An immunoturbidimetric kit for detecting ADP

Abstract

The invention relates to the technical field of adiponectin detection kits, and discloses an immunoturbidimetric kit for detecting ADP, wherein the kit comprises an R1 reagent and an R2 reagent; the R1 reagent comprises: a buffer solution, 10-100 mmol / L; NaCl, 0.1-1 mol / L; an anticoagulant, 10-100 mmol / L; an aggregation promoter, 0.1-1%; a surfactant, 0.1-1 g / L; a preservative, 0.5-1 g / L; and a blocking agent, 0.05-0.2 mg / mL; wherein the anticoagulant in the R1 reagent is EDTA-Na 2 , sodium heparin, sodium citrate or a combination thereof; R2 reagent: buffer and antibody-coated latex microspheres, the preparation process of which requires cross-linking liquid, blocking liquid and preservation liquid; the kit prepared by the present invention achieves the beneficial technical effects of reducing the difference in serum and plasma measurement values, reducing batch differences and improving stability.

Description

Technical Field

[0001] The invention relates to the technical field of adiponectin (ADP) detection kits, in particular to an immunoturbidimetric kit for detecting ADP. Background Art

[0002] Adiponectin, also known as ADP, Acrp30, AdipoQ, apM1 or GBP28, is an important regulatory factor of lipid or carbohydrate metabolism secreted by adipocytes. ADP exists in the blood circulation in the form of low molecular weight trimers, hexamers and high molecular weight complexes, and the high molecular weight complex is the main active form of ADP metabolism. Clinical studies have found that normal human blood circulation is rich in adiponectin, and its total plasma adiponectin concentration is 3-30.0 mg / L, accounting for about 0.01% of the total plasma protein. The adiponectin level in patients with obesity, diabetes and coronary heart disease is significantly reduced, and it is an independent risk factor for the onset of coronary heart disease. Adiponectin is the latest special protein discovered that is specifically expressed by adipocytes and secreted into the serum. Because of its important characteristics such as anti-diabetes, anti-atherosclerosis, anti-inflammation, prevention of liver fibrosis, inhibition of tumor growth, and influence on bone metabolism, it has become a hot topic for domestic and foreign scholars.

[0003] Adiponectin is a recently discovered predictive indicator for diabetes. Low serum adiponectin levels indicate that an individual has a high risk of developing diabetes. Dynamic monitoring of serum adiponectin levels may help understand the extent of development of type 2 diabetes. Low adiponectin levels are an independent predictor of diabetes risk excluding factors such as age, blood pressure, blood lipids, and smoking. Testing adiponectin can identify people at high risk of diabetes and manage their lives, which can effectively reduce the risk of developing type 2 diabetes. Therefore, clinical testing of adiponectin has huge market potential.

[0004] The adiponectin detection kits currently available on the market include enzyme-linked immunosorbent assay, chemiluminescence assay, and latex immunoturbidimetry assay, etc. The enzyme-linked immunosorbent assay has complicated operation steps, long detection time, and certain limitations in sensitivity and linear range, and cannot be tested in batches; the chemiluminescence assay requires expensive instruments and takes a long time to detect; and as an early predictive indicator of type 2 diabetes, the latex immunoturbidimetry assay kit is equipped with a fully automatic biochemical analyzer or a specific protein analyzer, which can achieve high-throughput and rapid testing, while taking into account the advantages of high precision and high sensitivity, and has great market prospects.

[0005] Existing technical solution: The adiponectin latex immunoturbidimetric assay kit is based on the combination of adiponectin in the sample with specific anti-adiponectin antibodies coated on latex particles to induce an agglutination reaction. The turbidity of the agglutination reaction is proportional to the adiponectin concentration in the sample. This method is an analytical method that does not require sample pretreatment, does not require high technology and equipment, and has higher precision and specificity. Since this method does not require expensive equipment, can be automated, and can simultaneously measure a large number of specimens, it is widely promoted in clinical practice. However, the commercially available latex immunoturbidimetric adiponectin detection kit has large differences in the measured values ​​of serum and plasma samples, large batch differences, and poor stability, which limits its promotion and application in clinical practice.

[0006] The present invention cites the following references:

[0007] (1) Fu Hanyue's master's thesis "Synthesis and Application Research of Pentaerythritol Tetrakis (3-mercaptopropionic acid) Ester" published by Beijing University of Chemical Technology in 2021 disclosed the structure and synthesis method of pentaerythritol tetrakis (3-mercaptopropionic acid) ester;

[0008] (2) Song Yanyan's master's thesis "POSS Particle Compatibilization of TPU / SiR System and Preparation of Reactive Blended Materials" published by Beijing University of Chemical Technology in 2022 disclosed the structure and synthesis method of octamercapto cage-type polysilsesquioxane (POSS-SH). Summary of the invention

[0009] In order to solve the technical problems of large differences in serum and plasma values, large reagent batch differences, and poor stability in the prior art adiponectin latex immunoturbidimetric detection kit, the present invention provides an immunoturbidimetric kit for detecting ADP, the kit comprising:

[0010] R1 reagent, including: buffer, 10-100mmol / L; NaCl, 0.1-1mol / L; anticoagulant, 10-100mmol / L; aggregation promoter, 0.1-1%; surfactant, 0.1-1g / L; preservative, 0.5-1g / L; blocking agent, 0.05-0.2mg / mL;

[0011] Wherein, the anticoagulant in the R1 reagent is one or a combination of EDTA-Na2, sodium heparin, sodium citrate;

[0012] R2 reagent, including: buffer and antibody-coated latex microspheres; its preparation process requires cross-linking solution, blocking solution and preservation solution;

[0013] Calibrators, including: adiponectin antigen and diluent, with concentrations of 0, 5, 10, 20, and 40 μg / mL;

[0014] Preferably, the R1 reagent includes: morpholineethanesulfonic acid (MES) buffer, 50mmol / L, pH=6.0; NaCl, 0.65mol / L; EDTA-Na2, 20mmoL / L; heparin sodium, 20mmoL / L; sodium citrate, 20mmoL / L; coagulant PEG20000, 4.5g / L; surfactant BrijL23, 0.1g / L; preservative NaN3, 1.0g / L; blocker, 0.05mg / mL; the blocker can be an IgG blocker, a Roche blocker or a Scantibody blocker.

[0015] The high concentration of NaCl in the R1 reagent combined with the anticoagulant can effectively reduce the difference in the measured values ​​of serum and plasma samples;

[0016] The antibody-coated latex microspheres in the R2 reagent are polystyrene latex microspheres-adiponectin antibody complexes; the particle size of the polystyrene latex microspheres is 200-400 nm;

[0017] Preferably, the buffer in the R2 reagent includes a glycine (Gly) buffer with a pH of 7.0 to 8.0; the cross-linking solution includes a 50-100mmol / L Hepes buffer with a pH of 6.0 to 8.0; and the blocking solution is composed of a 50-100mmoL / LGly buffer with a pH of 7.0 to 10.0, 6-12% AEO7, and 1-5% AEO3.

[0018] In some specific embodiments of the present invention, a specific range of the cross-linking system (ratio of latex, cross-linking liquid and blocking liquid) during the preparation of the R2 reagent can effectively control the batch difference.

[0019] In some specific embodiments of the present invention, the blocking solution required for the preparation of the R2 reagent is used in combination with AEO7 and AEO3 under high pH conditions to improve the stability of the reagent.

[0020] In some specific embodiments of the present invention, the preservation solution required for the preparation of the R2 reagent is composed of 50-100 mmol / L Gly buffer, pH 7.0-8.0, 0.1-1% BSA, 6-12% sucrose, and 0.05-0.1% NaN3.

[0021] In some specific embodiments of the present invention, the antibody-coated latex microspheres are aged at 37° C. for 1 to 3 days.

[0022] The present invention also provides a method for preparing a latex immunoturbidimetry kit for detecting adiponectin, comprising the following steps:

[0023] 1) Preparation of reagent R1:

[0024] According to the formula, MES, polyethylene glycol 20000, sodium chloride, EDTA-Na2, sodium heparin, sodium citrate, BrijL35, NaN3, and blocking agent rabbit IgG were weighed into a clean container, deionized water was added and stirred for 30 minutes to fully dissolve them, and the pH was adjusted to 6.00; deionized water was added to make up the volume, and the reagent R1 was filtered.

[0025] 2) Preparation of reagent R2:

[0026] Step 1: Add carboxylated polystyrene latex microspheres with a particle size of 325 nm and a solid content of 10% into the crosslinking solution to make the concentration 0.77%;

[0027] Step 2: Add 2 times the activation amount of EDC to the above latex microspheres, and stir and react at 37°C for 0.5h;

[0028] Step 3: Add adiponectin antibodies 1 and 2 to the activated polystyrene latex microspheres, respectively, and react at 37°C with stirring for 2 hours;

[0029] Step 4: Add blocking solution to make the concentration of polystyrene latex microsphere solution 0.44%, and block at 37 degrees overnight;

[0030] Step 5: Wash by centrifugation for 3 times, add preservation solution and ultrasonically disperse the precipitate; mix the two groups of latex evenly to obtain R2;

[0031] Step 6: Place the above R2 at 37°C for aging treatment for 1 to 3 days to obtain the final R2 reagent.

[0032] The present invention synthesizes a novel anticoagulant that can replace heparin sodium, and the specific synthesis process is as follows:

[0033] The heparin sodium in the R1 reagent can be replaced by sulfonic acid-polyethylene glycol mPEG-SO3H;

[0034] Wherein, the preparation method of sulfonic acid-polyethylene glycol mPEG-SO3H is as follows:

[0035] Step S1, synthesizing allyl-polyethylene glycol mPEG-CH2-CH=CH2: the hydroxyl functional group at the end of polyethylene glycol mPEG-OH and the epoxy functional group of allyl glycidyl ether undergo a ring-opening reaction under the action of an alkaline catalyst to generate allyl-polyethylene glycol mPEG-CH2-CH=CH2;

[0036] Step S2, synthesizing thiol-polyethylene glycol mPEG-SH: utilizing the thiol-ene click reaction mechanism, the allyl functional group of allyl-polyethylene glycol mPEG-CH2-CH=CH2 and the thiol functional group of the thiol-reactive agent undergo a thiol-ene click chemical reaction to generate thiol-polyethylene glycol mPEG-SH;

[0037] Step S3, synthesizing sulfonic acid-polyethylene glycol mPEG-SO3H: using hydrogen peroxide as an oxidizing agent, oxidizing the sulfhydryl functional group of sulfhydryl-polyethylene glycol mPEG-SH into a sulfonic acid functional group, thereby preparing sulfonic acid-polyethylene glycol mPEG-SO3H.

[0038] Preferably, the thiol-forming agent is one or a combination of pentaerythritol tetrakis(3-mercaptopropionate) and octathiocarbyl cage-type polysilsesquioxane POSS-SH.

[0039] Preferably, the sulfonic acid group-polyethylene glycol mPEG-SO3H is one or a combination of mPEG-SO3H-Ⅰ and mPEG-SO3H-Ⅱ.

[0040] Compared with the prior art, the present invention has the following beneficial technical effects:

[0041] (1) The high concentration of NaCl in reagent R1 combined with the anticoagulant can reduce the difference between serum and plasma values;

[0042] (2) Controlling the size of the cross-linking system during the preparation of reagent R2 (the microsphere concentration after adding the cross-linking solution is 0.77%, and the microsphere concentration after adding the blocking solution is 0.44%) can reduce the batch-to-batch variation of the kit;

[0043] (3) Using a blocking solution with high pH and 9% AEO7 and 3% AEO3 can improve the stability of the kit;

[0044] The invention provides an ADP immunoturbidimetric kit for detecting the ADP, which can reduce the difference in serum and plasma measurement values, reduce the batch difference and improve the stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is a calibration curve diagram of the immunoturbidimetric kit for detecting ADP in Example 1;

[0046] Figure 2 It is a linear curve diagram of the immunoturbidimetric kit for detecting ADP in Example 1;

[0047] Figure 3 is the chemical structural formula of allyl-polyethylene glycol (mPEG-CH2-CH=CH2);

[0048] Figure 4 is the chemical structural formula of thiol-polyethylene glycol (mPEG-SH-Ⅰ);

[0049] Figure 5 is the chemical structural formula of thiol-polyethylene glycol (mPEG-SH-Ⅱ);

[0050] Figure 6is the chemical structural formula of sulfonic acid-polyethylene glycol (mPEG-SO3H-Ⅰ);

[0051] Figure 7 It is the chemical structural formula of sulfonic acid-polyethylene glycol (mPEG-SO3H-Ⅱ). DETAILED DESCRIPTION

[0052] Embodiment 1:

[0053] An immunoturbidimetric kit for detecting ADP, the kit comprising an R1 reagent and an R2 reagent;

[0054] R1 reagent specifically includes: MES buffer 50mmol / L, pH=6.0; NaCl, 0.65mol / L; EDTA-Na2, 20mmol / L; heparin sodium, 20mmol / L; sodium citrate, 20mmol / L; coagulant PEG20000, 4.5g / L; surfactant BrijL23, 0.1g / L; preservative NaN3, 1.0g / L; blocking agent rabbit IgG, 0.05mg / mL;

[0055] R2 reagent specifically includes: polystyrene latex microspheres-adiponectin antibody complex with a particle size of 325nm, crosslinking solution 50mmol / L Hepes, pH = 7.0; blocking solution 50mmol / L Gly, pH = 9.5, 9% AEO7, 3% AEO3; preservation solution 50mmol / L Gly, pH = 8.0, 0.5% BSA, 10% sucrose, 0.1% NaN3;

[0056] The specific preparation steps of R2 reagent are as follows:

[0057] Step S1: adding carboxylated polystyrene latex microspheres with a particle size of 325 nm and a solid content of 10% to a crosslinking solution to make the concentration 0.77%;

[0058] Step S2: adding 2 times the activation amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide EDC to the above latex microspheres, stirring and reacting at 37° C. for 0.5 h;

[0059] Step S3: adding adiponectin antibody 1 and adiponectin antibody 2 to the activated polystyrene latex microspheres respectively, and stirring at 37° C. for 2 h;

[0060] Step S4: adding blocking solution to make the concentration of the polystyrene latex microsphere solution 0.44%, and blocking at 37° C. overnight;

[0061] Step S5: washing by centrifugation for 3 times, adding a preservation solution to ultrasonically disperse the precipitate; mixing the two groups of latex uniformly to obtain R2;

[0062] Step S6: placing the above R2 at 37° C. for aging for 3 days to obtain the final R2 reagent;

[0063] The calibrators specifically include: adiponectin antigen and diluent.

[0064] The kit in this example: the calibration curve is as follows Figure 1 The repeatability test is shown in Table 1, the linearity test is shown in Table 2, and the linear curve and linear equation are shown in Figure 2 As shown;

[0065] Table 1 Kit repeatability test

[0066] Experiment number Quality control 1 (μg / mL) Quality control 2 (μg / mL) Serum (μg / mL) 1 4.92 10.26 7.68 2 5.16 10.18 7.59 3 5.28 10.09 7.56 4 4.87 9.89 7.48 5 5.12 9.95 7.42 6 5.09 10.00 7.35 7 4.92 9.86 7.51 8 5.33 9.89 7.69 9 4.97 10.24 7.75 10 5.02 10.16 7.53 Mean 5.07 10.05 7.56 SD 0.15 0.15 0.12 CV 2.93％ 1.44％ 1.58％

[0067] Table 2 Kit linearity test

[0068]

[0069]

[0070] Embodiment 2:

[0071] An immunoturbidimetric kit for detecting ADP, specifically comprising:

[0072] R1 reagent: NaCl, 0.1 mol / L, other aspects are the same as in Example 1;

[0073] R2 reagent: same as in Example 1;

[0074] Calibrator, same as in Example 1.

[0075] Embodiment 3:

[0076] An immunoturbidimetric kit for detecting ADP, specifically comprising:

[0077] R1 reagent: does not contain anticoagulant (EDTA-Na2, 20mmol / L; heparin sodium, 20mmol / L; sodium citrate, 20mmol / L), and the rest is the same as in Example 1;

[0078] R2 reagent: same as in Example 1;

[0079] Calibrator: Same as Example 1.

[0080] Embodiment 4:

[0081] An immunoturbidimetric kit for detecting ADP, specifically comprising:

[0082] R1 reagent: does not contain anticoagulant (EDTA-Na2, 20mmol / L; heparin sodium, 20mmol / L; sodium citrate, 20mmol / L), and the rest is the same as in Example 1;

[0083] R2 reagent: same as in Example 1;

[0084] Calibrator: anticoagulant (EDTA-Na2, 20 mmol / L; heparin sodium, 20 mmol / L; sodium citrate, 20 mmol / L) was added to the diluent, and the rest was the same as in Example 1.

[0085] Embodiment 5:

[0086] An immunoturbidimetric kit for detecting ADP, specifically comprising:

[0087] R1 reagent: same as in Example 1;

[0088] R2 reagent: after adding the cross-linking solution, the microsphere concentration is 0.1%, after adding the blocking solution, the microsphere concentration is 0.091%, and the rest is the same as in Example 1;

[0089] Calibrator: Same as Example 1.

[0090] Embodiment 6:

[0091] An immunoturbidimetric kit for detecting ADP, specifically comprising:

[0092] R1 reagent: same as in Example 1;

[0093] R2 reagent: after adding the cross-linking solution, the microsphere concentration is 1.6%, after adding the blocking solution, the microsphere concentration is 0.6%, and the rest is the same as in Example 1;

[0094] Calibrator: Same as Example 1.

[0095] Embodiment 7:

[0096] An immunoturbidimetric kit for detecting ADP, specifically comprising:

[0097] R1 reagent: same as in Example 1;

[0098] R2 reagent: blocking solution pH = 7.0, other aspects are the same as in Example 1;

[0099] Calibrator, same as in Example 1.

[0100] Embodiment 8:

[0101] An immunoturbidimetric kit for detecting ADP, specifically comprising:

[0102] R1 reagent: same as in Example 1;

[0103] R2 reagent: the blocking solution does not contain AEO3, and the rest is the same as in Example 1;

[0104] Calibrator: Same as Example 1.

[0105] Embodiment 9:

[0106] An immunoturbidimetric kit for detecting ADP, specifically comprising:

[0107] R1 reagent: same as in Example 1;

[0108] R2 reagent: the blocking solution does not contain AEO7, and the rest is the same as in Example 1;

[0109] Calibrator: Same as Example 1.

[0110] Embodiment 10:

[0111] An immunoturbidimetric kit for detecting ADP, specifically comprising:

[0112] R1 reagent: same as in Example 1;

[0113] R2 reagent: the blocking solution does not contain AEO7 and AEO3, and the rest is the same as in Example 1;

[0114] Calibrator, same as in Example 1.

[0115] Test Example 1:

[0116] In order to investigate whether the combination of high concentration NaCl and anticoagulants can effectively reduce the difference in measured values ​​between serum and plasma samples, four groups of experiments were set up, namely, Example 1, Example 2, Example 3 and Example 4. The specific experimental process is as follows:

[0117] The test kits in Example 1, Example 2, Example 3 and Example 4 were used to measure serum and plasma samples of 18 subjects, and the measured results are shown in Table 3 below;

[0118] Table 3 Results of serum and plasma values ​​of subjects

[0119]

[0120]

[0121] According to Table 3, the difference in the measured values ​​of 18 test subjects' serum and plasma samples was the smallest when the kit in Example 1 was used to measure the serum and plasma samples. That is, the addition of 0.65 mol / L NaCl, 20 mmol / L EDTA-Na2, 20 mmol / L heparin sodium, and 20 mmol / L sodium citrate to the R1 reagent had the most obvious effect on reducing the difference in serum and plasma measured values.

[0122] Based on this, it can be concluded that the combination of high-concentration NaCl and composite anticoagulants can effectively reduce the difference in measured values ​​between serum and plasma samples.

[0123] Test Example 2:

[0124] In order to investigate whether the cross-linking system within a specific range during the preparation of the R2 reagent can effectively control the batch difference, three groups of experiments were set up, namely, Example 1, Example 5 and Example 6. The specific experimental process is as follows:

[0125] The kits in Example 1, Example 5 and Example 6 were used to measure 5 identical samples, and each kit was used to measure each group of samples 3 times. The measured results are shown in Table 4 below.

[0126] Table 4 Results of batch difference detection of the kits in Example 1, Example 5 and Example 6

[0127]

[0128]

[0129] According to Table 4, it can be seen that: using the kit in Example 1, 5 identical samples were measured, and each group of samples was measured 3 times, and the batch difference was the smallest, that is, when the microsphere concentration was 0.77% after adding the cross-linking solution during the preparation of the R2 reagent, and the microsphere concentration was 0.44% after adding the blocking solution, the batch difference of the kit was the smallest;

[0130] Based on this, it can be concluded that a specific range of cross-linking system in the preparation process of R2 reagent can effectively control batch differences.

[0131] Test Example 3:

[0132] In order to investigate whether the combined use of AEO7 and AEO3 in the blocking solution required for the preparation of R2 reagent under high pH conditions can improve the stability of the reagent, 5 groups of experiments were set up, namely, Example 1, Example 7, Example 8, Example 9 and Example 10. The specific experimental process is as follows:

[0133] The kits in Example 1, Example 7, Example 8, Example 9 and Example 10 were used to measure 10 frozen serum samples of different freezing ages, and the measured results are shown in Table 5 below;

[0134] Table 5 Long-term stability test of the test kit (the test results are expressed as deviation indicators, the unit is %)

[0135]

[0136]

[0137] According to Table 5, when the kit in Example 1 was used to measure 10 frozen serum samples of different freezing ages, the deviation was minimal, that is, the long-term stability was optimal, that is, when the blocking solution composition was 50 mmol / LGly, pH=9.5, 9% AEO7, 3% AEO3, the long-term stability of the kit was optimal;

[0138] Based on this, it can be concluded that the combined use of AEO7 and AEO3 in the blocking solution required for the preparation of R2 reagent under high pH conditions can improve the stability of the reagent.

[0139] The high concentration or high pH in the present invention includes but is not limited to the concentration and pH used in Example 1. The concentrations of the anticoagulant in R1 and AEO7+AEO3 in the blocking solution can be adjusted as needed. Any solution that is fine-tuned on this basis to achieve similar purposes belongs to the protection scope of the present invention, as follows:

[0140] First, the high concentration of NaCl in reagent R1 includes but is not limited to 0.65 mol / L, and the concentration of each anticoagulant includes but is not limited to 20 mmoL / L. The combination of NaCl greater than 0.3 mol / L and three anticoagulants used in R1 is within the scope of the present invention;

[0141] Second, in the preparation process of reagent R2, the concentration of microspheres after adding the cross-linking solution is, but not limited to, 0.77%, and the concentration of microspheres after adding the blocking solution is, but not limited to, 0.44%; in order to reduce the batch difference of the kit, the cross-linking system in which the concentration of microspheres after adding the cross-linking solution is controlled to be 0.60%-0.95% and the concentration of microspheres after adding the blocking solution is controlled to be 0.35%-0.5% are both within the scope of protection of the present invention;

[0142] Third, the high pH of the blocking solution includes but is not limited to 9.5, and the concentration of AEO7+AEO3 is not limited to Example 1. The average value of the blocking solution with a pH higher than 8.5 and a combination of AEO7+AEO3 is within the protection scope of the present invention.

[0143] Embodiment 11:

[0144] Compared with heparin sodium, heparin-mimicking polymers have the advantages of lower cost and easier control of chemical structure. It has been confirmed that the unique sulfonic acid sequence found in heparin sodium is the main reason for its anticoagulant activity, and studies have found that the presence of sulfonate groups in polymers can induce anticoagulant properties. Based on this, the present invention synthesizes a new anticoagulant that can replace heparin sodium. The specific synthesis process is as follows:

[0145] Step 1: Synthesize allyl-polyethylene glycol (mPEG-CH2-CH=CH2). The synthesis mechanism is as follows: the hydroxyl functional group at the end of polyethylene glycol (mPEG-OH) and the epoxy functional group of allyl glycidyl ether undergo a ring-opening reaction under the action of an alkaline catalyst to generate allyl-polyethylene glycol (mPEG-CH2-CH=CH2). The specific synthesis steps are as follows: 5gmPEG 2000Dissolve in 50 mL of dichloromethane, stir to dissolve, add 1.43 g of allyl glycidyl ether and 1.26 g of triethylamine catalyst, stir and react at room temperature for 12 h, after the reaction is completed, use 0.01 mol / L hydrochloric acid to extract, use saturated sodium chloride and deionized water to wash, use cold anhydrous ether to precipitate and wash the product, the product is vacuum freeze-dried for 24 h to obtain allyl-polyethylene glycol (mPEG-CH2-CH=CH2), its chemical structure is as follows Figure 3 As shown;

[0146] Step 2: synthesizing thiol-polyethylene glycol (mPEG-SH), the synthesis mechanism of which is as follows: utilizing the thiol-ene click reaction mechanism, the allyl functional group of allyl-polyethylene glycol (mPEG-CH2-CH=CH2) undergoes a thiol-ene click chemical reaction with the thiol functional group of the thiol-reactive agent to generate thiol-polyethylene glycol (mPEG-SH);

[0147] The thiol-forming agent is selected from one of pentaerythritol tetrakis (3-mercaptopropionate) ester and octathiocarbyl cage-type polysilsesquioxane (POSS-SH);

[0148] Pentaerythritol tetrakis (3-mercaptopropionic acid) ester was used as a thiol reagent, and the synthesized product was recorded as thiol-polyethylene glycol (mPEG-SH-Ⅰ). The specific synthesis steps were as follows: 2.12 g of thiol-polyethylene glycol (mPEG-SH), 0.75 g of pentaerythritol tetrakis (3-mercaptopropionic acid) ester, 50 mg of 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone ultraviolet initiator, and 25 mg of tris (2-carboxyethyl) phosphine reducing agent were added to a 100 mL centrifuge tube. After all of them were vortexed to dissolve, they were placed under a wavelength of 365 nm ultraviolet light and stirred for 48 hours. After the reaction was completed, the thiol-polyethylene glycol (mPEG-SH-Ⅰ) was dialyzed for 24 hours using a dialysis bag (MWCO: 5000) and then freeze-dried to obtain a thiol-polyethylene glycol (mPEG-SH-Ⅰ). Its chemical structure is as follows: Figure 4 As shown;

[0149] According to the synthesis steps of mercapto-polyethylene glycol (mPEG-SH-Ⅰ), octamercapto cage-type polysilsesquioxane (POSS-SH) is used to replace pentaerythritol tetrakis (3-mercaptopropionic acid) as a thiol-reactive agent, and the amount of the thiol-reactive agent is 1.5 g. The synthesized product is recorded as mercapto-polyethylene glycol (mPEG-SH-Ⅱ), and its chemical structure is as follows: Figure 5 As shown;

[0150] Step 3: synthesizing sulfonic acid-polyethylene glycol (mPEG-SO3H), the synthesis mechanism of which is as follows: using hydrogen peroxide as an oxidizing agent, the sulfhydryl functional group of sulfhydryl-polyethylene glycol (mPEG-SH) is oxidized to a sulfonic acid functional group to obtain sulfonic acid-polyethylene glycol (mPEG-SO3H);

[0151] The product obtained by oxidizing thiol-polyethylene glycol (mPEG-SH-Ⅰ) with hydrogen peroxide (H2O2) is recorded as sulfonic acid-polyethylene glycol (mPEG-SO3H-Ⅰ). The specific preparation steps are as follows: In a 100mL flask, 1.8g of thiol-polyethylene glycol (mPEG-SH-Ⅰ), 2mL of hydrogen peroxide (H2O2), and 20mL of ultrapure water are added. After all are dissolved, the mixture is placed at 0°C for reaction for 12h. After the reaction is completed, anhydrous ethanol is used to precipitate the precipitate, centrifugally filtered, and vacuum dried at 50°C for 24h to obtain sulfonic acid-polyethylene glycol (mPEG-SO3H-Ⅰ), whose chemical structure is as follows: Figure 6 As shown;

[0152] The product obtained by oxidizing thiol-polyethylene glycol (mPEG-SH-Ⅱ) with hydrogen peroxide (H2O2) is recorded as sulfonic acid-polyethylene glycol (mPEG-SO3H-Ⅱ). 2.35g of thiol-polyethylene glycol (mPEG-SH-Ⅱ) is used to replace thiol-polyethylene glycol (mPEG-SH-Ⅰ). According to the preparation steps of sulfonic acid-polyethylene glycol (mPEG-SO3H-Ⅰ), sulfonic acid-polyethylene glycol (mPEG-SO3H-Ⅱ) is prepared. Its chemical structure is as follows Figure 7 shown.

[0153] Embodiment 12:

[0154] In order to investigate whether sulfonic acid-polyethylene glycol (mPEG-SO3H) can replace heparin sodium, the following experiment was performed, which is as follows:

[0155] Experimental plan 1:

[0156] An immunoturbidimetric kit for detecting ADP, specifically comprising:

[0157] Reagent R1: 20 mmol / L sulfonic acid-polyethylene glycol (mPEG-SO3H-Ⅰ) was used to replace 20 mmol / L heparin sodium, and the rest was the same as in Example 1;

[0158] R2 reagent: same as in Example 1;

[0159] Calibrator, same as in Example 1.

[0160] Experimental plan 2:

[0161] An immunoturbidimetric kit for detecting ADP, specifically comprising:

[0162] Reagent R1: 20 mmol / L sulfonic acid-polyethylene glycol (mPEG-SO3H-Ⅱ) was used to replace 20 mmol / L heparin sodium, and the rest was the same as in Example 1;

[0163] R2 reagent: same as in Example 1;

[0164] Calibrator, same as in Example 1.

[0165] The quality control product (5 μg / mL) was measured using the kit in the above scheme, and the measured results are shown in Table 6 below;

[0166] Table 6 Experiments of different anticoagulant components in the kit

[0167] Experiment number Detection value (μg / mL) Theoretical value (μg / mL) Relative deviation Example 1 5.12 5.00 2.4％ Experimental plan 1 5.25 5.00 5.0％ Experimental plan 2 5.04 5.00 0.8％

[0168] It can be seen from the table that it is feasible to use sulfonic acid-polyethylene glycol (mPEG-SO3H) to replace the heparin sodium in the kit in Example 1 to measure the quality control samples.

Claims

1. An immunoturbidimetric kit for detecting ADP, characterized in that: The kit includes R1 reagent and R2 reagent; R1 reagent includes: buffer, 10~100mmol / L; NaCl, 0.1~1mol / L; anticoagulant, 10~100mmol / L; coagulant, 0.1~1%; surfactant, 0.1~1g / L; preservative, 0.5~1g / L; blocking agent, 0.05~0.2mg / mL; Among them, the anticoagulant in the R1 reagent is a combination of EDTA-Na2, sodium citrate and sulfonic acid-polyethylene glycol; R2 reagents include: buffer and antibody-coated latex microspheres; The R2 reagent is prepared using a cross-linking solution, a blocking solution and a preservation solution; The antibody-coated latex microspheres in the R2 reagent are polystyrene latex microsphere-adiponectin antibody complexes; The particle size of the polystyrene latex microspheres is 200-400 nm; The sulfonic acid group-polyethylene glycol mPEG-SO3H is one of mPEG-SO3H-Ⅰ and mPEG-SO3H-Ⅱ; The chemical structural formula of the sulfonic acid group-polyethylene glycol mPEG-SO3H-Ⅰ is: ； The chemical structural formula of the sulfonic acid group-polyethylene glycol mPEG-SO3H-Ⅱ is: 。 2. An immunoturbidimetric kit for detecting ADP according to claim 1, characterized in that: The buffer in the R2 reagent includes a Gly buffer with a pH of 7.0 to 8.0; The cross-linking solution includes 50-100 mmol / L Hepes buffer with a pH of 6.0-8.0; The blocking solution includes 50-100 mmoL / LGly buffer, 6-12% AEO7 and 1-5% AEO3, with a pH of 7.0-10.

0.

3. An immunoturbidimetric kit for detecting ADP according to claim 1, characterized in that: The preparation steps of the R1 reagent are as follows: weigh each component according to the R1 reagent formula into a clean container, add deionized water and stir to fully dissolve it, adjust the pH to 6.00, add deionized water to make up to volume, and filter to obtain the R1 reagent.

4. The immunoturbidimetric kit for detecting ADP according to claim 1, characterized in that: The preparation steps of the R2 reagent are as follows: Step S1: adding carboxylated polystyrene latex microspheres to the crosslinking solution to make the concentration 0.77%; Step S2: adding 2 times the activation amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide EDC to the above latex microspheres, stirring and reacting at 37° C.; Step S3: adding adiponectin antibody 1 and adiponectin antibody 2 to the activated polystyrene latex microspheres respectively, and reacting by stirring at 37° C.; Step S4: adding blocking solution to make the concentration of the polystyrene latex microsphere solution 0.44%, and blocking at 37° C. overnight; Step S5: washing by centrifugation, adding a preservation solution to ultrasonically disperse the precipitate; mixing the two groups of latex uniformly to obtain R2; Step S6: The above R2 is placed at 37° C. for aging for 1 to 3 days to obtain the final R2 reagent.

5. An immunoturbidimetric kit for detecting ADP according to claim 4, characterized in that: The step S5: the storage solution is composed of 50-100 mmol / L Gly buffer, pH 7.0-8.0, 0.1-1% BSA, 6-12% sucrose, and 0.05-0.1% NaN3.

6. The immunoturbidimetric kit for detecting ADP according to claim 1, characterized in that: The R1 reagent includes: 50mmol / L MES buffer with a pH of 6.0; 0.65mol / L NaCl, 20mmoL / L EDTA-Na2, 20mmoL / L sulfonic acid-polyethylene glycol, 20mmoL / L sodium citrate, 4.5g / L coagulant PEG20000, 0.1g / L surfactant BrijL23, 1.0g / L preservative NaN3 and 0.05mg / mL blocking agent.

7. An immunoturbidimetric kit for detecting ADP according to claim 6, characterized in that: The preparation method of sulfonic acid group-polyethylene glycol mPEG-SO3H in the R1 reagent is as follows: Step S1, synthesizing allyl-polyethylene glycol mPEG-CH2-CH=CH2: the hydroxyl functional group at the end of polyethylene glycol mPEG-OH and the epoxy functional group of allyl glycidyl ether undergo a ring-opening reaction under the action of an alkaline catalyst to generate allyl-polyethylene glycol mPEG-CH2-CH=CH2; Step S2, synthesizing thiol-polyethylene glycol mPEG-SH: utilizing the thiol-ene click reaction mechanism, the allyl functional group of allyl-polyethylene glycol mPEG-CH2-CH=CH2 and the thiol functional group of the thiol-reactive agent undergo a thiol-ene click chemical reaction to generate thiol-polyethylene glycol mPEG-SH; Step S3, synthesizing sulfonic acid-polyethylene glycol mPEG-SO3H: using hydrogen peroxide as an oxidizing agent, oxidizing the sulfhydryl functional group of sulfhydryl-polyethylene glycol mPEG-SH into a sulfonic acid functional group, thereby preparing sulfonic acid-polyethylene glycol mPEG-SO3H.

8. An immunoturbidimetric kit for detecting ADP according to claim 7, characterized in that: The thiol-forming agent is one or a combination of pentaerythritol tetrakis (3-mercaptopropionate) and octathiocarbyl cage-type polysilsesquioxane POSS-SH.

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