Heparin binding protein detection kit and preparation method thereof

Abstract

The invention discloses a heparin binding protein detection kit and a preparation method thereof. The detection kit comprises a first reagent and a second reagent. And the second reagent comprises latex microspheres B1, B2, B3 and B4 and a liposome. The latex microspheres B1 and B2 are loaded with a mouse anti-human heparin binding protein monoclonal antibody, and the latex microspheres B3 and B4 are loaded with a mouse anti-rabbit anti-human heparin binding protein monoclonal antibody-monoclonal antibody. Single surfaces of the latex microspheres B1 and B4 are covered with monoclonal antibodies, the whole surfaces of the latex microspheres B2 and B3 are covered with monoclonal antibodies, two groups of latex microspheres which have different diameters and different modification areas and are loaded with corresponding antibodies are arranged in the detection reagent, and the agglomeration diameter of the latex microspheres is controlled, so that the correlation between the turbidity of a to-be-detected system and the concentration of the heparin-binding protein in a to-be-detected sample is improved; the detection accuracy is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of clinical detection, and in particular to a heparin binding protein detection kit and a preparation method thereof. Background Art

[0002] Heparin Binding Protein (HBP) is a protein secreted by the azurophilic granules of mature neutrophils, with a relative molecular mass of 37KDa. HBP is both an inflammatory indicator and a pro-inflammatory factor. HBP activates monocytes and macrophages, releases inflammatory mediators such as tumor necrosis factor-α (TNF-α) and gamma-interferon (INF-γ), and enhances the body's immune response to infection or injury. HBP itself has a strong positive charge, which can affect the permeability of vascular endothelial cells, induce vascular leakage and tissue edema.

[0003] "Chinese Emergency Expert Consensus on Early Prevention and Blocking of Sepsis": HBP is the earliest and most accurate biomarker for diagnosing sepsis and septic shock. By dynamically monitoring the plasma HBP concentration, the shock that will occur in patients with severe infection can be predicted, thereby achieving early intervention. HBP can predict the occurrence of circulatory failure. At the same time, plasma HBP is a more sensitive marker of bacterial infection and has a high clinical value in distinguishing bacterial from non-bacterial pneumonia.

[0004] At present, the main methods for detecting heparin binding protein include colloidal gold method, enzyme-linked immunosorbent assay, magnetic microparticle chemiluminescence method, etc. These methods have the disadvantages of low measurement accuracy, high cost or long measurement time. Latex enhanced immunoturbidimetry is a simple, low-cost and highly versatile method, but the existing latex enhanced immunoturbidimetry for heparin binding protein has the problem of reduced accuracy of the measurement results due to the uneven diameter of latex microsphere aggregates.

[0005] Therefore, it is necessary to develop a heparin binding protein detection kit and a preparation method thereof that can achieve uniform latex microsphere agglomeration particles and stable and accurate reference values. Summary of the invention

[0006] In view of this, the present invention provides a heparin-binding protein detection kit and a preparation method thereof. By setting two groups of latex microspheres loaded with corresponding antibodies with different diameters and different modified regions in the detection reagent, and acting synergistically with liposomes also set in the detection reagent, when the concentration of heparin-binding protein in the sample to be tested is high, the liposomes are disintegrated to release the monoclonal antibodies therein, which can form latex microsphere clusters with the heparin-binding protein and latex microspheres in the sample, thereby achieving the improvement of the correlation between the turbidity of the system to be tested and the concentration of heparin-binding protein in the sample to be tested, and increasing the detection accuracy.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a heparin-binding protein detection kit, the heparin-binding protein detection kit comprising a first reagent, a second reagent, a first calibration sample and a second calibration sample;

[0009] The first reagent includes an anti-interference agent, a first protein stabilizer, a first surfactant, an inorganic salt, a first antibacterial preservative, and a buffer;

[0010] The second reagent includes latex microsphere B1, latex microsphere B2, latex microsphere B3, latex microsphere B4, liposomes, a second protein stabilizer, a second surfactant, a second antibacterial preservative, and a medium-chain dibasic acid;

[0011] The first calibration sample includes a standard solution with a heparin binding protein concentration of 20-50 ng / mL;

[0012] The second calibration sample includes a standard solution with a heparin binding protein concentration of 150-300 ng / mL;

[0013] The particle size of the latex microsphere B1 is 200-300 nm, the surface is loaded with mouse anti-human heparin binding protein monoclonal antibody, and the surface also has anion exchange groups. The latex microsphere B1 is a latex microsphere loaded with mouse anti-human heparin binding protein monoclonal antibody on one side;

[0014] The particle size of the latex microsphere B2 is 130-150nm, and the surface is loaded with mouse anti-human heparin binding protein monoclonal antibody. The latex microsphere B2 is a latex microsphere loaded with mouse anti-human heparin binding protein monoclonal antibody on the entire surface;

[0015] The particle size of the latex microsphere B3 is 200-250nm, and the surface is loaded with mouse anti-rabbit anti-human heparin binding protein monoclonal antibody-monoclonal antibody. The latex microsphere B3 is a latex microsphere loaded with mouse anti-rabbit anti-human heparin binding protein monoclonal antibody-monoclonal antibody on the entire surface;

[0016] The particle size of the latex microsphere B4 is 150-200 nm, and the surface is loaded with mouse anti-rabbit anti-human heparin binding protein monoclonal antibody-monoclonal antibody. The latex microsphere B4 is a latex microsphere loaded with mouse anti-rabbit anti-human heparin binding protein monoclonal antibody-monoclonal antibody on one side;

[0017] The mass ratio of the latex microspheres B1 to B2 is (20-48):1, the mass ratio of the latex microspheres B3 to B4 is 1:(6-7), the sum of the mass concentrations of the latex microspheres B1 and B2 in the second reagent is 0.38%-0.77%, and the sum of the mass concentrations of the latex microspheres B3 and B4 in the second reagent is 0.82%-1.46%;

[0018] The membrane of the liposome is composed of phosphatidylglycerol and cholesterol, and contains rabbit anti-human heparin binding protein monoclonal antibody and anion replacement agent. The diameter of the liposome is 30-60nm;

[0019] The pH of the first reagent is 6.6-6.8, and the pH of the second reagent is 6.7-7.0.

[0020] Preferably, the anion exchange groups on the latex microspheres B1 include primary amine groups and secondary amine groups, the molar ratio of the primary amine to the secondary amine is 5:(1-4), the exchange capacity on the latex microspheres B1 is 0.1-0.4 mmol / g, and the exchangeable groups on the anion exchange groups are hydroxides.

[0021] Preferably, the medium-chain dibasic acid comprises one of dibasic acids with 7-9 carbon atoms, with a concentration of 13-21 mmol / L.

[0022] Preferably, the latex microspheres B1, B2, B3, B4 include carboxyl polystyrene latex microspheres.

[0023] Preferably, the anti-interference agent includes at least one of anti-rheumatic factor antibodies and anti-rheumatoid factor antibodies; the first protein stabilizer and the second protein stabilizer include one of polyethylene glycol, polypropylene glycol, alkyl glycoside, oligoglucan, and oligoxylan; the first surfactant and the second surfactant include one of Tween 60 and lauroyl diethanolamine; the inorganic salt includes one of sodium bromide and sodium chloride; the first antibacterial preservative and the second antibacterial preservative include one of sodium azide and gentamicin; the anion replacer includes one of potassium chloride and potassium sulfate; the buffer includes one of glycine buffer and phosphate buffer.

[0024] In a second aspect, the present invention provides a method for preparing the heparin binding protein detection kit as described above, the preparation method comprising the following steps:

[0025] Preparation of latex microspheres B1: connecting a titanium-coated ruthenium electrode plate to the positive pole of a DC power supply, the power supply voltage is 20-40 volts, so that the titanium-coated ruthenium electrode plate carries a positive static charge; contacting blank latex microspheres with a diameter of 200-300 nm with a rubber plate with a negative static charge, the static voltage of the rubber plate is 150-220 volts, so that the blank latex microspheres carry a negative charge; using the positively charged titanium-coated ruthenium electrode plate to contact the negatively charged blank latex microspheres, so that the latex microspheres are adsorbed onto the titanium-coated ruthenium electrode plate; preparing a low-melting point agarose gel with a mass concentration of 1.3%-1.6%, heating it to 36-38°C, pouring it on the titanium-coated ruthenium electrode plate on which the blank latex microspheres are adsorbed, and the low-melting point agarose gel The liquid level is 60%-70% of the diameter of the latex microspheres, the temperature is reduced to below 30°C, and the solution is solidified for 5-6 hours to obtain a titanium-coated ruthenium electrode plate fixed with latex microspheres; a reaction solution of the latex microspheres coupled with the heparin-binding protein antibody is prepared, wherein the reaction solution comprises 2.4-5.1 g / L MES·H2O, 1.7-2.1 g / L boric acid, 0.7-0.8 g / L disodium hydrogen phosphate, 12-26 mg / mL mouse anti-human heparin-binding protein monoclonal antibody, 0.12-0.25 g / L EDC·HCI solution, and 0.4-0.5 g / L NHS solution; the reaction solution of the latex microspheres coupled with the heparin-binding protein antibody is coated on a hydrophilic treated glass plate, and An antibody coupling reaction liquid film is formed on the surface, and the height of the liquid film is 25%-35% of the diameter of the latex microspheres, to obtain a glass plate coated with the antibody coupling reaction liquid; the titanium-coated ruthenium electrode plate fixed with the latex microspheres is inverted on the glass plate coated with the antibody coupling reaction liquid, so that the exposed part of the latex microspheres is in contact with the coupling reaction liquid, and the antibody coupling reaction is carried out for 18-20 hours at a reaction temperature of 22-26°C; the titanium-coated ruthenium electrode plate fixed with the latex microspheres is separated from the glass plate coated with the antibody coupling reaction liquid, and a blocking solution containing 110-200 mL / L of Tween and 1.7-2.1 g / L of BSA is applied to the titanium-coated ruthenium electrode plate fixed with the latex microspheres, so that the exposed part of the latex microspheres is immersed In the blocking solution, a blocking reaction is carried out at 27-32° C. for 4-6 hours; proclin300 is added to the blocking solution to make its concentration in the system reach 0.3-0.8 mL / L, and the pH is adjusted to 6.8-7.0 using sodium hydroxide solution or hydrochloric acid, and soaked for 12-15 hours; the titanium-coated ruthenium electrode plate fixed with latex microspheres is immersed in a phosphate buffer at a temperature of 39-41° C. and a pH of 6.6-6.9 to liquefy the solidified low-melting point agarose gel and release the coupled latex microspheres, and the material containing the latex microspheres is centrifuged while hot for solid-liquid separation, and washed 3-4 times with the phosphate buffer at a pH of 6.9-7.2 to obtain the latex microspheres B1;

[0026] Preparation of latex microspheres B2: blank latex microspheres with a particle size of 130-150 nm are coupled with anion exchange groups according to a conventional method, and then MES·H2O 2.3-4.9 g / L is added, and the pH is adjusted to 6.7-6.8 with sodium hydroxide. After mixing evenly, 2.4-3.0 g / L of boric acid and 0.6-0.9 g / L of disodium hydrogen phosphate are added, and the pH is adjusted to 7.0-7.3 with sodium hydroxide. The mixture is mixed at 20-25° C. to obtain a latex microsphere suspension; then the mouse anti-human heparin binding protein monoclonal antibody is added to the latex microsphere suspension, and the concentration of the mouse anti-human heparin binding protein monoclonal antibody in the reaction system is 8-20 mg / mL, and 0.1-0.3 g / L of EDC·HCl solution and 0.2-0.6g / L NHS solution are stirred at 35-40°C for coupling reaction, and the reaction time is 7-10h; a blocking solution composed of 700-150mL / L Tween and 1.3-1.7g / L BSA is added, and the mixture is stirred and dispersed at 29-33°C for 4-9h blocking reaction; proclin300 is added to a concentration of 0.4-0.8mL / L in the system, and the pH is adjusted to 7.3-7.6 using sodium hydroxide solution or hydrochloric acid, and the mixture is stirred and soaked for 12-15h to obtain the latex microspheres B2;

[0027] Preparation of latex microspheres B3: The particle size of blank latex microspheres is 200-250 nm, and the surface thereof does not need to be coupled with anion exchange groups. The monoclonal antibody loaded on the surface is mouse anti-rabbit anti-human heparin binding protein monoclonal antibody-monoclonal antibody. The other steps are the same as those for preparing latex microspheres B2 to obtain the latex microspheres B3;

[0028] Preparation of latex microspheres B4: the particle size of blank latex microspheres is 150-200 nm, the monoclonal antibody loaded on the surface is mouse anti-rabbit anti-human heparin binding protein monoclonal antibody-monoclonal antibody, and the other steps are the same as those for preparing latex microspheres B1 to obtain the latex microspheres B4;

[0029] Preparation of liposomes: dissolving 7-10 parts by weight of phosphatidylglycerol and 0.7-1.2 parts by weight of cholesterol in 50-55 parts by weight of 80%-86% ethanol aqueous solution to obtain a liposome membrane preparation solution; dissolving 0.6-1 parts by weight of rabbit anti-human heparin binding protein monoclonal antibody and 0.3-0.5 parts by weight of anion replacement agent in 90-110 parts by weight of glycine buffer with a concentration of 100-120 mmol / L to obtain a liposome-containing solution; passing the liposome membrane preparation solution and the liposome-containing solution into a microfluidic device, and preparing the liposomes with a diameter of 30-60 nm according to conventional techniques;

[0030] Prepare the first reagent: prepare the volume of the first reagent as needed, calculate and weigh the ingredients according to the following ratio: 22-64 mg / L of the anti-interference agent, 530-1000 mg / L of the first protein stabilizer, 20-26 mmol / L of the first surfactant, 4-8 g / L of the inorganic salt, 34-56 mg / L of the first antibacterial preservative, and 75-160 mmol / L of the buffer solution, dissolve them in a corresponding volume of deionized water, and adjust the pH value to 6.6-6.8 with hydrochloric acid or sodium hydroxide solution to obtain the first reagent;

[0031] Prepare the second reagent: prepare the volume of the second reagent as needed, calculate and weigh the ingredients according to the following ratio, the mass ratio of the latex microspheres B1 and B2 is (20-48):1, the mass ratio of the latex microspheres B3 and B4 is 1:(6-7), the sum of the mass concentrations of the latex microspheres B1 and B2 in the second reagent is 0.38%-0.77%, the sum of the mass concentrations of the latex microspheres B3 and B4 in the second reagent is 0.82%-1.46%, the mass concentration of the liposomes in the second reagent is 0.5%-1%, the second protein stabilizer is 660-1200 mg / L, the second surfactant is 18-25 mmol / L, the second antibacterial preservative is 30-50 mg / L, the concentration of the medium-chain dibasic acid in the second reagent is 13-21 mmol / L, and they are dissolved in a corresponding volume of deionized water, and the pH value is adjusted to 6.7-7.0 with hydrochloric acid or sodium hydroxide solution to obtain the second reagent;

[0032] Preparation of calibration samples: The first calibration sample and the second calibration sample are prepared according to conventional methods. The heparin binding protein concentration of the first sample is 20-50 ng / mL, and the heparin binding protein concentration of the second calibration sample is 150-300 ng / mL.

[0033] Preferably, the blank latex microspheres for preparing the latex microspheres B1 and B4 include latex microspheres without surface water. First, the blank latex microspheres are immersed in ultrapure water for 90-110 minutes; then filtered and excess water is absorbed with filter paper; spread into a thin layer with a thickness of 0.4-0.6 mm, and placed in a sterile environment at 22-30° C. and a relative humidity of 55%-65% for 10-20 minutes to obtain the latex microspheres without surface water.

[0034] Preferably, the surface of the titanium-coated ruthenium electrode plate is coated with an insulating layer, the insulating layer comprises one of polyethylene, polypropylene and polyamide, and the thickness of the insulating layer is 10-30 nm.

[0035] Preferably, the hydrophilic treated glass plate comprises a glass plate treated by plasma cleaning.

[0036] Preferably, the relative humidity of the environment in which the latex microspheres B1 and B4 are prepared is 86%-92%.

[0037] Compared with the prior art, the present invention has the following beneficial technical effects: latex microspheres B1, B2, B3, and B4 are added to the second reagent, and when the concentration of heparin-binding protein in the sample to be tested is low, about 6 latex microspheres B1, 1 latex microsphere B2, and the heparin-binding protein-bound latex microspheres are aggregated 1; when the concentration of heparin-binding protein in the sample to be tested is high, the excess heparin-binding protein exchanges with the hydroxyl radicals on the latex microspheres B1, resulting in an increase in the pH of the detection system. Under the synergistic effect of the medium- and long-chain dibasic acids, the liposomes are disintegrated, and the rabbit anti-human heparin-binding protein monoclonal antibodies and The anion displacer is released and displaces the heparin-binding protein adsorbed on the primary amine or secondary amine group of latex microsphere B1, starting the process of forming latex microsphere cluster 2 with 1 latex microsphere B3, 14 latex microspheres B4, rabbit anti-human heparin-binding protein monoclonal antibody and heparin-binding protein. The diameters of latex microsphere clusters 1 and 2 are similar, but the corresponding heparin-binding protein concentration coefficients are different, the latter is about 2.3 times that of the former. Bringing it into the calculation process can improve the correlation between the turbidity of the system to be tested and the heparin-binding protein concentration in the sample to be tested, thereby increasing the detection accuracy. DETAILED DESCRIPTION

[0038] The technical solution of the present invention is described in detail below in conjunction with embodiments.

[0039] It should be noted that the following embodiments and features in the embodiments may be combined with each other in the absence of conflict; and, based on the embodiments in the present disclosure, all other embodiments obtained by ordinary technicians in the field without making any creative work are within the scope of protection of the present disclosure.

[0040] It should be noted that various aspects of the embodiments within the scope of the appended claims are described below. It should be apparent that the aspects described herein may be embodied in a wide variety of forms, and any specific structures and / or functions described herein are merely illustrative. Based on the present disclosure, it should be understood by those skilled in the art that an aspect described herein may be implemented independently of any other aspect, and two or more of these aspects may be combined in various ways. For example, any number of aspects described herein may be used to implement the device and / or practice method. It should be noted that the serial numbers themselves, such as "first", "second", "third", etc., in the present application document only distinguish the objects described and do not have any order or technical meaning.

[0041] The present invention provides a heparin-binding protein detection kit and a preparation method thereof. By arranging two latex microspheres loaded with corresponding antibodies with different diameters and different modified regions in the detection reagent, the diameter of the latex microsphere agglomeration is controlled, thereby improving the correlation between the turbidity of the system to be detected and the heparin-binding protein concentration in the sample to be detected, and increasing the detection accuracy; by arranging a third reagent with the same composition but the latex microspheres are not modified as a strict reference absorbance value, the detection stability is improved.

[0042] The present invention provides a heparin-binding protein detection kit, which comprises a first reagent, a second reagent, a first calibration sample and a second calibration sample;

[0043] The first reagent includes an anti-interference agent, a first protein stabilizer, a first surfactant, an inorganic salt, a first antibacterial preservative, and a buffer;

[0044] The second reagent includes latex microsphere B1, latex microsphere B2, latex microsphere B3, latex microsphere B4, liposomes, a second protein stabilizer, a second surfactant, a second antimicrobial preservative, and a medium-chain dibasic acid;

[0045] The first calibration sample includes a standard solution with a heparin binding protein concentration of 20-50 ng / mL;

[0046] The second calibration sample includes a standard solution with a heparin binding protein concentration of 150-300 ng / mL;

[0047] The particle size of the latex microsphere B1 is 200-300 nm, the surface of which is loaded with mouse anti-human heparin binding protein monoclonal antibody, and the surface also has an anion exchange group, and the latex microsphere B1 is a latex microsphere loaded with mouse anti-human heparin binding protein monoclonal antibody on one side;

[0048] The particle size of latex microsphere B2 is 130-150nm, and the surface is loaded with mouse anti-human heparin binding protein monoclonal antibody. The latex microsphere B2 is a latex microsphere with mouse anti-human heparin binding protein monoclonal antibody loaded on the entire surface;

[0049] The particle size of latex microsphere B3 is 200-250nm, and the surface is loaded with mouse anti-rabbit anti-human heparin binding protein monoclonal antibody-monoclonal antibody. The latex microsphere B3 is a latex microsphere with mouse anti-rabbit anti-human heparin binding protein monoclonal antibody-monoclonal antibody loaded on the entire surface;

[0050] The particle size of latex microsphere B4 is 150-200 nm, and the surface is loaded with mouse anti-rabbit anti-human heparin binding protein monoclonal antibody-monoclonal antibody. The latex microsphere B4 is a latex microsphere loaded with mouse anti-rabbit anti-human heparin binding protein monoclonal antibody-monoclonal antibody on one side;

[0051] The mass ratio of latex microspheres B1 to B2 is (20-48):1, the mass ratio of latex microspheres B3 to B4 is 1:(6-7), the sum of the mass concentrations of latex microspheres B1 and B2 in the second reagent is 0.38%-0.77%, and the sum of the mass concentrations of latex microspheres B3 and B4 in the second reagent is 0.82%-1.46%;

[0052] The membrane of the liposome is composed of phosphatidylglycerol and cholesterol, and contains rabbit anti-human heparin binding protein monoclonal antibody and anion displacer. The diameter of the liposome is 30-60nm.

[0053] The pH of the first reagent is 6.6-6.8, and the pH of the second reagent is 6.7-7.0.

[0054] Preferably, the anion exchange groups on the latex microspheres B1 include primary amine groups and secondary amine groups, the molar ratio of primary amine to secondary amine is 5:(1-4), the exchange capacity on the latex microspheres B1 is 0.1-0.4 mmol / g, and the exchangeable groups on the anion exchange groups are hydroxides.

[0055] Preferably, the medium-chain dibasic acid includes one of dibasic acids with 7-9 carbon atoms, with a concentration of 13-21 mmol / L.

[0056] Preferably, the latex microspheres B1, B2, B3, B4 comprise carboxylated polystyrene latex microspheres.

[0057] Preferably, the anti-interference agent includes at least one of anti-rheumatic factor antibodies and anti-rheumatoid factor antibodies; the first protein stabilizer and the second protein stabilizer include one of polyethylene glycol, polypropylene glycol, alkyl glycoside, oligoglucosaccharide, and oligoxylan; the first surfactant and the second surfactant include one of Tween 60 and lauroyl diethanolamine; the inorganic salt includes one of sodium bromide and sodium chloride; the first antibacterial preservative and the second antibacterial preservative include one of sodium azide and gentamicin; the anion replacer includes one of potassium chloride and potassium sulfate; the buffer includes one of glycine buffer and phosphate buffer.

[0058] It should be noted that the heparin binding protein concentration of the first calibration sample is less than the maximum value determined by the combination of latex microspheres B1 and B2, and the heparin binding protein concentration of the second calibrator is greater than the maximum value determined by the combination of latex microspheres B1 and B2.

[0059] It should be noted that the carbon chain of the medium-chain dibasic acid is a straight chain without branches, and the two carboxyl groups are located at the two ends of the carbon chain.

[0060] It should be noted that the role of the combination of latex microspheres B1 and B2 is that when the concentration of heparin binding protein in the sample to be tested is low, latex microspheres B1, B2 and heparin binding protein form a composite microsphere of a certain diameter. For example, 6 latex microspheres B1 and 1 latex microsphere B2 form a 7-microsphere combination with a diameter of about 600-850nm. Since latex microsphere B1 is a single-sided loaded heparin binding protein monoclonal antibody, it has been combined with heparin binding protein and latex microsphere B2 to form a microsphere combination. The latex microsphere B1 located outside the combination has no monoclonal antibody and cannot be combined with other heparin binding proteins in the sample, thereby preventing the microsphere combination from further increasing, achieving a small particle size distribution range of the microsphere combination, and improving its detection accuracy.

[0061] It should be noted that the effect of the combination of latex microspheres B3 and B4 is basically the same as that of latex microspheres B1 and B2. The difference is that the surface load is not a monoclonal antibody that can directly bind to heparin binding protein, but a monoclonal antibody of another heparin binding protein monoclonal antibody. When the other heparin binding protein monoclonal antibody is released from the liposome, latex microspheres B3, B4, heparin binding protein, and heparin binding protein monoclonal antibody aggregate with each other to form a composite microsphere of 1 latex microsphere B3 and about 14 latex microspheres B4, with a diameter of about 500-650nm.

[0062] Latex microspheres B1 (200-300nm) + latex microspheres B2 (130-150nm), latex microspheres B3 (200-250nm) + latex microspheres B4 (150-200nm) are combined to form composite microspheres with similar particle sizes, but the corresponding HBP amounts are different, and the slope of the working curve is 2.3 times.

[0063] It should be noted that the function of liposomes is to release the heparin-binding protein monoclonal antibody and anion displacer therein under specific conditions.

[0064] More preferably, the molecular weight of the polyethylene glycol is in the range of 2000-5000.

[0065] More preferably, the molecular weight of the polypropylene glycol is in the range of 1500-4500.

[0066] More preferably, the alkyl glycoside includes alkyl glucoside, which is produced by reacting fatty alcohol and glucose in the presence of a catalyst.

[0067] More preferably, the oligosaccharide comprises an oligosaccharide formed by polymerization of 8 to 18 glucose monomers.

[0068] More preferably, the xylo-oligosaccharide comprises an oligosaccharide formed by polymerization of 5-10 xylose monomers.

[0069] It should be noted that the reasons for selecting the pH range are as follows: under pH 6.7-6.9, heparin-binding protein preferentially binds to the monoclonal antibody coupled to the B1 and B2 latex microspheres, which will not cause a significant increase in the pH of the system. When the heparin-binding protein is in excess relative to the heparin-binding protein monoclonal antibody on the latex microspheres B1 and B2, it can undergo ion exchange with the heparin-binding protein to release hydroxide, thereby raising the pH of the system to above 7.9, and synergizing with the medium- and long-chain dibasic acid in the second reagent to trigger the disintegration of liposomes and release the intermediary substance that condenses the heparin-binding protein with the latex microspheres B3 and B4.

[0070] The present invention also provides a method for preparing the above-mentioned heparin-binding protein detection kit, which comprises the following steps:

[0071] Preparation of latex microspheres B1: connecting a titanium-coated ruthenium electrode plate to the positive pole of a DC power supply, the power supply voltage is 20-40 volts, so that the titanium-coated ruthenium electrode plate is positively charged with static electricity; contacting blank latex microspheres with a diameter of 200-300 nm with a rubber plate with negative static electricity, the static voltage of the rubber plate is 150-220 volts, so that the blank latex microspheres are negatively charged; using the positively charged titanium-coated ruthenium electrode plate to contact the negatively charged blank latex microspheres, so that the latex microspheres are adsorbed onto the titanium-coated ruthenium electrode plate; preparing a low-melting point agarose gel with a mass concentration of 1.3%-1.6%, heating it to 36-38°C, pouring it on the titanium-coated ruthenium electrode plate on which the blank latex microspheres are adsorbed, and the liquid level of the low-melting point agarose gel is high The degree is 60%-70% of the diameter of the latex microspheres, the temperature is reduced to below 30°C, and solidified for 5-6 hours to obtain a titanium-coated ruthenium electrode plate with latex microspheres fixed thereon; a reaction solution of latex microspheres coupled with heparin-binding protein antibodies is prepared, the reaction solution composition is MES·H2O2.4-5.1g / L, boric acid 1.7-2.1g / L, disodium hydrogen phosphate 0.7-0.8g / L, mouse anti-human heparin-binding protein monoclonal antibody 12-26mg / mL, EDC·HCI solution 0.12-0.25g / L, NHS solution 0.4-0.5g / L, the reaction solution of latex microspheres coupled with heparin-binding protein antibodies is coated on a hydrophilic treated glass plate, and The antibody coupling reaction liquid film is formed on the surface of the glass plate, and the height of the liquid film is 25%-35% of the diameter of the latex microspheres, thereby obtaining a glass plate coated with the antibody coupling reaction liquid; the titanium-coated ruthenium electrode plate fixed with the latex microspheres is inverted on the glass plate coated with the antibody coupling reaction liquid, so that the exposed part of the latex microspheres is in contact with the coupling reaction liquid, and the antibody coupling reaction is carried out for 18-20 hours at a reaction temperature of 22-26°C; the titanium-coated ruthenium electrode plate fixed with the latex microspheres is separated from the glass plate coated with the antibody coupling reaction liquid, and a blocking liquid containing 110-200 mL / L of Tween and 1.7-2.1 g / L of BSA is applied to the titanium-coated ruthenium electrode plate fixed with the latex microspheres, so that the exposed part of the latex microspheres is immersed in the glass plate. The method comprises the following steps: performing a blocking reaction at 27-32° C. for 4-6 hours in a blocking solution; adding proclin300 to the blocking solution so that its concentration in the system reaches 0.3-0.8 mL / L, adjusting the pH to 6.8-7.0 using a sodium hydroxide solution or hydrochloric acid, and soaking for 12-15 hours; immersing the titanium-coated ruthenium electrode plate with the latex microspheres fixed thereon in a phosphate buffer at a temperature of 39-41° C. and a pH of 6.6-6.9 to liquefy the solidified low-melting point agarose gel and release the coupled latex microspheres; centrifuging the material containing the latex microspheres while hot for solid-liquid separation, and washing 3-4 times with a phosphate buffer at a pH of 6.9-7.2 to obtain latex microspheres B1;

[0072] Preparation of latex microspheres B2: blank latex microspheres with a particle size of 130-150 nm are coupled with anion exchange groups according to a conventional method, and then MES·H2O 2.3-4.9 g / L is added, and the pH is adjusted to 6.7-6.8 with sodium hydroxide, and then 2.4-3.0 g / L of boric acid and 0.6-0.9 g / L of disodium hydrogen phosphate are added, and the pH is adjusted to 7.0-7.3 with sodium hydroxide, and the mixture is mixed at 20-25° C. to obtain a latex microsphere suspension; then mouse anti-human heparin binding protein monoclonal antibody is added to the latex microsphere suspension, and the concentration of the mouse anti-human heparin binding protein monoclonal antibody in the reaction system is 8-20 mg / mL, and then 0.1-0.3 g / L of EDTA is added. C·HCl solution and 0.2-0.6g / L NHS solution, stirring at 35-40°C for coupling reaction, the reaction time is 7-10h; adding blocking solution composed of 700-150mL / L Tween and 1.3-1.7g / L BSA, stirring and dispersing at 29-33°C, and performing blocking reaction for 4-9h; adding proclin300 to make its concentration in the system reach 0.4-0.8mL / L, and adjusting the pH to 7.3-7.6 with sodium hydroxide solution or hydrochloric acid, stirring and soaking for 12-15h, to obtain latex microspheres B2;

[0073] Preparation of latex microspheres B3: The particle size of blank latex microspheres is 200-250 nm, and the surface thereof does not need to be coupled with anion exchange groups. The monoclonal antibody loaded on the surface is mouse anti-rabbit anti-human heparin binding protein monoclonal antibody-monoclonal antibody. The other steps are the same as those for preparing latex microspheres B2 to obtain latex microspheres B3;

[0074] Preparation of latex microspheres B4: the particle size of blank latex microspheres is 150-200 nm, the monoclonal antibody loaded on the surface is mouse anti-rabbit anti-human heparin binding protein monoclonal antibody-monoclonal antibody, and the other steps are the same as those for preparing latex microspheres B1 to obtain latex microspheres B4;

[0075] Preparation of liposomes: dissolving 7-10 parts by weight of phosphatidylglycerol and 0.7-1.2 parts by weight of cholesterol in 50-55 parts by weight of 80%-86% ethanol aqueous solution to obtain a liposome membrane preparation solution; dissolving 0.6-1 parts by weight of rabbit anti-human heparin binding protein monoclonal antibody and 0.3-0.5 parts by weight of anion replacement agent in 90-110 parts by weight of 100-120 mmol / L glycine buffer to obtain a liposome-containing solution; passing the liposome membrane preparation solution and the liposome-containing solution into a microfluidic device, and preparing the liposomes with a diameter of 30-60 nm according to conventional techniques;

[0076] Prepare the first reagent: prepare the volume of the first reagent as needed, calculate and weigh the ingredients according to the following ratio: 22-64 mg / L of anti-interference agent, 530-1000 mg / L of first protein stabilizer, 20-26 mmol / L of first surfactant, 4-8 g / L of inorganic salt, 34-56 mg / L of first antibacterial preservative, 75-160 mmol / L of buffer, dissolve in corresponding volume of deionized water, adjust the pH value to 6.6-6.8 with hydrochloric acid or sodium hydroxide solution, and obtain the first reagent;

[0077] Prepare the second reagent: prepare the volume of the second reagent as needed, calculate and weigh the ingredients according to the following ratio, the mass ratio of latex microspheres B1 and B2 is (20-48):1, the mass ratio of latex microspheres B3 and B4 is 1:(6-7), the sum of the mass concentrations of latex microspheres B1 and B2 in the second reagent is 0.38%-0.77%, the sum of the mass concentrations of latex microspheres B3 and B4 in the second reagent is 0.82%-1.46%, the mass concentration of liposomes in the second reagent is 0.5%-1%, the second protein stabilizer is 660-1200 mg / L, the second surfactant is 18-25 mmol / L, the second antibacterial preservative is 30-50 mg / L, the concentration of medium-chain dibasic acid in the second reagent is 13-21 mmol / L, and they are dissolved in a corresponding volume of deionized water, and the pH value is adjusted to 6.7-7.0 with hydrochloric acid or sodium hydroxide solution to obtain the second reagent;

[0078] Preparation of calibration samples: The first calibration sample and the second calibration sample were prepared according to conventional methods. The heparin binding protein concentration of the first sample was 20-50 ng / mL, and the heparin binding protein concentration of the second calibration sample was 150-300 ng / mL.

[0079] Preferably, the blank latex microspheres for preparing latex microspheres B1 and B4 include latex microspheres without surface water. First, the blank latex microspheres are immersed in ultrapure water for 90-110 minutes; then filtered and excess water is absorbed with filter paper; spread into a thin layer with a thickness of 0.4-0.6 mm, and placed in a sterile environment at 22-30° C. and a relative humidity of 55%-65% for 10-20 minutes to obtain latex microspheres without surface water.

[0080] Preferably, the surface of the titanium-coated ruthenium electrode plate is coated with an insulating layer, the insulating layer comprises one of polyethylene, polypropylene and polyamide, and the thickness of the insulating layer is 10-30 nm.

[0081] Preferably, the hydrophilic treated glass plate comprises a glass plate treated by plasma cleaning.

[0082] Preferably, the relative humidity of the environment in which the latex microspheres B1 and B4 are prepared is 86%-92%.

[0083] It should be noted that the method for preparing monoclonal antibodies is a method of the prior art.

[0084] Further preferably, the microfluidic device for preparing liposomes is a microfluidic device of prior art.

[0085] In a specific embodiment, the application process of the heparin binding protein detection kit is as follows:

[0086] (1) Take fresh blood and prepare a sample liquid;

[0087] (2) adding 10-20 μl of the sample liquid to 120-300 μl of the first reagent, mixing well, and incubating at 35-38° C. for 3-10 minutes to obtain a mixed solution;

[0088] (3) Take 100-200 μl of the second reagent, add it to the mixed solution, mix well, and incubate for 3-7 minutes to obtain a second reagent mixed solution;

[0089] (4) Read the absorbance of the mixed solution after adding the second reagent at a wavelength of 600 nm, recorded as A;

[0090] (5) Using the first calibrator, a first working curve with a slope of k1 is obtained, and the maximum absorbance value corresponding to the first working curve is recorded as A0. Using the second calibrator, a second working curve with a slope of k2 is obtained.

[0091] (6) When A<A0, the first working curve is used to calculate the result; when A>A0, the first working curve and the second working curve are used to calculate the result.

[0092] In order to further understand the present invention, the technical solution of the present invention is described below in conjunction with embodiments, but it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the present invention.

[0093] Example 1

[0094] The heparin binding protein detection kit of the present invention is prepared according to the following steps and conditions.

[0095] Preparation of latex microspheres B1: Connect a titanium-coated ruthenium electrode plate with a polyethylene insulating layer of 10 nm thickness to the positive pole of a DC power supply with a power supply voltage of 20 volts, so that the titanium-coated ruthenium electrode plate carries a positive static charge; immerse blank latex microspheres with a diameter of 200 nm in ultrapure water for 90 minutes; then filter and absorb excess water with filter paper; spread into a 0.5 mm thick layer, place in a sterile environment at 22°C and a relative humidity of 60% for 10 minutes to obtain latex microspheres without surface water, and then contact with a rubber plate with a negative static charge, the static voltage of the rubber plate is 180 volts, so that the blank latex microspheres carry a negative charge; use the positively charged titanium-coated ruthenium electrode plate to contact the negatively charged blank latex microspheres, so that The latex microspheres are adsorbed onto a titanium-coated ruthenium electrode plate; a low-melting point agarose gel with a mass concentration of 1.3% is prepared, heated to 36°C, and poured onto the titanium-coated ruthenium electrode plate on which blank latex microspheres are adsorbed, the liquid level of the low-melting point agarose gel is 65% of the diameter of the latex microspheres, the temperature is reduced to below 30°C, and solidified for 5 hours to obtain a titanium-coated ruthenium electrode plate on which latex microspheres are fixed; a reaction solution of latex microspheres coupled with heparin-binding protein antibodies is prepared, and the reaction solution comprises 2.4 g / L MES·H2O, 1.9 g / L boric acid, 0.7 g / L disodium hydrogen phosphate, 12 mg / mL mouse anti-human heparin-binding protein monoclonal antibody, 0.18 g / L EDC·HCI solution, and NH S solution 0.4g / L, the reaction solution of latex microspheres coupled with heparin binding protein antibody is coated on the hydrophilic treated glass plate, and an antibody coupling reaction liquid film is formed on the surface of the glass plate, and the height of the liquid film is 30% of the diameter of the latex microspheres, thereby obtaining a glass plate coated with the antibody coupling reaction liquid; the titanium-coated ruthenium electrode plate fixed with latex microspheres is inverted on the glass plate coated with the antibody coupling reaction liquid, so that the exposed part of the latex microspheres is in contact with the coupling reaction liquid, and the antibody coupling reaction is carried out for 18 hours at a reaction temperature of 22°C; the titanium-coated ruthenium electrode plate fixed with latex microspheres is separated from the glass plate coated with the antibody coupling reaction liquid, and a blocking solution containing 110mL / L of Tween and 1.9g / L of BSA is coated on the solid On the titanium-coated ruthenium electrode plate with latex microspheres fixed thereon, the exposed part of the latex microspheres is immersed in a blocking solution, and a blocking reaction is carried out at 29°C for 4 hours; proclin300 is added to the blocking solution so that its concentration in the system reaches 0.5 mL / L, and the pH is adjusted to 6.8 using a sodium hydroxide solution or hydrochloric acid, and the solution is soaked for 12 hours; the titanium-coated ruthenium electrode plate with latex microspheres fixed thereon is immersed in a phosphate buffer solution at a temperature of 39°C and a pH of 6.75 to liquefy the solidified low-melting point agarose gel and release the coupled latex microspheres, and the material containing the latex microspheres is centrifuged while hot for solid-liquid separation, and washed three times with a phosphate buffer solution at a pH of 6.9 to obtain latex microspheres B1;

[0096] Preparation of latex microspheres B2: blank latex microspheres with a particle size of 140 nm were coupled with anion exchange groups according to conventional methods, and then MES·H2O 2.3 g / L was added to a buffer solution adjusted to pH 6.7 with sodium hydroxide, and mixed evenly, and then 2.7 g / L boric acid and 0.6 g / L disodium hydrogen phosphate were added to a buffer solution adjusted to pH 7.2 with sodium hydroxide, and mixed at 20°C to obtain a latex microsphere suspension; then mouse anti-human heparin binding protein monoclonal antibody was added to the latex microsphere suspension, and the concentration of mouse anti-human heparin binding protein monoclonal antibody in the reaction system was 10 mg / mL, and then 0.1 g / L EDC·HCI solution and 0.2 g / L NHS solution were added, and the coupling reaction was stirred at 35°C for 8.5 h; Tween 20 0mL / L and BSA1.3g / L blocking solution, stirred and dispersed at 31°C, and carried out blocking reaction for 4h; proclin300 was added to make its concentration in the system reach 0.4mL / L, and the pH was adjusted to 7.4 with sodium hydroxide solution or hydrochloric acid, and stirred and soaked for 14h to obtain latex microspheres B2;

[0097] Preparation of latex microspheres B3: The particle size of blank latex microspheres is 200 nm, and the surface thereof does not need to be coupled with anion exchange groups. The monoclonal antibody loaded on the surface is mouse anti-rabbit anti-human heparin binding protein monoclonal antibody-monoclonal antibody. The other steps are the same as those for preparing latex microspheres B2 to obtain latex microspheres B3;

[0098] Preparation of latex microspheres B4: the particle size of blank latex microspheres is 150 nm, the monoclonal antibody loaded on the surface is mouse anti-rabbit anti-human heparin binding protein monoclonal antibody-monoclonal antibody, and the other steps are the same as those for preparing latex microspheres B1 to obtain latex microspheres B4;

[0099] Preparation of liposomes: dissolving 7 parts by weight of phosphatidylglycerol and 1.0 parts by weight of cholesterol in 50 parts by weight of 83% ethanol aqueous solution to obtain a liposome membrane preparation solution; dissolving 0.6 parts by weight of rabbit anti-human heparin binding protein monoclonal antibody and 0.4 parts by weight of potassium chloride in 90 parts by weight of 110 mmol / L glycine buffer to obtain a liposome-containing solution; passing the liposome membrane preparation solution and the liposome-containing solution into a microfluidic device, and preparing the liposome with a diameter of 30 nm according to conventional techniques;

[0100] Prepare the first reagent: prepare the volume of the first reagent as needed, calculate and weigh the ingredients according to the following ratio: rheumatoid factor antibody 22 mg / L, polyethylene glycol 750 mg / L, Tween 60, 20 mmol / L, sodium bromide 6 g / L, sodium azide 34 mg / L, glycine buffer 120 mmol / L, dissolve in the corresponding volume of deionized water, adjust the pH value to 6.6 with hydrochloric acid or sodium hydroxide solution, and obtain the first reagent;

[0101] Prepare the second reagent: prepare the volume of the second reagent as needed, calculate and weigh the ingredients according to the following ratio, the mass ratio of latex microspheres B1 and B2 is 20:1, the mass ratio of latex microspheres B3 and B4 is 1:6., the sum of the mass concentrations of latex microspheres B1 and B2 in the second reagent is 0.38%, the sum of the mass concentrations of latex microspheres B3 and B4 in the second reagent is 0.82%, the mass concentration of liposomes in the second reagent is 0.75%, polypropylene glycol 660 mg / L, lauroyl diethanolamine mmol / L, sodium azide 30 mg / L, the concentration of medium and long chain dibasic acid in the second reagent is 17 mmol / L, dissolve in a corresponding volume of deionized water, adjust the pH value to 6.7 with hydrochloric acid or sodium hydroxide solution, and obtain the second reagent;

[0102] Preparation of calibration samples: The first calibration sample and the second calibration sample were prepared according to conventional methods. The heparin binding protein concentration of the first sample was 20 ng / mL, and the heparin binding protein concentration of the second calibration sample was 150 ng / mL.

[0103] Example 2

[0104] Preparation of latex microspheres B1: Connect a titanium-coated ruthenium electrode plate with a polypropylene insulating layer of 30 nm thickness to the positive pole of a DC power supply with a power supply voltage of 40 volts, so that the titanium-coated ruthenium electrode plate carries a positive static charge; immerse blank latex microspheres with a diameter of 250 nm in ultrapure water for 100 minutes; then filter and remove excess water with filter paper; spread into a 0.4 mm thick layer, place in a sterile environment at 30°C and a relative humidity of 55% for 20 minutes to obtain latex microspheres without surface water, and then contact with a rubber plate with a negative static charge with an electrostatic voltage of 150 volts to make the blank latex microspheres The microspheres are negatively charged; a positively charged titanium-coated ruthenium electrode plate is used to contact a negatively charged blank latex microsphere, so that the latex microspheres are adsorbed onto the titanium-coated ruthenium electrode plate; a low-melting point agarose gel with a mass concentration of 1.6% is prepared, heated to 37°C, and poured onto the titanium-coated ruthenium electrode plate adsorbing the blank latex microspheres, the liquid level of the low-melting point agarose gel is 60% of the diameter of the latex microspheres, the temperature is reduced to below 30°C, and solidified for 5.5 hours to obtain a titanium-coated ruthenium electrode plate with latex microspheres fixed thereon; a reaction solution of latex microspheres coupled with heparin-binding protein antibodies is prepared, and the reaction solution composition is MES·H2O 5.1g / L, boric acid 1.7g / L, disodium hydrogen phosphate 0.8g / L, mouse anti-human heparin binding protein monoclonal antibody 26mg / mL, EDC·HCI solution 0.12g / L, NHS solution 0.45g / L, the reaction solution of latex microspheres coupling heparin binding protein antibody is coated on the glass plate after hydrophilic treatment, and an antibody coupling reaction liquid film is formed on the surface of the glass plate, and the height of the liquid film is 25% of the diameter of the latex microspheres, so as to obtain a glass plate coated with the antibody coupling reaction liquid; the titanium-coated ruthenium electrode plate fixed with latex microspheres is inverted on the glass plate coated with the antibody coupling reaction liquid, so that the exposed part of the latex microspheres is in contact with the coupling reaction liquid, and the antibody coupling reaction is carried out for 20h at a reaction temperature of 24°C; the titanium-coated ruthenium electrode plate fixed with latex microspheres is separated from the glass plate coated with the antibody coupling reaction liquid, and Tween 20 is added to the titanium-coated ruthenium electrode plate fixed with latex microspheres. 0mL / L and 1.7g / L BSA blocking solution are coated on the titanium-coated ruthenium electrode plate fixed with latex microspheres, and the exposed part of the latex microspheres is immersed in the blocking solution, and the blocking reaction is carried out at 27°C for 6 hours; proclin300 is added to the blocking solution to make its concentration in the system reach 0.3mL / L, and the pH is adjusted to 7.0 with sodium hydroxide solution or hydrochloric acid, and soaked for 13 hours; the titanium-coated ruthenium electrode plate fixed with latex microspheres is immersed in a phosphate buffer at a temperature of 41°C and a pH of 6.6 to liquefy the solidified low-melting point agarose gel and release the coupled latex microspheres, and the material containing the latex microspheres is centrifuged while hot for solid-liquid separation, and washed 4 times with a pH of 7.0 phosphate buffer to obtain latex microspheres B1;

[0105] Preparation of latex microspheres B2: Blank latex microspheres with a particle size of 130 nm were coupled with anion exchange groups according to conventional methods, and then MES·H2O was added. 3.6g / L, use sodium hydroxide to adjust the pH to 6.8 in a buffer, mix well, add 2.4g / L boric acid and 0.9g / L disodium hydrogen phosphate, use sodium hydroxide to adjust the pH to 7.0 in a buffer, mix at 25°C to obtain a latex microsphere suspension; then add mouse anti-human heparin binding protein monoclonal antibody to the latex microsphere suspension, the concentration of mouse anti-human heparin binding protein monoclonal antibody in the reaction system is 8mg / mL, then add 0.3g / L EDC·HCl solution and 0.4g / L NHS solution, stir at 40°C for coupling reaction, the reaction time is 7h; add blocking solution composed of 175mL / L Tween and 1.7g / L BSA, stir and disperse at 29°C, and perform a blocking reaction for 6h; add proclin300 to make its concentration in the system reach 0.8mL / L, and use sodium hydroxide solution or hydrochloric acid to adjust the pH to 7.3, stir and soak for 12h to obtain latex microsphere B2;

[0106] Preparation of latex microspheres B3: The particle size of blank latex microspheres is 230 nm, and anion exchange groups do not need to be coupled to the surface thereof. The monoclonal antibody loaded on the surface is mouse anti-rabbit anti-human heparin binding protein monoclonal antibody-monoclonal antibody. The other steps are the same as those for preparing latex microspheres B2 to obtain latex microspheres B3;

[0107] Preparation of latex microspheres B4: the particle size of blank latex microspheres is 200 nm, the monoclonal antibody loaded on the surface is mouse anti-rabbit anti-human heparin binding protein monoclonal antibody-monoclonal antibody, and the other steps are the same as those for preparing latex microspheres B1 to obtain latex microspheres B4;

[0108] Preparation of liposomes: dissolving 9 parts by weight of phosphatidylglycerol and 0.7 parts by weight of cholesterol in 55 parts by weight of 80% ethanol aqueous solution to obtain a liposome membrane preparation solution; dissolving 0.8 parts by weight of rabbit anti-human heparin binding protein monoclonal antibody and 0.3 parts by weight of potassium sulfate in 110 parts by weight of 100 mmol / L glycine buffer to obtain a liposome-containing solution; passing the liposome membrane preparation solution and the liposome-containing solution into a microfluidic device, and preparing the liposomes with a diameter of 45 nm according to conventional techniques;

[0109] Prepare the first reagent: prepare the volume of the first reagent as needed, calculate and weigh the ingredients according to the following ratio: 43 mg / L anti-rheumatoid factor antibody, 530 mg / L polypropylene glycol, 26 mmol / L lauroyl diethanolamine, 4 g / L sodium chloride, 56 mg / L sodium azide, 75 mmol / L phosphate buffer, dissolve in the corresponding volume of deionized water, adjust the pH value to 6.7 with hydrochloric acid or sodium hydroxide solution, and obtain the first reagent;

[0110] Prepare the second reagent: prepare the volume of the second reagent as needed, calculate and weigh the ingredients according to the following ratio, the mass ratio of latex microspheres B1 and B2 is 48:1, the mass ratio of latex microspheres B3 and B4 is 1:6, the sum of the mass concentrations of latex microspheres B1 and B2 in the second reagent is 0.6%, the sum of the mass concentrations of latex microspheres B3 and B4 in the second reagent is 1.46%, the mass concentration of liposomes in the second reagent is 0.5%, alkyl glycoside 1200 mg / L, Tween 60, 18 mmol / L, gentamicin 50 mg / L, the concentration of medium-chain dibasic acid in the second reagent is 13 mmol / L, dissolve in a corresponding volume of deionized water, adjust the pH to 6.9 with hydrochloric acid or sodium hydroxide solution, and obtain the second reagent;

[0111] Preparation of calibration samples: The first calibration sample and the second calibration sample were prepared according to conventional methods. The heparin-binding protein concentration of the first sample was 35 ng / mL, and the heparin-binding protein concentration of the second calibration sample was 300 ng / mL.

[0112] Example 3

[0113] Preparation of latex microspheres B1: Connect a titanium-coated ruthenium electrode plate with a polyamide insulating layer of 15 nm thickness to the positive pole of a DC power supply with a power supply voltage of 30 volts, so that the titanium-coated ruthenium electrode plate carries a positive charge static electricity; immerse blank latex microspheres with a diameter of 300 nm in ultrapure water for 110 minutes; then filter and absorb excess water with filter paper; spread into a 0.6 mm thick thin layer, place in a sterile environment at 26°C and a relative humidity of 65% for 15 minutes to obtain latex microspheres without surface water, and then contact with a rubber plate with a negative charge static electricity with an electrostatic voltage of 220 volts to make the blank latex microspheres The latex microspheres are negatively charged; a positively charged titanium-coated ruthenium electrode plate is used to contact the negatively charged blank latex microspheres, so that the latex microspheres are adsorbed on the titanium-coated ruthenium electrode plate; a low-melting point agarose gel with a mass concentration of 1.45% is prepared, heated to 38°C, and poured on the titanium-coated ruthenium electrode plate adsorbing the blank latex microspheres, the liquid level of the low-melting point agarose gel is 70% of the diameter of the latex microspheres, the temperature is reduced to below 30°C, and solidified for 6 hours to obtain a titanium-coated ruthenium electrode plate with latex microspheres fixed thereon; a reaction solution of latex microspheres coupled with heparin-binding protein antibodies is prepared, and the reaction solution composition is MES·H2O 3.6g / L, boric acid 2.1g / L, disodium hydrogen phosphate 0.75g / L, mouse anti-human heparin binding protein monoclonal antibody 22mg / mL, EDC·HCI solution 0.25g / L, NHS solution 0.5g / L, the reaction solution of latex microspheres coupling heparin binding protein antibody is coated on the glass plate after hydrophilic treatment, and an antibody coupling reaction liquid film is formed on the surface of the glass plate, and the height of the liquid film is 35% of the diameter of the latex microspheres, so as to obtain a glass plate coated with the antibody coupling reaction liquid; the titanium-coated ruthenium electrode plate fixed with latex microspheres is inverted on the glass plate coated with the antibody coupling reaction liquid, so that the exposed part of the latex microspheres is in contact with the coupling reaction liquid, and the antibody coupling reaction is carried out for 19h at a reaction temperature of 26°C; the titanium-coated ruthenium electrode plate fixed with latex microspheres and the glass plate coated with the antibody coupling reaction liquid are placed in a vacuum chamber ... The glass plates were separated, and a blocking solution containing 150 mL / L of Tween and 2.1 g / L of BSA was applied to the titanium-coated ruthenium electrode plate fixed with latex microspheres, and the exposed part of the latex microspheres was immersed in the blocking solution, and a blocking reaction was carried out at 32°C for 5 hours; proclin300 was added to the blocking solution to make its concentration in the system reach 0.8 mL / L, and the pH was adjusted to 6.9 with sodium hydroxide solution or hydrochloric acid, and soaked for 15 hours; the titanium-coated ruthenium electrode plate fixed with latex microspheres was immersed in a phosphate buffer at a temperature of 40°C and a pH of 6.9 to liquefy the solidified low-melting point agarose gel and release the coupled latex microspheres, and the material containing the latex microspheres was centrifuged while hot for solid-liquid separation, and washed three times with a phosphate buffer at a pH of 7.2 to obtain latex microspheres B1;

[0114] Preparation of latex microspheres B2: blank latex microspheres with a particle size of 150 nm were coupled with anion exchange groups according to conventional methods, and then 4.9 g / L of MES·H2O was added to a buffer solution adjusted to pH 6.75 with sodium hydroxide, and after mixing evenly, 3.0 g / L of boric acid and 0.75 g / L of disodium hydrogen phosphate were added to a buffer solution adjusted to pH 7.3 with sodium hydroxide, and mixed at 23°C to obtain a latex microsphere suspension; then mouse anti-human heparin binding protein monoclonal antibody was added to the latex microsphere suspension, and the concentration of mouse anti-human heparin binding protein monoclonal antibody in the reaction system was 8 mg / mL, and then 0.2 g / L of EDC·HCl solution and 0.6g / L NHS solution were stirred at 37°C for coupling reaction, and the reaction time was 10h; a blocking solution consisting of 150mL / L Tween and 1.5g / L BSA was added, and the mixture was stirred and dispersed at 33°C for 9h blocking reaction; proclin300 was added to a concentration of 0.6mL / L in the system, and the pH was adjusted to 7.6 using sodium hydroxide solution or hydrochloric acid, and the mixture was stirred and soaked for 15h to obtain latex microspheres B2;

[0115] Preparation of latex microspheres B3: The particle size of blank latex microspheres is 250 nm, and anion exchange groups do not need to be coupled to the surface thereof. The monoclonal antibody loaded on the surface is mouse anti-rabbit anti-human heparin binding protein monoclonal antibody-monoclonal antibody. The other steps are the same as those for preparing latex microspheres B2 to obtain latex microspheres B3;

[0116] Preparation of latex microspheres B4: The particle size of the blank latex microspheres is 180 nm, the monoclonal antibody loaded on the surface is mouse anti-rabbit anti-human heparin binding protein monoclonal antibody-monoclonal antibody, and the other steps are the same as those for preparing latex microspheres B1 to obtain latex microspheres B4;

[0117] Preparation of liposomes: dissolving 10 parts by weight of phosphatidylglycerol and 1.2 parts by weight of cholesterol in 53 parts by weight of 86% ethanol aqueous solution to obtain a liposome membrane preparation solution; dissolving 1 part by weight of rabbit anti-human heparin binding protein monoclonal antibody and 0.5 parts by weight of potassium chloride in 100 parts by weight of 120 mmol / L glycine buffer to obtain a liposome-containing solution; passing the liposome membrane preparation solution and the liposome-containing solution into a microfluidic device, and preparing the liposome with a diameter of 60 nm according to conventional techniques;

[0118] Prepare the first reagent: prepare the volume of the first reagent as needed, calculate and weigh the ingredients according to the following ratio: 64 mg / L anti-rheumatoid factor antibody, 1000 mg / L oligoxylan, 23 mmol / L lauroyl diethanolamine, 8 g / L sodium chloride, 45 mg / L gentamicin, 160 mmol / L glycine buffer, dissolve in the corresponding volume of deionized water, adjust the pH value to 6.8 with hydrochloric acid or sodium hydroxide solution, and obtain the first reagent;

[0119] Prepare the second reagent: prepare the volume of the second reagent as needed, calculate and weigh the ingredients according to the following ratio, the mass ratio of latex microspheres B1 and B2 is 34:1, the mass ratio of latex microspheres B3 and B4 is 1:7, the sum of the mass concentrations of latex microspheres B1 and B2 in the second reagent is 0.77%, the sum of the mass concentrations of latex microspheres B3 and B4 in the second reagent is 1.1%, the mass concentration of liposomes in the second reagent is 1%, oligodextran 900 mg / L, lauroyl diethanolamine 25 mmol / L, sodium azide 40 mg / L, the concentration of medium-chain dibasic acid in the second reagent is 21 mmol / L, dissolve in a corresponding volume of deionized water, adjust the pH to 7.0 with hydrochloric acid or sodium hydroxide solution, and obtain the second reagent;

[0120] Preparation of calibration samples: The first calibration sample and the second calibration sample were prepared according to conventional methods. The heparin binding protein concentration of the first sample was 50 ng / mL, and the heparin binding protein concentration of the second calibration sample was 230 ng / mL. Comparative Example 1-Setting the second reagent of the prior art, the second reagent does not contain latex microspheres B1, B3, B4, and liposomes

[0121] The preparation method of this comparative example does not include the steps of preparing latex microspheres B1, B3, B4, and liposomes. The second reagent does not include latex microspheres B1, B3, B4, and liposomes, and the addition amount of latex microspheres B2 is 2%. The first calibrator is not provided. The other parameters are the same as those in Example 2. The detection kit of this comparative example is obtained.

[0122] The heparin binding protein detection kit obtained in Examples 1-3 was tested and compared using the following detection method: (1) fresh blood was taken to prepare a sample liquid; (2) 15 μL of the sample liquid was added to 200 μL of the first reagent, mixed evenly, and incubated at 35-38° C. for 5 minutes to obtain a mixed solution; (3) 200 μL of the second reagent was taken, added to the mixed solution, mixed evenly, and incubated for 4 minutes to obtain a second reagent mixed solution; (4) the absorbance value of the mixed solution after adding the second reagent was read at a wavelength of 600 nm, which was recorded as A; (5) a first working curve with a slope of k1 was obtained using the first calibrator, and the maximum absorbance value corresponding to the first working curve was recorded as A0, and a second working curve with a slope of k2 was obtained using the second calibrator; (6) when A<A0, the first working curve was used to calculate the result, and when A>A0, the first working curve and the second working curve were used to calculate the result.

[0123] The test steps (1) to (4) of the detection kit of comparative example 1 are the same as those of the above method; (5) 200 μl of the second reagent is taken, added to the mixed solution and mixed evenly, incubated at 37° C. for 5 minutes, and the absorbance A is read. The standard curve is used to calculate the concentration of heparin-binding protein in the sample to be tested.

[0124] A batch of samples to be tested was tested 9 times using the detection kit corresponding to each embodiment and comparative example.

[0125] The standard deviation of the test results of multiple tests for each embodiment and comparative example was statistically calculated, and the results showed that the standard deviation of the test results of embodiments 1-3 was between 1.5% and 2.8%, and the standard deviation of comparative example 1 was 8.1%, indicating that the addition of the latex microspheres B1-B4 combination and the liposome technology solution in the second reagent of the present invention can significantly improve the stability and accuracy of the detection kit of the present application.

Claims

1. A heparin binding protein detection kit, characterized in that: The heparin binding protein detection kit comprises a first reagent, a second reagent, a first calibration sample and a second calibration sample; The first reagent includes an anti-interference agent, a first protein stabilizer, a first surfactant, an inorganic salt, a first antibacterial preservative, and a buffer; The second reagent includes latex microsphere B1, latex microsphere B2, latex microsphere B3, latex microsphere B4, liposomes, a second protein stabilizer, a second surfactant, a second antibacterial preservative, and a medium-chain dibasic acid; The first calibration sample includes a standard solution with a heparin binding protein concentration of 20-50 ng / mL; The second calibration sample includes a standard solution with a heparin binding protein concentration of 150-300 ng / mL; The particle size of the latex microsphere B1 is 200-300 nm, the surface is loaded with mouse anti-human heparin binding protein monoclonal antibody, and the surface also has anion exchange groups. The latex microsphere B1 is a latex microsphere loaded with mouse anti-human heparin binding protein monoclonal antibody on one side; The particle size of the latex microsphere B2 is 130-150nm, and the surface is loaded with mouse anti-human heparin binding protein monoclonal antibody. The latex microsphere B2 is a latex microsphere loaded with mouse anti-human heparin binding protein monoclonal antibody on the entire surface; The particle size of the latex microsphere B3 is 200-250nm, and the surface is loaded with mouse anti-rabbit anti-human heparin binding protein monoclonal antibody-monoclonal antibody. The latex microsphere B3 is a latex microsphere loaded with mouse anti-rabbit anti-human heparin binding protein monoclonal antibody-monoclonal antibody on the entire surface; The particle size of the latex microsphere B4 is 150-200 nm, and the surface is loaded with mouse anti-rabbit anti-human heparin binding protein monoclonal antibody-monoclonal antibody. The latex microsphere B4 is a latex microsphere loaded with mouse anti-rabbit anti-human heparin binding protein monoclonal antibody-monoclonal antibody on one side; The mass ratio of the latex microspheres B1 to B2 is (20-48):1, the mass ratio of the latex microspheres B3 to B4 is 1:(6-7), the sum of the mass concentrations of the latex microspheres B1 and B2 in the second reagent is 0.38%-0.77%, and the sum of the mass concentrations of the latex microspheres B3 and B4 in the second reagent is 0.82%-1.46%; The membrane of the liposome is composed of phosphatidylglycerol and cholesterol, and contains rabbit anti-human heparin binding protein monoclonal antibody and anion replacement agent. The diameter of the liposome is 30-60nm; The pH of the first reagent is 6.6-6.8, and the pH of the second reagent is 6.7-7.

0.

2. The heparin binding protein detection kit according to claim 1, characterized in that: The anion exchange groups on the latex microspheres B1 include primary amine groups and secondary amine groups, the molar ratio of the primary amine to the secondary amine is 5:(1-4), the exchange capacity on the latex microspheres B1 is 0.1-0.4 mmol / g, and the exchangeable groups on the anion exchange groups are hydroxides.

3. The heparin binding protein detection kit according to claim 1, characterized in that: The medium-chain dibasic acid includes one of dibasic acids with 7 to 9 carbon atoms, and the concentration is 13 to 21 mmol / L.

4. The heparin binding protein detection kit according to claim 1, characterized in that: The latex microspheres B1, B2, B3, and B4 include carboxyl polystyrene latex microspheres.

5. The heparin binding protein detection kit according to claim 1, characterized in that: The anti-interference agent includes at least one of anti-rheumatic factor antibody and anti-rheumatoid factor antibody; The first protein stabilizer and the second protein stabilizer include one of polyethylene glycol, polypropylene glycol, alkyl glycoside, oligoglucan, and oligoxylan; The first surfactant and the second surfactant include one of Tween 60 and lauroyl diethanolamine; The inorganic salt includes one of sodium bromide and sodium chloride; The first antimicrobial preservative and the second antimicrobial preservative include one of sodium azide and gentamicin; The anion replacement agent includes one of potassium chloride and potassium sulfate; The buffer comprises one of a glycine buffer and a phosphate buffer.

6. A method for preparing the heparin binding protein detection kit according to any one of claims 1 to 5, characterized in that: The preparation method comprises the following steps: Preparation of latex microspheres B1: connecting a titanium-coated ruthenium electrode plate to the positive pole of a DC power supply, the power supply voltage is 20-40 volts, so that the titanium-coated ruthenium electrode plate carries a positive static charge; contacting blank latex microspheres with a diameter of 200-300 nm with a rubber plate with a negative static charge, the static voltage of the rubber plate is 150-220 volts, so that the blank latex microspheres carry a negative charge; using the positively charged titanium-coated ruthenium electrode plate to contact the negatively charged blank latex microspheres, so that the latex microspheres are adsorbed to the titanium-coated ruthenium electrode plate. The method comprises the following steps: preparing a low melting point agarose gel with a mass concentration of 1.3% to 1.6%, heating the gel to 36 to 38° C., and pouring the gel on the titanium-coated ruthenium electrode plate on which the blank latex microspheres are adsorbed, wherein the liquid level of the low melting point agarose gel is 60% to 70% of the diameter of the latex microspheres, and the temperature is lowered to below 30° C. and solidified for 5 to 6 hours to obtain a titanium-coated ruthenium electrode plate on which the latex microspheres are fixed; preparing a reaction solution for coupling the latex microspheres with the heparin binding protein antibody, wherein the reaction solution is composed of MES·H2O 2.4-5.1g / L, boric acid 1.7-2.1g / L, disodium hydrogen phosphate 0.7-0.8g / L, the mouse anti-human heparin binding protein monoclonal antibody 12-26mg / mL, EDC·HCI solution 0.12-0.25g / L, NHS solution 0.4-0.5g / L, the reaction solution of the latex microspheres coupled with the heparin binding protein antibody is coated on a hydrophilic treated glass plate to form an antibody coupling reaction solution film on the surface thereof. , the height of the liquid film is 25%-35% of the diameter of the latex microspheres, and a glass plate coated with an antibody coupling reaction solution is obtained; the titanium-coated ruthenium electrode plate with the latex microspheres fixed is turned upside down on the glass plate coated with the antibody coupling reaction solution, so that the exposed part of the latex microspheres is in contact with the coupling reaction solution, and the antibody coupling reaction is carried out for 18-20 hours, and the reaction temperature is 22-26°C; the titanium-coated ruthenium electrode plate with the latex microspheres fixed is separated from the glass plate coated with the antibody coupling reaction solution, and a blocking solution containing Tween 110-200mL / L and BSA 1.7-2.1g / L is applied to the titanium-coated ruthenium electrode plate with the latex microspheres fixed, so that the exposed part of the latex microspheres is immersed in the blocking solution, and a blocking reaction is carried out at 27-32°C for 4-6h; proclin300 is added to the blocking solution to make its concentration in the system reach 0.3-0.8mL / L, and sodium hydroxide solution or hydrochloric acid is used to remove p H is adjusted to 6.8-7.0, and soaked for 12-15 hours; the titanium-coated ruthenium electrode plate fixed with latex microspheres is immersed in a phosphate buffer at a temperature of 39-41°C and a pH of 6.6-6.9 to liquefy the solidified low-melting point agarose gel and release the coupled latex microspheres, and the material containing the latex microspheres is centrifuged while hot for solid-liquid separation, and washed 3-4 times with the phosphate buffer at a pH of 6.9-7.2 to obtain the latex microspheres B1; Preparation of latex microspheres B2: blank latex microspheres with a particle size of 130-150 nm are coupled with anion exchange groups according to a conventional method, and then MES·H2O 2.3-4.9 g / L is added, and the pH is adjusted to 6.7-6.8 with sodium hydroxide. After mixing evenly, 2.4-3.0 g / L of boric acid and 0.6-0.9 g / L of disodium hydrogen phosphate are added, and the pH is adjusted to 7.0-7.3 with sodium hydroxide. The mixture is mixed at 20-25° C. to obtain a latex microsphere suspension; then the mouse anti-human heparin binding protein monoclonal antibody is added to the latex microsphere suspension, and the concentration of the mouse anti-human heparin binding protein monoclonal antibody in the reaction system is 8-20 mg / mL, and 0.1-0.3 g / L of EDC·HCl solution and 0.2-0.6g / L NHS solution are stirred at 35-40°C for coupling reaction, and the reaction time is 7-10h; a blocking solution composed of 700-150mL / L Tween and 1.3-1.7g / L BSA is added, and the mixture is stirred and dispersed at 29-33°C for 4-9h blocking reaction; proclin300 is added to a concentration of 0.4-0.8mL / L in the system, and the pH is adjusted to 7.3-7.6 using sodium hydroxide solution or hydrochloric acid, and the mixture is stirred and soaked for 12-15h to obtain the latex microspheres B2; Preparation of latex microspheres B3: The particle size of blank latex microspheres is 200-250 nm, and the surface thereof does not need to be coupled with anion exchange groups. The monoclonal antibody loaded on the surface is mouse anti-rabbit anti-human heparin binding protein monoclonal antibody-monoclonal antibody. The other steps are the same as those for preparing latex microspheres B2 to obtain the latex microspheres B3; Preparation of latex microspheres B4: the particle size of blank latex microspheres is 150-200 nm, the monoclonal antibody loaded on the surface is mouse anti-rabbit anti-human heparin binding protein monoclonal antibody-monoclonal antibody, and the other steps are the same as those for preparing latex microspheres B1 to obtain the latex microspheres B4; Preparation of liposomes: dissolving 7-10 parts by weight of phosphatidylglycerol and 0.7-1.2 parts by weight of cholesterol in 50-55 parts by weight of 80%-86% ethanol aqueous solution to obtain a liposome membrane preparation solution; dissolving 0.6-1 parts by weight of rabbit anti-human heparin binding protein monoclonal antibody and 0.3-0.5 parts by weight of anion replacement agent in 90-110 parts by weight of 100-120 mmol / L glycine buffer to obtain a liposome-containing solution; passing the liposome membrane preparation solution and the liposome-containing solution into a microfluidic device, and preparing the liposomes with a diameter of 30-60 nm according to conventional techniques; Prepare the first reagent: prepare the volume of the first reagent as needed, calculate and weigh the ingredients according to the following ratio: 22-64 mg / L of the anti-interference agent, 530-1000 mg / L of the first protein stabilizer, 20-26 mmol / L of the first surfactant, 4-8 g / L of the inorganic salt, 34-56 mg / L of the first antibacterial preservative, and 75-160 mmol / L of the buffer solution, dissolve them in a corresponding volume of deionized water, and adjust the pH value to 6.6-6.8 with hydrochloric acid or sodium hydroxide solution to obtain the first reagent; Prepare the second reagent: prepare the volume of the second reagent as needed, calculate and weigh the ingredients according to the following ratio, the mass ratio of the latex microspheres B1 and B2 is (20-48):1, the mass ratio of the latex microspheres B3 and B4 is 1:(6-7), the sum of the mass concentrations of the latex microspheres B1 and B2 in the second reagent is 0.38%-0.77%, the sum of the mass concentrations of the latex microspheres B3 and B4 in the second reagent is 0.82%-1.46%, the mass concentration of the liposomes in the second reagent is 0.5%-1%, the second protein stabilizer is 660-1200 mg / L, the second surfactant is 18-25 mmol / L, the second antibacterial preservative is 30-50 mg / L, the concentration of the medium-chain dibasic acid in the second reagent is 13-21 mmol / L, and they are dissolved in a corresponding volume of deionized water, and the pH value is adjusted to 6.7-7.0 with hydrochloric acid or sodium hydroxide solution to obtain the second reagent; Preparation of calibration samples: The first calibration sample and the second calibration sample are prepared according to conventional methods. The heparin binding protein concentration of the first sample is 20-50 ng / mL, and the heparin binding protein concentration of the second calibration sample is 150-300 ng / mL.

7. The method for preparing the heparin binding protein detection kit according to claim 6, characterized in that: The blank latex microspheres for preparing the latex microspheres B1 and B4 include latex microspheres without surface water. The blank latex microspheres are first immersed in ultrapure water for 90-110 minutes; then filtered and excess water is absorbed with filter paper; spread into a thin layer with a thickness of 0.4-0.6 mm, and placed in a sterile environment at 22-30° C. and a relative humidity of 55%-65% for 10-20 minutes to obtain the latex microspheres without surface water.

8. The method for preparing the heparin binding protein detection kit according to claim 6, characterized in that: The surface of the titanium-coated ruthenium electrode plate is coated with an insulating layer, the insulating layer comprises one of polyethylene, polypropylene and polyamide, and the thickness of the insulating layer is 10-30 nm.

9. The method for preparing the heparin binding protein detection kit according to claim 6, characterized in that: The hydrophilic treated glass plate includes a glass plate treated by plasma cleaning.

10. The method for preparing the heparin binding protein detection kit according to claim 6, characterized in that: The relative humidity of the environment in which the latex microspheres B1 and B4 are prepared is 86%-92%.