilute native or recombinant ADPN protein to the target concentration with lyophilization diluent, lyophilize, and store at 4°C;

[0045] VI. Composition of the kit:

[0046] The reagent R1 and reagent R2 prepared above were divided into bottles at a volume ratio of R1:R2=4:1, and formed a test kit with the calibrator and quality control product.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] The present invention comprises EMAL 20C, OP-10, tritonx-405 by adding anti-interference agent in reagent R1, adjusts its usage ratio, can significantly improve the anti-interference ability of reagent, keeps the correlation good with existing reagent while expanding applicable population.Anti-interference is also to be derived from the stability accuracy of reagent and substantially promotes, the anti-interference agent of these three kinds of material compositions selected by the present invention, and the unsealing stability of mixed reagent can be promoted to about 40 days from unsealing 20 days in the system of the present invention.

[0049] The kit of the present invention has high sensitivity, strong specificity and good stability, has good clinical application prospects, is simple to prepare, is suitable for industrial production, and has great application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is the linear test graph of the kit;

[0051] Figure 2 This is a test chart for correlation with imported reagents;

[0052] Figure 3 This is the anti-interference test diagram of serum samples;

[0053] Figure 4 This is a reagent stability test chart. DETAILED DESCRIPTION

[0054] The present invention is further described in detail below with reference to the examples, but is not limited to the following examples. The raw materials used in the examples are commercially available.

[0055] The ADPN latex immunoturbidimetric assay kit provided by the present invention comprises reagent R1, reagent R2, a calibrator, and a quality control product, wherein the volume ratio of reagent R1 to reagent R2 is 4:1.

[0056] The reagent R1 includes a conventional buffer, an anti-interference agent, a sensitizer, an electrolyte, and a preservative. The reagent R2 includes latex microspheres connected to ADPN antibodies, a buffer, a stabilizer, and a preservative. The buffer can be a common buffer in this research field, such as phosphate buffer, HEPES buffer, TRIS buffer, MOPSO buffer, etc., with a concentration of 25-150mM and a pH of 6.5-9.0. Preferably, the buffer has a pH of 7.0-8.0. The sensitizer is one or more of polyethylene glycol 6000, polyethylene glycol 8000, or PAA, with a content of 0.5%-10%. The electrolyte is common potassium chloride, with a content of 0.5%-5%. The preservative is one or both of sodium azide or Proclin300, with a content of 0.02%-2%.

[0057] The anti-interference agent includes Kao EMAL 20C, the content of which is 0.1%-0.5%, and emulsifier OP-10, the content of which is 0.2%-1%, and tritonx-405, the content of which is 0.2%-1%.

[0058] The latex microspheres linked to ADPN antibodies in reagent R2 are ADPN monoclonal or polyclonal antibodies linked to polystyrene carboxyl microspheres. Both the antibody and microspheres are commercially available. The diameter of the latex microspheres is 100-300 nm, and the content of the latex particles linked to the antibodies is 2% to 10%. The buffer is selected from one or more of phosphate buffered saline (PBS), tris (Tris) buffer, 2-(N-morpholino)ethanesulfonic acid buffer (MES), borate buffer, glycine buffer, or hydroxyethylpiperazineethanesulfonic acid buffer; the buffer has a pH of 6.5-9.0, preferably 7.0-8.0. The stabilizer is one or more of sorbitol, bovine serum albumin, and trehalose, with a content of 0.5%-5%. The preservative is one or both of sodium azide and Proclin 300, with a content of 0.02%-2%.

[0059] The calibrators and quality control materials are recombinant or natural ADPN proteins.

[0060] The following are more detailed implementation cases, which further illustrate the technical solutions of the present invention and the technical effects that can be obtained.

[0061] Example 1

[0062] Preparation of Reagent R1: To 995 g of water, add 4.8 g of sodium dihydrogen phosphate, 22.8 g of sodium dihydrogen phosphate, 10.0 g of PAA (polyacrylic acid), 30.0 g of NaCl, 2 g of EMAL 20c, 2 g of OP-10, 2 g of triton X-405, and 0.5 g of Proclin 300, stirring each time until completely dissolved. Adjust the pH to 7.4 and filter through a 0.22 μm filter to obtain 1 L of Reagent R1.

[0063] Prepare reagent R2 as follows:

[0064] (1) Activation of polystyrene latex microspheres: Take 10 ml of 200 nm latex microspheres (100 mg / mL), add 10 mL of 500 mM HEPES solution (pH 7.4), 1.5 mL of freshly prepared 20 mg / mL EDC solution, and add purified water to a final volume of 100 mL. Mix thoroughly and mix at room temperature (20-25°C) for 30 min.

[0065] (2) Coupling: Add ADPN antibody (antibody product from Medix, catalog number 100255) to the reaction mixture at a microsphere:antibody mass ratio of 18:1, and mix and couple at room temperature for 2 h.

[0066] (3) Washing: Select a suitable hollow fiber column (mPES / 500KD / 790cm2 , SPECTRUM Company), filter the solution in step (2) using a tangential flow filtration system (KrosFlo research IIi TFF system, SPECTRUM Company), replace the reaction buffer with a storage buffer 100mM Tris 8.0, control the shear force value to 3000-4500, filter out 5 times the volume of step (2) 100mM Tris buffer and wash;

[0067] (4) Blocking: Add 20 mL of 20% bovine serum albumin solution to the system in step (3) and mix at room temperature for 1 h;

[0068] (5) Mixing and storage: The initial reaction solution of latex microspheres obtained in step (4) was diluted to a final volume of 1000 mL with R2 diluent (containing 100 mM glycine buffer, 4% bovine serum albumin, 0.5% trehalose, 0.05% sodium azide, pH 8.0) to obtain reagent R2.

[0069] III. Preparation of ADPN Calibrator: To 995 mL of water, add 100 mM phosphate buffer, 1% sodium chloride, 0.2% disodium EDTA, 3.0% bovine serum albumin, and 0.1% sodium azide, stirring each time until completely dissolved. Adjust the pH to 8.0. After filtering the solution with a 0.22 μm filter, add 40 mg of commercially available recombinant ADPN antigen (Medix, Cat. No. 710013) to obtain a final concentration of 40 mg / L ADPN calibrator solution. Assign values ​​to the calibrator using a commercially available ADPN assay kit (latex immunoturbidimetry).

[0070] Preparation of ADPN quality control: Take 995 mL of water and add 100 mM phosphate buffer, 1% sodium chloride, 0.2% disodium ethylenediaminetetraacetic acid, 3.0% bovine serum albumin, and 0.1% sodium azide in sequence. Stir each addition until completely dissolved. Adjust the pH to 8.0. After the solution is filtered through a 0.22 μm membrane, take 500 mL and add 15 mg of commercially available ADPN recombinant antigen (product number: 710013 produced by Medix) to obtain an ADPN quality control solution with a final concentration of 30 mg / L. Take another 500 mL and add 2.5 mg of commercially available ADPN recombinant antigen to obtain an ADPN quality control solution with a final concentration of 5 mg / L. Use a commercially available ADPN assay kit (latex immunoturbidimetry (produced by Diazyme, USA) to assign values ​​to the calibrator.

[0071] Composition of the kit: The prepared R1 reagent and R2 reagent are matched according to the volume ratio of R1:R2 of 4:1 to form a kit. The packaging specifications of the kit are: R1 (4×45ml / per bottle), R2 (4×12ml / per bottle).

[0072] Example 2

[0073] Preparation of Reagent R1: To 995 g of water, add 4.8 g of sodium dihydrogen phosphate, 22.8 g of sodium dihydrogen phosphate, 10.0 g of polyethylene glycol 8000, 30.0 g of NaCl, 5 g of EMAL 20c, 2 g of OP-10, 2 g of triton X-405, and 0.5 g of Proclin 300, stirring each addition until completely dissolved. Adjust the pH to 7.4 and filter through a 0.22 μm filter to obtain 1 L of Reagent R1.

[0074] Prepare reagent R2 as follows:

[0075] (1) Activation of polystyrene latex microspheres: Take 10 ml of 200 nm latex microspheres (100 mg / mL), add 10 mL of 500 mM HEPES solution (pH 7.4), 2 mL of freshly prepared EDC solution (20 mg / mL), and make up to a final volume of 100 mL with purified water. Mix thoroughly and mix at room temperature (20-25°C) for 30 min.

[0076] (2) Coupling: Add ADPN antibody (antibody product from Medix, catalog number 100255) to the reaction solution at a microsphere:antibody mass ratio of 15:1, and mix and couple at room temperature for 2 h.

[0077] (3) Washing: Select a suitable hollow fiber column (mPES / 500KD / 790cm2, SPECTRUM), filter the solution in step (2) using a tangential flow filtration system (KrosFlo research IIi TFF system, SPECTRUM), replace the reaction buffer with a storage buffer of 100mM Tris 8.0, control the shear force value to 3000-4500, and filter out 5 times the volume of step (2) 100mM Tris buffer to complete the washing;

[0078] (4) Blocking: Add 20 mL of 20% bovine serum albumin solution to the system in step (3) and mix at room temperature for 1 h;

[0079] (5) Mixing and storage: The initial reaction solution of latex microspheres obtained in step (4) was diluted to a final volume of 1000 mL with R2 diluent (containing 100 mM glycine buffer, 4% bovine serum albumin, 0.5% trehalose, 0.05% sodium azide, pH 8.0) to obtain reagent R2.

[0080] III. Preparation of ADPN Calibrator: 100 mM Tris buffer, 1% sodium chloride, 0.2% disodium EDTA, 3.0% bovine serum albumin, and 0.1% sodium azide were added to 995 mL of water, stirring each time until completely dissolved. The pH was adjusted to 8.0. After filtering the solution with a 0.22 μm filter, 40 mg of commercially available recombinant ADPN antigen (Medix) was added to obtain a final concentration of 40 mg / mL ADPN calibrator solution. The calibrator was assigned a value using a commercially available ADPN assay kit (latex immunoturbidimetry).

[0081] Preparation of ADPN quality control: Take 995 mL of water and add 100 mM Tris buffer, 1% sodium chloride, 0.2% disodium ethylenediaminetetraacetic acid, 3.0% bovine serum albumin, and 0.1% sodium azide in sequence. Stir each time the material is added until it is completely dissolved. Adjust the pH to 8.0. After the solution is filtered with a 0.22 μm membrane, take 500 mL and add 15 mg of commercially available ADPN recombinant antigen to obtain an ADPN quality control solution with a final concentration of 30 mg / mL. Take another 500 mL and add 2.5 mg of commercially available ADPN recombinant antigen (Medix) to obtain an ADPN quality control solution with a final concentration of 5 mg / mL. Use a commercially available ADPN assay kit (latex immunoturbidimetry DIAZYME) to assign values ​​to the calibrator.

[0082] Composition of the kit: The prepared R1 reagent and R2 reagent are matched according to the volume ratio of R1:R2 of 4:1 to form a kit. The packaging specifications of the kit are: R1 (4×45ml / per bottle), R2 (4×12ml / per bottle).

[0083] Example 3

[0084] Preparation of Reagent R1: To 995 g of water, add 4.8 g of sodium dihydrogen phosphate, 22.8 g of sodium dihydrogen phosphate, 10.0 g of polyethylene glycol 8000, 30.0 g of NaCl, 5 g of EMAL 20c, 2 g of OP-10, 2 g of triton X-405, and 0.5 g of Proclin 300, stirring each addition until completely dissolved. Adjust the pH to 7.4 and filter through a 0.22 μm filter to obtain 1 L of Reagent R1.

[0085] Prepare reagent R2 as follows:

[0086] (1) Activation of polystyrene latex microspheres: Take 10 ml of 200 nm latex microspheres (100 mg / mL), add 10 mL of 500 mM HEPES solution (pH 7.4), 2 mL of freshly prepared EDC solution (20 mg / mL), and make up to a final volume of 100 mL with purified water. Mix thoroughly and mix at room temperature (20-25°C) for 30 min.

[0087] (2) Coupling: Add ADPN antibody (Medix) to the reaction solution in the previous step at a microsphere:antibody mass ratio of 15:1, mix and couple at room temperature for 2 h;

[0088] (3) Washing: Select a suitable hollow fiber column (mPES / 500KD / 790cm2, SPECTRUM), filter the solution in step (2) using a tangential flow filtration system (KrosFlo research IIi TFF system, SPECTRUM), replace the reaction buffer with a storage buffer of 100mM Tris 8.0, control the shear force value to 3000-4500, and filter out 5 times the volume of step (2) 100mM Tris buffer to complete the washing;

[0089] (4) Blocking: Add 20 mL of 20% bovine serum albumin solution to the system in step (3) and mix at room temperature for 1 h;

[0090] (5) Mixing and storage: The initial reaction solution of latex microspheres obtained in step (4) was diluted to a final volume of 1000 mL with R2 diluent (containing 100 mM glycine buffer, 4% bovine serum albumin, 0.5% trehalose, 0.05% sodium azide, pH 8.0) to obtain reagent R2.

[0091] III. Preparation of ADPN Calibrator: 100 mM Tris buffer, 1% sodium chloride, 0.2% disodium ethylenediaminetetraacetic acid, 3.0% bovine serum albumin, and 0.1% sodium azide were added to 995 mL of water, stirring each time until completely dissolved. The pH was adjusted to 8.0. After filtering the solution with a 0.22 μm filter, 20 mg of commercially available ADPN recombinant antigen (Medix) was added to obtain an ADPN calibrator solution with a final concentration of 40 mg / L. The calibrator was assigned a value using a commercially available ADPN assay kit (latex immunoturbidimetry (DIAZYME)).

[0092] Preparation of ADPN quality control: Take 995 mL of water and add 100 mM Tris buffer, 1% sodium chloride, 0.2% disodium ethylenediaminetetraacetic acid, 3.0% bovine serum albumin, and 0.1% sodium azide in sequence. Stir each addition until completely dissolved. Adjust the pH to 8.0. After the solution is filtered through a 0.22 μm membrane, take 500 mL and add 15 mg of commercially available ADPN recombinant antigen (Medix) to obtain an ADPN quality control solution with a final concentration of 30 mg / L. Take another 500 mL and add 2.5 mg of commercially available ADPN recombinant antigen (Medix) to obtain an ADPN quality control solution with a final concentration of 5 mg / L. Use a commercially available ADPN assay kit (latex immunoturbidimetry (DIAZYME)) to assign values ​​to the calibrator.

[0093] Composition of the kit: The prepared R1 reagent and R2 reagent are matched according to the volume ratio of R1:R2 of 4:1 to form a kit. The packaging specifications of the kit are: R1 (4×45ml / per bottle), R2 (4×12ml / per bottle).

[0094] Example 4

[0095] Preparation of Reagent R1: To 995 g of water, add 4.8 g of sodium dihydrogen phosphate, 22.8 g of sodium dihydrogen phosphate, 10.0 g of polyethylene glycol 8000, 30.0 g of NaCl, 2 g of EMAL 20c, 2 g of OP-10, 10 g of triton X-405, and 0.5 g of Proclin 300, stirring each addition until completely dissolved. Adjust the pH to 7.4 and filter through a 0.22 μm filter to obtain 1 L of Reagent R1.

[0096] Prepare reagent R2 as follows:

[0097] (1) Activation of polystyrene latex microspheres: Take 10 ml of 200 nm latex microspheres (100 mg / mL), add 10 mL of 500 mM HEPES solution (pH 7.4), 2 mL of freshly prepared EDC solution (20 mg / mL), and make up to a final volume of 100 mL with purified water. Mix thoroughly and mix at room temperature (20-25°C) for 30 min.

[0098] (2) Coupling: Add ADPN antibody (Medix) to the reaction solution in the previous step at a microsphere:antibody mass ratio of 12:1, mix and couple at room temperature for 2 h;

[0099] (3) Washing: Select a suitable hollow fiber column (mPES / 500KD / 790cm2, SPECTRUM), filter the solution in step (2) using a tangential flow filtration system (KrosFlo research IIi TFF system, SPECTRUM), replace the reaction buffer with a storage buffer of 100mM Tris 8.0, control the shear force value to 3000-4500, and filter out 5 times the volume of step (2) 100mM Tris buffer to complete the washing;

[0100] (4) Blocking: Add 20 mL of 20% bovine serum albumin solution to the system in step (3) and mix at room temperature for 1 h;

[0101] (5) Mixing and storage: The initial reaction solution of latex microspheres obtained in step (4) was diluted to a final volume of 1000 mL with R2 diluent (containing 100 mM glycine buffer, 4% bovine serum albumin, 0.5% trehalose, 0.05% sodium azide, pH 8.0) to obtain reagent R2.

[0102] III. Preparation of ADPN Calibrator: 100 mM Tris buffer, 1% sodium chloride, 0.2% disodium ethylenediaminetetraacetic acid, 3.0% bovine serum albumin, and 0.1% sodium azide were added to 995 mL of water, stirring each time until completely dissolved. The pH was adjusted to 8.0. After filtering the solution with a 0.22 μm filter, 40 mg of commercially available recombinant ADPN antigen (Medix) was added to obtain a final concentration of 40 mg / L ADPN calibrator solution. The calibrator was assigned a value using a commercially available ADPN assay kit (latex immunoturbidimetry (DIAZYME)).

[0103] Preparation of ADPN quality control: Take 995 mL of water and add 100 mM Tris buffer, 1% sodium chloride, 0.2% disodium ethylenediaminetetraacetic acid, 3.0% bovine serum albumin, and 0.1% sodium azide in sequence. Stir each addition until completely dissolved. Adjust the pH to 8.0. After the solution is filtered through a 0.22 μm membrane, take 500 mL and add 15 mg of commercially available ADPN recombinant antigen to obtain an ADPN quality control solution with a final concentration of 30 mg / L. Take another 500 mL and add 2.5 mg of commercially available ADPN recombinant antigen (Medix) to obtain an ADPN quality control solution with a final concentration of 5 mg / L. Use a commercially available ADPN assay kit (latex immunoturbidimetry (DIAZYME)) to assign values ​​to the calibrator.

[0104] Composition of the kit: The prepared R1 reagent and R2 reagent are matched according to the volume ratio of R1:R2 of 4:1 to form a kit. The packaging specifications of the kit are: R1 (4×45ml / per bottle), R2 (4×12ml / per bottle).

[0105] Performance test of the ADPN determination kit of the present invention

[0106] Detection tool: Dirui AU680 fully automatic biochemical analyzer.

[0107] Analytical method: Two-point endpoint method. Main wavelength: 700 nm; secondary wavelength: none. Add 2 μl of serum sample to 160 μl of each prepared reagent R1. Incubate at 37°C for 5 minutes, then add 40 μl of reagent R2. Read the absorbance A1 after a 30-second delay. Read the absorbance A2 after 270 seconds. The final absorbance is calculated as the difference between A2 and A1.

[0108] Calibration mode: Multi-point nonlinear. Response direction: Ascending.

[0109] Calculation method: Create a calibration curve based on the absorbance and different concentrations of the calibrator. Test the serum sample to obtain the absorbance, and calculate the sample content based on the calibration curve.

[0110] Reference range: ≥5mg / L.

[0111] I. Linearity test of the kit of the present invention

[0112] The ADPN calibrators prepared in each example were diluted with water to 0 mg / mL, 6.25 mg / L, 12.5 mg / L, 25 mg / L, and 40 mg / L, and the calibration curves were tested using the corresponding reagents R1 and R2. Figure 1 The figure shows the linear side view of the kit of Example 4, in which the X axis represents the theoretical value (mg / L), the Y axis represents the measured value (mg / L), and R 2 =0.999, Y=1.003X+0.056. This indicates that the kit of the present invention has a good linear relationship within the concentration range of 0-40 mg / L.

[0113] II. Sensitivity and correlation test of the kit of the present invention and the imported control kit

[0114] The kits of each embodiment and the imported control kit (latex immunoturbidimetry (DIAZYME) were tested for different concentrations of calibrants, and the absorbance comparison is shown in Table 1. This shows that the kit of the present invention has high sensitivity.

[0115] Table 1

[0116]

[0117] The kit of Example 4 and the imported control kit (latex immunoturbidimetry) were used to test the calibration curve. 40 clinical serum samples of different concentrations were tested, and the test results were as follows: Figure 2 As shown, the linear correlation coefficient R 2 =0.999, good correlation.

[0118] Reagent 1 uses phosphate buffer, and polyacrylic acid has the best sensitization effect, significantly improving sensitivity at both low and high values. However, sensitivity differentiation in the 20-40 range is weak, so comprehensive judgment is required. Combinations of different surfactant concentrations all significantly improved sensitivity compared to the control reagent.

[0119] III. Batch Difference Test of the Kit of the Present Invention

[0120] According to the experimental method described in Example 4, three batches of ADPN kits were prepared repeatedly, and different concentrations of calibrators were tested respectively. The absorbance comparison is shown in Table 2. The results show that the ADPN latex immunoturbidimetric kit used in the present invention has small inter-batch difference and good repeatability.

[0121] Table 2

[0122] Calibrator concentration (mg / L) First batch absorbance The second batch of absorbance The third batch of absorbance 2.5 0.0463 0.0442 0.0469 5 0.0925 0.0936 0.0929 10 0.1859 0.1846 0.1862 20 0.3362 0.3295 0.3378 40 0.5186 0.5123 0.5198

[0123] IV. Anti-interference test of the kit of the present invention

[0124] Blood samples of healthy individuals with normal physical examination results (samples were collected by our employees) were taken at 15 mg / L. High-concentration interfering substances were accurately measured and mixed with serum to prepare mixed samples with different interfering substance concentrations (as shown in Table 3). Each sample was measured three times and the average value was taken. The interference degree was calculated as follows:

[0125] Interference degree = mean value of samples with interference / mean value of samples without interference × 100%

[0126] The acceptable interference range is between 90% and 110%.

[0127] Table 3

[0128]

[0129] Result description: Figure 3 and Figure 4 As shown, the low value quality control is 5 mg / L, the high value quality control is 30 mg / L, and different concentrations of anti-interference substances are added as shown in the above figure. Compared with the samples without added interference substances, the measured values ​​after addition can be controlled within ±5%. At the same time, compared with the previous invention, this kit can accept more interference substances and has stronger anti-interference ability.

[0130] V. Stability test of the kit of the present invention during shelf life

[0131] Under storage conditions of 2-8°C, the kit prepared in Example 2 was tested on the same high-value quality control product (30 mg / L) and blood low-value quality control product (5 mg / L) at 0, 3, 6, 9, 12, and 14 months, and each sample was measured 5 times and the average value was taken (the results are shown in Figure 2). Figure 4 The results show that the kit of the present invention can be stably stored at 2-8°C for one year.

[0132] From the above examples, it can be seen that the ADPN determination kit prepared by the present invention has the advantages of strong anti-interference ability, good linearity, good stability, small batch difference, etc., can be produced in large quantities industrially, and has good clinical application prospects.

[0133] It should be noted that the above descriptions are merely embodiments of the present invention and do not limit the patent scope of the present invention. Any direct or indirect application of the present invention's description and drawings to other related technical fields shall be included in the patent protection scope of the present invention.

Claims

1. A latex immunoturbidimetric assay kit for adiponectin, comprising reagent R1, reagent R2, a calibrator, and a quality control product, characterized in that: The reagent R1 contains an anti-interference agent, which includes polyoxyethylene lauryl ether sulfate sodium salt, with a mass content of 0.1%-0.5%; an emulsifier, with a mass content of 0.2%-1%; and triaton, with a mass content of 0.2%-1%; The polyoxyethylene lauryl ether sulfate sodium salt includes Kao EMAL 20C; The emulsifier includes emulsifier OP-10; The Triton includes tritonx-405; Reagent R1 further comprises a buffer solution and a sensitizer. The pH of the buffer solution is 7.0-8.0, and the sensitizer is polyacrylic acid.

2. The adiponectin latex immunoturbidimetric assay kit according to claim 1, characterized in that: The reagent R1 also includes an electrolyte and a preservative; The buffer solution is used at a concentration of 25-150 mM; The mass content of the sensitizer is 0.5%-10%; The electrolyte mass content is 0.5%-5%; The mass content of the preservative is 0.02%-2%.

3. The adiponectin latex immunoturbidimetric assay kit according to claim 2, wherein: The buffer described in reagent R1 includes phosphate buffer, HEPES buffer, TRIS buffer or MOPSO buffer; The electrolyte includes potassium chloride; The preservative comprises one or both of sodium azide and Proclin 300.

4. The adiponectin latex immunoturbidimetric assay kit according to claim 1, wherein: The reagent R2 includes latex microspheres connected to ADPN antibodies, a buffer, a stabilizer and a preservative; The diameter of the latex microspheres connected with ADPN antibodies is 100-300 nm, and the mass content of the latex particles connected with ADPN antibodies is 2%-10%. The pH value of the buffer solution is 6.5-9.0; The mass content of the stabilizer is 0.5%-5%; The mass content of the preservative is 0.02%-2%.

5. The adiponectin latex immunoturbidimetric assay kit according to claim 4, characterized in that: In reagent R2, The buffer is selected from one or more of phosphate buffered saline (PBS), tris (Tris), 2-(N-morpholino)ethanesulfonic acid buffer (MES), borate buffer, glycine buffer or hydroxyethylpiperazineethanesulfonic acid buffer; the pH value of the buffer is 7.0-8.0; The latex microspheres connected with ADPN antibodies are ADPN monoclonal antibodies or polyclonal antibodies connected to polystyrene carboxyl microspheres; The stabilizer is one or more of sorbitol, bovine serum albumin, and trehalose. The preservative is one or both of sodium azide and Proclin 300.

6. The adiponectin latex immunoturbidimetric assay kit according to claim 1, characterized in that: The calibrators and quality control products are recombinant or natural ADPN proteins.

7. A method for preparing the adiponectin latex immunoturbidimetric assay kit according to any one of claims 1 to 6, characterized in that: The following steps are involved: I. Preparation of reagent R1: Take an appropriate amount of purified water, then add buffer, anti-interference agent, sensitizer, electrolyte, and preservative in sequence. Stir each time until the material is completely dissolved, adjust the pH value to 7.0-8.0, and filter; II. Preparation of reagent R2: Take latex microspheres with a particle size of 120-250 nm, add 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, mix and activate at 20°C-25°C for 30 minutes, then add adiponectin antibody and react at 20°C-25°C for 2 hours for covalent coupling. After washing the obtained latex microsphere reaction solution, add bovine serum albumin to block the carboxyl sites of the uncoupled antibody on the microspheres, and use R2 diluent containing stabilizers, preservatives, and buffer to dilute 3-15 times to finally obtain reagent R2, which is stored at 4°C; III. Preparation of calibrators and quality control products: Dilute native or recombinant ADPN protein to the target concentration with lyophilization diluent, lyophilize, and store at 4°C; VI. Composition of the kit: The reagent R1 and reagent R2 prepared above were divided into bottles at a volume ratio of R1:R2=4:1, and formed a test kit with the calibrator and quality control product.

8. The method for preparing the adiponectin latex immunoturbidimetric assay kit according to claim 7, wherein: In the preparation of reagent R1, 0.22 μm filter membrane was used for filtration.

9. The method for preparing the adiponectin latex immunoturbidimetric assay kit according to claim 7, wherein: The latex microsphere reaction solution is washed by centrifugation to remove the supernatant and remove free antibodies and small molecule impurities; The time for blocking the carboxyl sites on the microspheres that were not coupled to antibodies was 1 h.

Images