A high-hook capability, anti-interference HBP detection kit and a preparation method thereof

Abstract

The present application relates to the technical field of medical detection, and particularly relates to a high-hook-capability and anti-interference HBP detection kit, which comprises R1 reagent and R2 reagent; 3-[3-(cholylamido propyl) dimethylamino] propanesulfonic acid inner salt and sodium dodecyl sulfate are used to prepare the reagent R1, and by adjusting the use ratio of the two, the mutual cooperation of the two can ensure the reaction sensitivity of the reagent and improve the anti-interference performance of the reagent; two kinds of latex microspheres with different particle sizes are used to coat two kinds of HBP antibodies with different antigen recognition epitopes in the reagent R2, which can not only ensure that the reagent has a wider detection range in the high value area and improve the hook capability, but also ensure the sensitivity of the kit; the activation system adopts the method of combining two activators of NHS and EDC, which can effectively improve the coupling efficiency. Meanwhile, the present application also provides a preparation method of the high-hook-capability and anti-interference HBP detection kit, which does not need centrifugation and resuspension operation steps, and the method operation is simpler and the production cost is lower.

Description

Technical Field

[0001] This invention relates to the field of medical testing technology, specifically to an HBP detection kit with high hook capability and anti-interference properties. Background Technology

[0002] Heparin-binding protein (HBP) is an inactive serine protease homolog derived from neutrophils. It is named for its strong ability to bind heparin. HBP is a particulate glycoprotein with a relative molecular weight of 37,000, composed of 222 amino acids, and is also known as CAP37 / azurocidin. Due to its broad-spectrum antibacterial activity, chemotactic properties, and ability to promote vascular leakage, HBP has become an emerging biomarker for infectious diseases. HBP is particularly advantageous in the early diagnosis and monitoring of treatment efficacy in patients with sepsis; elevated HBP concentrations are associated with the severity of severe sepsis and septic shock. It also has some application value in other infectious diseases such as urinary tract infections, bacterial meningitis, and pulmonary infections, as well as in ICU monitoring, guiding antibiotic use, and prognostic evaluation. In normal individuals, the plasma HBP level is <11.4 ng / mL. A level of 20–30 ng / mL indicates a general infection, while a concentration exceeding 100 ng / mL indicates a severe infection.

[0003] Currently, the main immunoassay methods for heparin-binding protein (HBP) detection include latex-enhanced immunoturbidimetry (LETIA), enzyme-linked immunosorbent assay (ELISA), colloidal gold immunochromatography, and fluorescence immunochromatography. Among these, ELISA is inexpensive, uses simple instruments, and has high selectivity, making it suitable for on-site testing; however, it is cumbersome to operate, susceptible to external factors, has low sensitivity, a narrow linear range, and a long testing cycle. Accurate and rapid test results are required in the development of acute infections and sepsis, thus limiting the application of ELISA. Colloidal gold immunochromatography and fluorescence immunochromatography, as point-of-care testing (POCT) methods, have the advantages of small sample volumes and rapid processing; however, their quantification accuracy is not high enough, and their sensitivity is relatively poor, further limiting their application in clinical HBP detection.

[0004] Latex-enhanced immunoturbidimetric assay reagents can be operated fully automatically on biochemical analyzers with small systematic errors and good precision. However, existing reagents have weak hook capabilities, easily leading to false negatives when testing high-value samples, affecting diagnosis. The reagents also have poor anti-interference capabilities. Hook capability is a crucial performance indicator for reagent kits; when testing samples with excessively high concentrations, even if the kit cannot report the accurate target value, it should clearly indicate that the sample has exceeded the kit's detection limit to alert the user. Clinical samples contain many special types, such as lipid turbidity and hemolysis, which contain interfering substances that can easily produce false positives. Furthermore, anticoagulants and rheumatoid factor also significantly affect the results of latex-enhanced immunoturbidimetric assays. Therefore, the hook capability and anti-interference capabilities of existing reagent kits need further improvement. Summary of the Invention

[0005] To address the technical problems in existing technologies, the present invention aims to provide a high-hook, interference-resistant HBP detection kit. This kit uses latex immunoturbidimetry as the detection principle to determine the HBP content in the human body, and has advantages such as high hooking ability, good stability, strong interference resistance, and wide applicability. The present invention also provides its preparation method.

[0006] To achieve the above objectives, the technical solution provided by the present invention is as follows:

[0007] This invention provides an HBP detection kit with high hook capability and anti-interference, comprising reagent R1 and reagent R2;

[0008] The reagent R1 comprises the following components at the following concentrations: buffer solution: 20-60 mM / L; inorganic salt: 40-60 g / L; anti-interference agent: 10-20 g / L; polymerization accelerator: 3-15 g / L; wherein the pH value of the reagent R1 is 7.0-8.0; more preferably, the pH adjustment solution is a 6 mol / L hydrochloric acid solution, and when the pH value of the reagent R1 is adjusted to 7.0-8.0, it can provide a suitable buffer range for the reaction and achieve the best effect.

[0009] The reagent R2 comprises the following components at the following concentrations: latex microspheres 1: 0.5-1.2 mg / L; latex microspheres 2: 0.5-1.2 mg / L; activation system: 0.006-0.030 g / mL; buffer: 20-50 mM / L; HBP monoclonal antibody 1: 0.04-0.06 mg / mL; HBP monoclonal antibody 2: 0.04-0.06 mg / mL; blocking agent: 0.02-0.06 g / mL; preservative: 0.8-1.2 g / L; stabilizer: 0.04-0.08 g / mL.

[0010] It should be noted that the component concentration in reagent R1 is the final concentration of the component in reagent R1; the component concentration in reagent R2 is the final concentration of the component in reagent R2.

[0011] Based on the above technical solution, the anti-interference agent is prepared by mixing 3-[3-(cholephthalamide propyl)dimethylamino]propanesulfonic acid inner salt and sodium dodecyl sulfate in a mass ratio of 1:3 to 1:5. In reagent R1, the anti-interference agent utilizes the synergistic effect of 3-[3-(cholephthalamide propyl)dimethylamino]propanesulfonic acid inner salt and sodium dodecyl sulfate; preferably, they are prepared in a mass ratio of 1:3. By reasonably adjusting the ratio of the two, both the reagent's reaction sensitivity and its anti-interference performance can be guaranteed. Specifically, 3-[3-(cholephthalamide propyl)dimethylamino]propanesulfonic acid inner salt can maintain the native state of the protein, ensuring the stability of key components during detection, and can reduce the interference of competitive inhibition by reactant analogs (interfering proteins). However, its anti-interference ability is limited for samples with matrix effects (e.g., viscous, lipemic, chylous samples). Sodium dodecyl sulfate (CLS) is an anionic surfactant with excellent emulsifying and dispersing properties, enabling it to promote the uniform dispersion of particles in a sample within the medium. It exhibits exceptional anti-interference capabilities for viscous samples, lipemic samples, and other samples exhibiting matrix effects. Therefore, this invention improves the anti-interference performance of the reagent by using CHAPS and CLS in combination and adjusting their ratio appropriately.

[0012] Based on the above technical solution, the latex microspheres 1 have a particle size of 60-180 nm; the latex microspheres 2 have a particle size of 180-350 nm. In this invention, latex microspheres 1 with a particle size of 60-180 nm are used to coat HBP antibody 1. The relatively smaller particle size of latex microspheres 1 results in a larger number of carboxyl groups on their surface, enabling them to bind more antibodies and providing a significant advantage in high-value sample regions. This can improve the hook effect caused by an inappropriate antigen-antibody ratio. Lactus microspheres 2 with a particle size of 180-350 nm are used to coat HBP antibody 2. The relatively larger particle size of latex microspheres 2 can improve the sensitivity of the reagent. When the two are mixed in a certain ratio, both the hooking ability is improved, giving the reagent a wider detection range in the high-value region, while also ensuring the reagent's sensitivity.

[0013] It should be noted that Hook capability refers to the ability to resolve false negatives caused by inappropriate antigen-antibody ratios in existing technologies, or the tendency for false negatives to occur when testing high-value samples.

[0014] Based on the above technical solution, the activation system includes one or both of EDC and NHS, with the concentration of EDC being 0.006-0.008 g / mL and the concentration of NHS being 0.01-0.03 g / mL. The activation system in reagent R2 uses a combination of NHS and EDC as activators. The addition of NHS improves the coupling efficiency mediated by EDC, while the content of EDC directly affects the coupling efficiency. This invention improves the coupling efficiency between the antibody and latex microspheres through precise dosage of EDC and effectively prevents latex microsphere aggregation, making the detection kit more sensitive and stable. Specifically, in the activation system of this invention, EDC plays the main role in coupling. The dosage of EDC is precisely calculated based on the carboxyl group concentration on the microsphere surface, i.e., 2-3 mol of EDC is added for every 1 mol of carboxyl group. Since the content of EDC directly affects the coupling efficiency between the antibody and latex microspheres, excessive EDC can cause antibody coupling on adjacent microspheres, leading to microsphere aggregation or neutralization of the carboxyl groups on the microsphere surface, affecting reagent stability. More preferably, NHS is added to the activation system of the present invention, which can convert the carboxyl groups on the surface of latex microspheres into amine-reactive NHS esters, thereby improving the coupling efficiency mediated by EDC. Therefore, the reagent sensitivity and stability of the NHS / EDC activation system are higher. It should be noted that the NHS is N-hydroxysuccinimide, and the EDC is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0015] Based on the above technical solution, both HBP monoclonal antibody 1 and HBP monoclonal antibody 2 are mouse anti-human HBP antibodies, and their antigen recognition epitopes are different. In this invention, when preparing reagent R2, two monoclonal antibodies with different antigen recognition epitopes are used simultaneously. The two monoclonal antibodies label latex microspheres of different particle sizes, and more preferably, they are mixed in a 5:1 ratio, which can improve the hooking ability while ensuring the sensitivity of the reagent.

[0016] Based on the above technical solution, the buffer solutions in reagents R1 and R2 are one or more of Tris buffer, borate-borax buffer, HEPES buffer, and MES buffer.

[0017] Based on the above technical solution, the inorganic salt in reagent R1 is one or more of sodium chloride, calcium chloride, and magnesium chloride; the polymerization promoter is one or more of PEG6000, PEG8000, and PEG12000.

[0018] Based on the above technical solution, the blocking agent in reagent R2 is one or more of BSA, casein, and ethanolamine; the preservative is one or more of Proclin 300, sodium azide, and thimerosal; and the stabilizer is one or more of trehalose, sucrose, casein, gelatin, and EDTA-2Na.

[0019] This invention provides a method for preparing an HBP detection kit with high hooking capability and anti-interference, comprising the following steps:

[0020] Step 1, preparation of reagent R1: Dissolve the buffer solution in water according to the component content of reagent R1 and adjust the pH value of reagent R1 to 7.0-8.0; then add inorganic salt, anti-interference agent and polymerization accelerator in sequence and stir evenly. After stirring evenly, reagent R1 is obtained.

[0021] Step two, preparation of reagent R2;

[0022] (1) Preparation of solution 1: Take latex microspheres 1 and add them to an appropriate amount of pure water. Place them on a shaker at 37°C and shake at 200 r / min for 5 min. Then add the prepared activation system solution and activate the latex for 20 min. Then add the mixture of buffer solution and HBP monoclonal antibody 1 on a magnetic stirrer and couple for 2 h to obtain solution 1.

[0023] (2) Preparation of solution 2: Replace latex microspheres 1 and HBP monoclonal antibody 1 in step (1) with latex microspheres 2 and HBP monoclonal antibody 2, and prepare solution 2 using the same preparation method as in step (1);

[0024] (3) Take out the solutions 1 and 2 prepared above from the shaker, place them on the stirrer, add buffer solution, incubate at 37°C for 30 min, add blocking agent, and incubate for 1 h; finally add stabilizer and preservative, stabilize at 37°C overnight, and obtain antibody-loaded latex microsphere reagent 1 and antibody-loaded latex microsphere reagent 2 respectively.

[0025] (4) The antibody-loaded latex microsphere reagent 1 and antibody-loaded latex microsphere reagent 2 prepared in step (3) are mixed in a certain proportion and mixed evenly to obtain reagent R2.

[0026] The preparation method and process of reagent R2 are simpler than existing technologies, and do not require multiple preparation steps such as ultrasound and centrifugation.

[0027] Based on the above technical solution, in step (4), the mass ratio between antibody-loaded latex microsphere reagent 1 and antibody-loaded latex microsphere reagent 2 is 5:1; the above ratio can improve the hooking ability of the reagent kit and ensure the sensitivity of the reagent.

[0028] The beneficial effects of the technical solution provided by this invention are as follows:

[0029] 1. In this invention, reagent R1 is prepared using 3-[3-(cholephthalamide propyl)dimethylamino]propanesulfonic acid inner salt and sodium dodecyl sulfate. By adjusting the ratio of these two components, their synergistic effect ensures both the reagent's reaction sensitivity and its anti-interference performance. Reagent R2 uses two different particle sizes of latex microspheres coated with two HBP antibodies that recognize different antigen epitopes. This not only ensures a wider detection range in the high-value region and improves the hooking ability, but also guarantees the sensitivity of the kit. The activation system uses a combination of NHS and EDC activators, which effectively improves the coupling efficiency. Furthermore, this invention improves the coupling efficiency between the antibody and latex microspheres through precise dosage of EDC and effectively prevents latex microsphere aggregation, making the detection kit more sensitive and stable. Therefore, this invention provides a high-hook, anti-interference HBP detection kit with excellent hooking ability, strong anti-interference ability, and superior reaction sensitivity.

[0030] 2. This invention also provides a method for preparing the above-mentioned high-hook, interference-resistant HBP detection kit, which eliminates the need for centrifugation and resuspending operations, making the method simpler and reducing production costs. Attached Figure Description

[0031] Figure 1 This is the calibration curve of the detection reagent in Example 1 of this invention;

[0032] Figure 2 This is a linear fitting curve of the detection reagent in Example 1 of this invention; Detailed Implementation

[0033] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this specification includes any and all combinations of one or more of the associated listed items. It should be understood that, unless otherwise specified, all the various materials used in this invention are commercially available.

[0035] Example 1

[0036] This invention provides an HBP detection kit with high hook capability and anti-interference, comprising reagent R1 and reagent R2;

[0037] The reagent R1 comprises the following components at the following concentrations: a Tris buffer solution of 50 mM / L; an inorganic salt of 50 g / L sodium chloride; an anti-interference agent of 1:3 (by mass) 3-[3-(cholephthalamide propyl)dimethylamino]propanesulfonic acid inner salt (CHAPS) and sodium dodecyl sulfate of 16 g / L; and a polymerization promoter of PEG6000 of 10 g / L; wherein the pH of the reagent R1 is 7.0-8.0.

[0038] The reagent R2 comprises the following components at the following concentrations: latex microspheres 1: 0.9 mg / L; latex microspheres 2: 0.9 mg / L; activation system consisting of NHS and EDC: 0.0192 g / mL; buffer solution consisting of HEPES buffer and borate-borax buffer: 50 mM / L; HBP monoclonal antibody 1: 0.05 mg / mL; HBP monoclonal antibody 2: 0.05 mg / mL; blocking agent consisting of 2% BSA: 0.05 g / mL; preservative consisting of Proclin 300: 1.0 g / L; and stabilizers consisting of sucrose, trehalose, and EDTA-2Na: 0.06 g / mL.

[0039] This invention provides a method for preparing an HBP detection kit with high hooking capability and anti-interference, comprising the following steps:

[0040] Step 1, preparation of reagent R1: Dissolve the buffer solution in water according to the component content of reagent R1 and adjust the pH value of reagent R1 to 7.0-8.0; then add inorganic salt, anti-interference agent and polymerization accelerator in sequence and stir evenly. After stirring evenly, reagent R1 is obtained.

[0041] Step two, preparation of reagent R2;

[0042] (1) Preparation of solution 1: Take latex microspheres 1 and add them to an appropriate amount of pure water. Place them on a shaker at 37°C and shake at 200 r / min for 5 min. Then add the prepared activation system solution and activate the latex for 20 min. Then add the mixture of HEPES buffer and HBP monoclonal antibody 1 on a magnetic stirrer and couple for 2 h to obtain solution 1.

[0043] (2) Preparation of solution 2: Replace latex microspheres 1 and HBP monoclonal antibody 1 in step (1) with latex microspheres 2 and HBP monoclonal antibody 2, and prepare solution 2 using the same preparation method as in step (1);

[0044] (3) Take out the solutions 1 and 2 prepared above from the shaker and place them on the stirrer respectively. Add boric acid-borax buffer to each solution and incubate at 37°C for 30 min. Then add blocking agent and incubate for 1 h. Finally, add stabilizer and preservative and stabilize at 37°C overnight to obtain antibody-loaded latex microsphere reagent 1 and antibody-loaded latex microsphere reagent 2.

[0045] (4) The antibody-loaded latex microsphere reagent 1 and antibody-loaded latex microsphere reagent 2 prepared in step (3) are mixed in a ratio of 5:1 and mixed evenly to obtain reagent R2.

[0046] Example 2

[0047] This invention provides an HBP detection kit with high hook capability and anti-interference, comprising reagent R1 and reagent R2;

[0048] The reagent R1 comprises the following components at the following concentrations: a Tris buffer solution of 50 mM / L; an inorganic salt of 50 g / L sodium chloride; an anti-interference agent of 16 g / L of 3-[3-(cholephthalamide propyl)dimethylamino]propanesulfonic acid inner salt (CHAPS) and sodium dodecyl sulfate in a mass ratio of 1:5; and a polymerization accelerator of PEG6000 of 10 g / L; wherein the pH of the reagent R1 is 7.0-8.0.

[0049] The reagent R2 comprises the following components at the following concentrations: latex microspheres 1: 0.9 mg / L; latex microspheres 2: 0.9 mg / L; activation system consisting of NHS and EDC: 0.0192 g / mL; buffer solution consisting of HEPES buffer and borate-borax buffer: 50 mM / L; HBP monoclonal antibody 1: 0.05 mg / mL; HBP monoclonal antibody 2: 0.05 mg / mL; blocking agent consisting of 2% BSA: 0.05 g / mL; preservative consisting of Proclin 300: 1.0 g / L; and stabilizers consisting of sucrose, trehalose, and EDTA-2Na: 0.06 g / mL.

[0050] This invention provides a method for preparing an HBP detection kit with high hooking capability and anti-interference, comprising the following steps:

[0051] Step 1, preparation of reagent R1: Dissolve the buffer solution in water according to the component content of reagent R1 and adjust the pH value of reagent R1 to 7.0-8.0; then add inorganic salt, anti-interference agent and polymerization accelerator in sequence and stir evenly. After stirring evenly, reagent R1 is obtained.

[0052] Step two, preparation of reagent R2;

[0053] (1) Preparation of solution 1: Take latex microspheres 1 and add them to an appropriate amount of pure water. Place them on a shaker at 37°C and shake at 200 r / min for 5 min. Then add the prepared activation system solution and activate the latex for 20 min. Then add the mixture of HEPES buffer and HBP monoclonal antibody 1 on a magnetic stirrer and couple for 2 h to obtain solution 1.

[0054] (2) Preparation of solution 2: Replace latex microspheres 1 and HBP monoclonal antibody 1 in step (1) with latex microspheres 2 and HBP monoclonal antibody 2, and prepare solution 2 using the same preparation method as in step (1);

[0055] (3) Take out the solutions 1 and 2 prepared above from the shaker and place them on the stirrer respectively. Add boric acid-borax buffer to each solution and incubate at 37°C for 30 min. Then add blocking agent and incubate for 1 h. Finally, add stabilizer and preservative and stabilize at 37°C overnight to obtain antibody-loaded latex microsphere reagent 1 and antibody-loaded latex microsphere reagent 2.

[0056] (4) The antibody-loaded latex microsphere reagent 1 and antibody-loaded latex microsphere reagent 2 prepared in step (3) are mixed in a ratio of 5:1 and mixed evenly to obtain reagent R2.

[0057] Example 3

[0058] This invention provides an HBP detection kit with high hook capability and anti-interference, comprising reagent R1 and reagent R2;

[0059] The reagent R1 comprises the following components at the following concentrations: a Tris buffer solution of 50 mM / L; an inorganic salt of 50 g / L sodium chloride; an anti-interference agent of 1:0 ratio of 3-[3-(cholephthalamide propyl)dimethylamino]propanesulfonic acid inner salt (CHAPS) and sodium dodecyl sulfate of 16 g / L; and a polymerization promoter of PEG6000 of 10 g / L; wherein the pH of the reagent R1 is 7.0-8.0.

[0060] The reagent R2 comprises the following components at the following concentrations: latex microspheres 1: 0.9 mg / L; latex microspheres 2: 0.9 mg / L; activation system consisting of NHS and EDC: 0.0192 g / mL; buffer solution consisting of HEPES buffer and borate-borax buffer: 50 mM / L; HBP monoclonal antibody 1: 0.05 mg / mL; HBP monoclonal antibody 2: 0.05 mg / mL; blocking agent consisting of 2% BSA: 0.05 g / mL; preservative consisting of Proclin 300: 1.0 g / L; and stabilizers consisting of sucrose, trehalose, and EDTA-2Na: 0.06 g / mL.

[0061] This invention provides a method for preparing an HBP detection kit with high hooking capability and anti-interference, comprising the following steps:

[0062] Step 1, preparation of reagent R1: Dissolve the buffer solution in water according to the component content of reagent R1 and adjust the pH value of reagent R1 to 7.0-8.0; then add inorganic salt, anti-interference agent and polymerization accelerator in sequence and stir evenly. After stirring evenly, reagent R1 is obtained.

[0063] Step two, preparation of reagent R2;

[0064] (1) Preparation of solution 1: Take latex microspheres 1 and add them to an appropriate amount of pure water. Place them on a shaker at 37°C and shake at 200 r / min for 5 min. Then add the prepared activation system solution and activate the latex for 20 min. Then add the mixture of HEPES buffer and HBP monoclonal antibody 1 on a magnetic stirrer and couple for 2 h to obtain solution 1.

[0065] (2) Preparation of solution 2: Replace latex microspheres 1 and HBP monoclonal antibody 1 in step (1) with latex microspheres 2 and HBP monoclonal antibody 2, and prepare solution 2 using the same preparation method as in step (1);

[0066] (3) Take out the solutions 1 and 2 prepared above from the shaker and place them on the stirrer respectively. Add boric acid-borax buffer to each solution and incubate at 37°C for 30 min. Then add blocking agent and incubate for 1 h. Finally, add stabilizer and preservative and stabilize at 37°C overnight to obtain antibody-loaded latex microsphere reagent 1 and antibody-loaded latex microsphere reagent 2.

[0067] (4) The antibody-loaded latex microsphere reagent 1 and antibody-loaded latex microsphere reagent 2 prepared in step (3) are mixed in a ratio of 5:1 and mixed evenly to obtain reagent R2.

[0068] Example 4

[0069] This invention provides an HBP detection kit with high hook capability and anti-interference, comprising reagent R1 and reagent R2;

[0070] The reagent R1 comprises the following components at the following concentrations: a Tris buffer solution of 50 mM / L; an inorganic salt of 50 g / L sodium chloride; an anti-interference agent of 1:3 (by mass) 3-[3-(cholephthalamide propyl)dimethylamino]propanesulfonic acid inner salt (CHAPS) and sodium dodecyl sulfate of 16 g / L; and a polymerization promoter of PEG6000 of 10 g / L; wherein the pH of the reagent R1 is 7.0-8.0.

[0071] The reagent R2 comprises the following components at the following concentrations: latex microspheres 1: 0.9 mg / L; latex microspheres 2: 0.9 mg / L; activation system consisting of NHS and EDC: 0.0192 g / mL; buffer solution consisting of HEPES buffer and borate-borax buffer: 50 mM / L; HBP monoclonal antibody 1: 0.05 mg / mL; HBP monoclonal antibody 2: 0.05 mg / mL; blocking agent consisting of 2% BSA: 0.05 g / mL; preservative consisting of Proclin 300: 1.0 g / L; and stabilizers consisting of sucrose, trehalose, and EDTA-2Na: 0.06 g / mL.

[0072] This invention provides a method for preparing an HBP detection kit with high hooking capability and anti-interference, comprising the following steps:

[0073] Step 1, preparation of reagent R1: Dissolve the buffer solution in water according to the component content of reagent R1 and adjust the pH value of reagent R1 to 7.0-8.0; then add inorganic salt, anti-interference agent and polymerization accelerator in sequence and stir evenly. After stirring evenly, reagent R1 is obtained.

[0074] Step two, preparation of reagent R2;

[0075] (1) Preparation of solution 1: Take latex microspheres 1 and add them to an appropriate amount of pure water. Place them on a shaker at 37°C and shake at 200 r / min for 5 min. Then add the prepared activation system solution and activate the latex for 20 min. Then add the mixture of HEPES buffer and HBP monoclonal antibody 1 on a magnetic stirrer and couple for 2 h to obtain solution 1.

[0076] (2) Preparation of solution 2: Replace latex microspheres 1 and HBP monoclonal antibody 1 in step (1) with latex microspheres 2 and HBP monoclonal antibody 2, and prepare solution 2 using the same preparation method as in step (1);

[0077] (3) Take out the solutions 1 and 2 prepared above from the shaker and place them on the stirrer respectively. Add boric acid-borax buffer to each solution and incubate at 37°C for 30 min. Then add blocking agent and incubate for 1 h. Finally, add stabilizer and preservative and stabilize at 37°C overnight to obtain antibody-loaded latex microsphere reagent 1 and antibody-loaded latex microsphere reagent 2.

[0078] (4) The antibody-loaded latex microsphere reagent 1 and antibody-loaded latex microsphere reagent 2 prepared in step (3) are mixed in a ratio of 0:1 and mixed evenly to obtain reagent R2.

[0079] Example 5

[0080] This invention provides an HBP detection kit with high hook capability and anti-interference, comprising reagent R1 and reagent R2;

[0081] The reagent R1 comprises the following components at the following concentrations: a Tris buffer solution of 50 mM / L; an inorganic salt of 50 g / L sodium chloride; an anti-interference agent of 1:3 (by mass) 3-[3-(cholephthalamide propyl)dimethylamino]propanesulfonic acid inner salt (CHAPS) and sodium dodecyl sulfate of 16 g / L; and a polymerization promoter of PEG6000 of 10 g / L; wherein the pH of the reagent R1 is 7.0-8.0.

[0082] The reagent R2 comprises the following components at the following concentrations: latex microspheres 1: 0.9 mg / L; latex microspheres 2: 0.9 mg / L; activation system consisting of NHS and EDC: 0.0192 g / mL; buffer solution consisting of HEPES buffer and borate-borax buffer: 50 mM / L; HBP monoclonal antibody 1: 0.05 mg / mL; HBP monoclonal antibody 2: 0.05 mg / mL; blocking agent consisting of 2% BSA: 0.05 g / mL; preservative consisting of Proclin 300: 1.0 g / L; and stabilizers consisting of sucrose, trehalose, and EDTA-2Na: 0.06 g / mL.

[0083] This invention provides a method for preparing an HBP detection kit with high hooking capability and anti-interference, comprising the following steps:

[0084] Step 1, preparation of reagent R1: Dissolve the buffer solution in water according to the component content of reagent R1 and adjust the pH value of reagent R1 to 7.0-8.0; then add inorganic salt, anti-interference agent and polymerization accelerator in sequence and stir evenly. After stirring evenly, reagent R1 is obtained.

[0085] Step two, preparation of reagent R2;

[0086] (1) Preparation of solution 1: Take latex microspheres 1 and add them to an appropriate amount of pure water. Place them on a shaker at 37°C and shake at 200 r / min for 5 min. Then add the prepared activation system solution and activate the latex for 20 min. Then add the mixture of HEPES buffer and HBP monoclonal antibody 1 on a magnetic stirrer and couple for 2 h to obtain solution 1.

[0087] (2) Preparation of solution 2: Replace latex microspheres 1 and HBP monoclonal antibody 1 in step (1) with latex microspheres 2 and HBP monoclonal antibody 2, and prepare solution 2 using the same preparation method as in step (1);

[0088] (3) Take out the solutions 1 and 2 prepared above from the shaker and place them on the stirrer respectively. Add boric acid-borax buffer to each solution and incubate at 37°C for 30 min. Then add blocking agent and incubate for 1 h. Finally, add stabilizer and preservative and stabilize at 37°C overnight to obtain antibody-loaded latex microsphere reagent 1 and antibody-loaded latex microsphere reagent 2.

[0089] (4) The antibody-loaded latex microsphere reagent 1 and antibody-loaded latex microsphere reagent 2 prepared in step (3) are mixed in a ratio of 5:0 and mixed evenly to obtain reagent R2.

[0090]

[0091] Among them, the kits of Example 1, Example 2 and Example 3 were used together for subsequent kit anti-interference experiments; the kits of Example 1, Example 4 and Example 5 were used together for subsequent kit hooking ability experiments.

[0092] Example 6

[0093] This invention provides an HBP detection kit with high hook capability and anti-interference, comprising reagent R1 and reagent R2;

[0094] The reagent R1 comprises the following components at the following concentrations: a Tris buffer solution of 20 mM / L; an inorganic salt of 40 g / L; an anti-interference agent of 1:3 (by mass) 3-[3-(cholephthalamide propyl)dimethylamino]propanesulfonic acid inner salt (CHAPS) and sodium dodecyl sulfate of 10 g / L; and a polymerization promoter of PEG6000 of 3 g / L. The pH of the reagent R1 is 7.0-8.0.

[0095] The reagent R2 comprises the following components at the following concentrations: latex microspheres 1: 0.5 mg / L; latex microspheres 2: 0.5 mg / L; activation system consisting of NHS and EDC: 0.006 g / mL; buffer consisting of HEPES buffer and borate-borax buffer: 20 mM / L; HBP monoclonal antibody 1: 0.04 mg / mL; HBP monoclonal antibody 2: 0.04 mg / mL; blocking agent consisting of 2% BSA: 0.02 g / mL; preservative consisting of Proclin 300: 0.8 g / L; and stabilizers consisting of sucrose, trehalose, and EDTA-2Na: 0.04 g / mL.

[0096] This invention provides a method for preparing an HBP detection kit with high hooking capability and anti-interference, comprising the following steps:

[0097] Step 1, preparation of reagent R1: Dissolve the buffer solution in water according to the component content of reagent R1 and adjust the pH value of reagent R1 to 7.0-8.0; then add inorganic salt, anti-interference agent and polymerization accelerator in sequence and stir evenly. After stirring evenly, reagent R1 is obtained.

[0098] Step two, preparation of reagent R2;

[0099] (1) Preparation of solution 1: Take latex microspheres 1 and add them to an appropriate amount of pure water. Place them on a shaker at 37°C and shake at 200 r / min for 5 min. Then add the prepared activation system solution and activate the latex for 20 min. Then add the mixture of HEPES buffer and HBP monoclonal antibody 1 on a magnetic stirrer and couple for 2 h to obtain solution 1.

[0100] (2) Preparation of solution 2: Replace latex microspheres 1 and HBP monoclonal antibody 1 in step (1) with latex microspheres 2 and HBP monoclonal antibody 2, and prepare solution 2 using the same preparation method as in step (1);

[0101] (3) Take out the solutions 1 and 2 prepared above from the shaker and place them on the stirrer respectively. Add boric acid-borax buffer to each solution and incubate at 37°C for 30 min. Then add blocking agent and incubate for 1 h. Finally, add stabilizer and preservative and stabilize at 37°C overnight to obtain antibody-loaded latex microsphere reagent 1 and antibody-loaded latex microsphere reagent 2.

[0102] (4) The antibody-loaded latex microsphere reagent 1 and antibody-loaded latex microsphere reagent 2 prepared in step (3) are mixed in a ratio of 5:1 and mixed evenly to obtain reagent R2.

[0103] Example 7

[0104] This invention provides an HBP detection kit with high hook capability and anti-interference, comprising reagent R1 and reagent R2;

[0105] The reagent R1 comprises the following components at the following concentrations: a Tris buffer solution of 60 mM / L; an inorganic salt of 60 g / L; an anti-interference agent of 20 g / L (1:3 mass ratio of 3-[3-(cholephthalamide propyl)dimethylamino]propanesulfonic acid inner salt (CHAPS) and sodium dodecyl sulfate); and a polymerization accelerator of PEG6000 of 15 g / L. The pH of the reagent R1 is 7.0-8.0.

[0106] The reagent R2 comprises the following components at the following concentrations: latex microspheres 1: 1.2 mg / L; latex microspheres 2: 1.2 mg / L; activation system consisting of NHS and EDC: 0.030 g / mL; buffer consisting of HEPES buffer and borate-borax buffer: 50 mM / L; HBP monoclonal antibody 1: 0.06 mg / mL; HBP monoclonal antibody 2: 0.06 mg / mL; blocking agent consisting of 2% BSA: 0.06 g / mL; preservative consisting of Proclin 300: 1.2 g / L; and stabilizers consisting of sucrose, trehalose, and EDTA-2Na: 0.08 g / mL.

[0107] This invention provides a method for preparing an HBP detection kit with high hooking capability and anti-interference, comprising the following steps:

[0108] Step 1, preparation of reagent R1: Dissolve the buffer solution in water according to the component content of reagent R1 and adjust the pH value of reagent R1 to 7.0-8.0; then add inorganic salt, anti-interference agent and polymerization accelerator in sequence and stir evenly. After stirring evenly, reagent R1 is obtained.

[0109] Step two, preparation of reagent R2;

[0110] (1) Preparation of solution 1: Take latex microspheres 1 and add them to an appropriate amount of pure water. Place them on a shaker at 37°C and shake at 200 r / min for 5 min. Then add the prepared activation system solution and activate the latex for 20 min. Then add the mixture of HEPES buffer and HBP monoclonal antibody 1 on a magnetic stirrer and couple for 2 h to obtain solution 1.

[0111] (2) Preparation of solution 2: Replace latex microspheres 1 and HBP monoclonal antibody 1 in step (1) with latex microspheres 2 and HBP monoclonal antibody 2, and prepare solution 2 using the same preparation method as in step (1);

[0112] (3) Take out the solutions 1 and 2 prepared above from the shaker and place them on the stirrer respectively. Add boric acid-borax buffer to each solution and incubate at 37°C for 30 min. Then add blocking agent and incubate for 1 h. Finally, add stabilizer and preservative and stabilize at 37°C overnight to obtain antibody-loaded latex microsphere reagent 1 and antibody-loaded latex microsphere reagent 2.

[0113] (4) The antibody-loaded latex microsphere reagent 1 and antibody-loaded latex microsphere reagent 2 prepared in step (3) are mixed in a ratio of 5:1 and mixed evenly to obtain reagent R2.

[0114] Example 8

[0115] This invention provides an HBP detection kit with high hook capability and anti-interference, comprising reagent R1 and reagent R2;

[0116] The reagent R1 comprises the following components at the following concentrations: a Tris buffer solution of 50 mM / L; an inorganic salt of 50 g / L; an anti-interference agent of 1:3 (by weight) 3-[3-(cholephthalamide propyl)dimethylamino]propanesulfonic acid inner salt (CHAPS) and sodium dodecyl sulfate of 16 g / L; and a polymerization promoter of PEG8000 of 10 g / L. The pH of the reagent R1 is 7.0-8.0.

[0117] The reagent R2 comprises the following components at the following concentrations: latex microspheres 1: 0.9 mg / L; latex microspheres 2: 0.9 mg / L; activation system consisting of NHS and EDC: 0.0192 g / mL; buffer consisting of HEPES buffer and borate-borax buffer: 50 mM / L; HBP monoclonal antibody 1: 0.05 mg / mL; HBP monoclonal antibody 2: 0.05 mg / mL; blocking agent consisting of casein: 0.05 g / mL; preservative consisting of sodium azide: 1.0 g / L; and stabilizers consisting of sucrose, trehalose, and EDTA-2Na: 0.06 g / mL.

[0118] This invention provides a method for preparing an HBP detection kit with high hooking capability and anti-interference, comprising the following steps:

[0119] Step 1, preparation of reagent R1: Dissolve the buffer solution in water according to the component content of reagent R1 and adjust the pH value of reagent R1 to 7.0-8.0; then add inorganic salt, anti-interference agent and polymerization accelerator in sequence and stir evenly. After stirring evenly, reagent R1 is obtained.

[0120] Step two, preparation of reagent R2;

[0121] (1) Preparation of solution 1: Take latex microspheres 1 and add them to an appropriate amount of pure water. Place them on a shaker at 37°C and shake at 200 r / min for 5 min. Then add the prepared activation system solution and activate the latex for 20 min. Then add the mixture of HEPES buffer and HBP monoclonal antibody 1 on a magnetic stirrer and couple for 2 h to obtain solution 1.

[0122] (2) Preparation of solution 2: Replace latex microspheres 1 and HBP monoclonal antibody 1 in step (1) with latex microspheres 2 and HBP monoclonal antibody 2, and prepare solution 2 using the same preparation method as in step (1);

[0123] (3) Take out the solutions 1 and 2 prepared above from the shaker and place them on the stirrer respectively. Add boric acid-borax buffer to each solution and incubate at 37°C for 30 min. Then add blocking agent and incubate for 1 h. Finally, add stabilizer and preservative and stabilize at 37°C overnight to obtain antibody-loaded latex microsphere reagent 1 and antibody-loaded latex microsphere reagent 2.

[0124] (4) The antibody-loaded latex microsphere reagent 1 and antibody-loaded latex microsphere reagent 2 prepared in step (3) are mixed in a ratio of 5:1 and mixed evenly to obtain reagent R2.

[0125] Example 9

[0126] This invention provides an HBP detection kit with high hook capability and anti-interference, comprising reagent R1 and reagent R2;

[0127] The reagent R1 comprises the following components at the following concentrations: a Tris buffer solution of 50 mM / L; an inorganic salt of 50 g / L; an anti-interference agent of 1:3 (by mass) 3-[3-(cholephthalamide propyl)dimethylamino]propanesulfonic acid inner salt (CHAPS) and sodium dodecyl sulfate of 16 g / L; and a polymerization promoter of PEG12000 of 10 g / L. The pH of the reagent R1 is 7.0-8.0.

[0128] The reagent R2 comprises the following components at the following concentrations: latex microspheres 1: 0.9 mg / L; latex microspheres 2: 0.9 mg / L; activation system consisting of NHS and EDC: 0.0192 g / mL; buffer solution consisting of HEPES buffer and borate-borax buffer: 50 mM / L; HBP monoclonal antibody 1: 0.05 mg / mL; HBP monoclonal antibody 2: 0.05 mg / mL; blocking agent consisting of ethanolamine: 0.05 g / mL; preservative consisting of thimerosal: 1.0 g / L; and stabilizers consisting of sucrose, trehalose, and EDTA-2Na: 0.06 g / mL.

[0129] This invention provides a method for preparing an HBP detection kit with high hooking capability and anti-interference, comprising the following steps:

[0130] Step 1, preparation of reagent R1: Dissolve the buffer solution in water according to the component content of reagent R1 and adjust the pH value of reagent R1 to 7.0-8.0; then add inorganic salt, anti-interference agent and polymerization accelerator in sequence and stir evenly. After stirring evenly, reagent R1 is obtained.

[0131] Step two, preparation of reagent R2;

[0132] (1) Preparation of solution 1: Take latex microspheres 1 and add them to an appropriate amount of pure water. Place them on a shaker at 37°C and shake at 200 r / min for 5 min. Then add the prepared activation system solution and activate the latex for 20 min. Then add the mixture of HEPES buffer and HBP monoclonal antibody 1 on a magnetic stirrer and couple for 2 h to obtain solution 1.

[0133] (2) Preparation of solution 2: Replace latex microspheres 1 and HBP monoclonal antibody 1 in step (1) with latex microspheres 2 and HBP monoclonal antibody 2, and prepare solution 2 using the same preparation method as in step (1);

[0134] (3) Take out the solutions 1 and 2 prepared above from the shaker and place them on the stirrer respectively. Add boric acid-borax buffer to each solution and incubate at 37°C for 30 min. Then add blocking agent and incubate for 1 h. Finally, add stabilizer and preservative and stabilize at 37°C overnight to obtain antibody-loaded latex microsphere reagent 1 and antibody-loaded latex microsphere reagent 2.

[0135] (4) The antibody-loaded latex microsphere reagent 1 and antibody-loaded latex microsphere reagent 2 prepared in step (3) are mixed in a ratio of 5:1 and mixed evenly to obtain reagent R2.

[0136] Experimental Results and Performance Evaluation Analysis

[0137] This application also provides an application of the HBP detection kit, i.e., a detection method, with the following steps:

[0138] Taking the Hitachi 7180 as an example: The measurement wavelength is 600nm. Take calibrator solutions of different concentrations (8µL), add reagent R1 (160µL), mix well, incubate at 37℃ for 5 minutes, then add reagent R2 (40µL), mix well, incubate at 37℃ for 30 seconds, and read the absorbance value A1 for each tube. After reacting for 5 minutes, read the absorbance value A2 for each tube. Calculate the absorbance difference ΔA = A2 - A1. Repeat the measurement twice for each tube. Plot a "concentration-absorbance difference" calibration curve with the average of the two absorbance differences ΔA for each calibration tube as the ordinate and the corresponding calibrator concentration as the abscissa. Take the serum or plasma sample to be tested, measure the absorbance difference of the sample using the same method, and substitute it into the calibration curve to calculate the HBP content in the sample. If the HBP concentration in the sample exceeds the range of the calibration curve, the sample needs to be diluted before testing to ensure the accuracy of the test results. This kit is not only compatible with Hitachi 7180, but also with other brands and models of semi-automatic and fully automated biochemical analyzers. Specific parameters can be adjusted according to the instrument.

[0139] (1) Development of the standard curve

[0140] A. Experimental Methods

[0141] The wavelength was measured at 600 nm. Calibration solutions of different concentrations (8 μL) were taken, and reagent R1 (160 μL) was added. After mixing and incubation at 37°C for 5 minutes, reagent R2 (40 μL) was added, mixed, and incubated at 37°C for 30 seconds. The absorbance value A1 of each tube was read. After reacting for 5 minutes, the absorbance value A2 of each tube was read. The absorbance difference ΔA = A2 - A1 was calculated. Each tube was measured twice. The average of the absorbance difference ΔA measured twice for each calibration tube was plotted on the ordinate, and the corresponding calibrator concentration was plotted on the abscissa to create a "concentration-absorbance difference" calibration curve.

[0142] B. Experimental Results and Analysis

[0143] The calibration curve of the HBP detection kit in Example 1 is shown below. Figure 1 As shown.

[0144] (2) Linear range

[0145] A. Experimental Methods

[0146] The linear range is 8-400 ng / mL. Samples with near-high linear values ​​and near-low linear values ​​are mixed in ratios of 1:0, 5:1, 1:1, 1:5, and 0:1, respectively, to obtain a batch of mixed samples. The concentration is determined using the above detection procedure. Each sample is measured 2-3 times, and the average value is taken. A linear graph is plotted based on the average value and the theoretical value, and the correlation coefficient r and linear deviation are calculated. The calculation method is described in "EP6-A: Linearity evaluation of quantitative measurement methods: a statistical method, approval guidance".

[0147] The correlation coefficient |r| ≥ 0.9900; the absolute deviation of linearity should not exceed ±5 ng / mL in the range of [8.0, 50.0] ng / mL; and the relative deviation of linearity should not exceed ±10% in the range of (50.0, 400.0] ng / mL.

[0148] B. Experimental Results and Analysis

[0149] The linearity test results of the HBP detection kits of Examples 1, 6, 7, 8, and 9 are shown in Table 1. The linear fitting graph of Example 1 is shown in the figure. Figure 2 As shown.

[0150] Table 1. Linearity test results of the HBP detection kit

[0151]

[0152] As shown in Table 1, the linear correlation coefficients and deviations of the five embodiments are all within acceptable ranges, and the linearity meets the requirements. The results of Embodiment 1 are better than those of Embodiments 6, 7, 8 and 9.

[0153] (3) Analytical sensitivity

[0154] A. Experimental Methods

[0155] Based on the calibration data, calculate the change in absorbance caused by a 10 ng / mL sample, and the absolute value should be ≥0.001.

[0156] B. Experimental Results and Analysis

[0157] Table 2. Results of sensitivity testing of HBP detection kit

[0158] Example 1 Example 6 Example 7 Example 8 Example 9 Analytical sensitivity 0.0313 0.0105 0.0157 0.0119 0.0144

[0159] As shown in Table 2, the analytical sensitivity of all five examples met the requirements, with Example 1 exhibiting the highest sensitivity.

[0160] (4) Repeatability testing

[0161] A. Experimental Methods

[0162] Using HBP quality control material with a labeled value of 15 ng / mL from Hangzhou Yibaixin Biotechnology Co., Ltd. as a low-value sample, the assay was performed according to the biochemical analyzer detection method described above. Each concentration was measured 10 times, and the mean and standard deviation (SD) were calculated. The coefficient of variation (CV) was then used to assess the repeatability of the kits in Examples 1-4. The acceptable range of CV was ±10%.

[0163] B. Experimental Results and Analysis

[0164] Table 3. Repeatability test results of the HBP detection kit

[0165] Example 1 Example 6 Example 7 Example 8 Example 9 Mean 14.89 16.22 16.34 15.46 15.69 SD 0.58 1.02 1.07 0.98 1.01 CV 3.89％ 6.28％ 6.55％ 6.34％ 6.44％

[0166] As shown in Table 3, the coefficients of variation (CV) of the five examples are all within an acceptable range, indicating that the components and their concentration ranges involved in this invention can have good in-bottle homogeneity. The CV results of the components and concentrations of the reagent in Example 1 are better than those of Examples 6, 7, 8 and 9.

[0167] (5) Anti-interference experiment

[0168] A. Experimental Methods

[0169] Healthy plasma samples were collected, and high concentrations of interfering substances were precisely measured and mixed with the samples to prepare mixed samples with different concentrations of interfering substances. Each sample was measured three times, and the average value was taken. The interfering substances used in this experiment were 0.3 g / L bilirubin, 4.5 g / L hemoglobin, and 550 IU / mL rheumatoid factor. The reagent preparation processes used were those in Examples 1, 2, and 3.

[0170] The method for calculating interference is as follows:

[0171] Interference level = (mean of sample with interference - mean of sample without interference) / mean of sample without interference × 100%; an interference level of ±10% is considered acceptable.

[0172] B. Experimental Results and Analysis

[0173] Table 4. Interference (%) Test Results of HBP Detection Kit

[0174] Example 1 Example 2 Example 3 bilirubin -2.61 -5.31 15.26 hemoglobin -1.05 -4.42 18.69 rheumatoid factor -2.04 -5.33 16.36

[0175] As shown in Table 4, the interference levels of samples with added interfering substances in Examples 1 and 2 were controlled within ±10% compared to those without, indicating that the reagent has good anti-interference ability. The data shows that Example 1 has better anti-interference ability. When a certain concentration of interfering substances such as bilirubin, hemoglobin, and rheumatoid factor is present in the test sample, the impact on the measurement results of the kit of this invention is relatively small. Example 3 did not add sodium dodecyl sulfate, and its anti-interference ability did not meet the requirements.

[0176] (6) Hook Ability Experiment

[0177] A. Experimental Methods

[0178] High-value samples with a concentration of 500 ng / mL were prepared using Ebaixin antigen and serially diluted 1 / 2, 1 / 4, 1 / 8, 1 / 16, and 1 / 32. The reagent preparation processes used were those in Examples 1, 4, and 5. Each sample was measured 5 times and the average value was taken. The relative deviation between the average value and the corresponding theoretical value should be ≤ ±10%.

[0179] B. Experimental Results and Analysis

[0180] The average results of the high-value samples for the HBP detection kit's hook capability are shown in Table 5, and the relative deviations between the measured results and the theoretical values ​​are shown in Table 6. Table 5 shows that no false negatives were observed in the high-value samples of Examples 1 and 5 (detecting the upper limit of linearity), while Example 4 showed false negatives. This is because Example 4 did not add microspheres with smaller particle sizes, resulting in an unsatisfactory hook capability. Table 6 shows that the relative deviations between the measured values ​​and the theoretical values ​​of Examples 1 and 4 are ≤ ±10% (excluding false negatives). Example 5 has a larger deviation, and this is because Example 5 did not add microspheres with larger particle sizes, resulting in low sensitivity and inaccurate measurements. In conclusion, Example 1 has better hook capability and higher accuracy. Adding microspheres of different particle sizes to the experimental system can improve both hook capability and accuracy.

[0181] Table 5. Sample average of HBP detection kit hook capability test results

[0182] Dilution factor Example 1 Example 4 Example 5 1 509.8 0.26 552.6 1 / 2 251.3 250.8 278.6 1 / 4 124.8 127.6 138.5 1 / 8 60.9 60.8 69.4 1 / 16 31.5 32.8 35.9 1 / 32 15.3 16.3 17.2

[0183] Table 6. Relative deviation of samples with high Hook capability of HBP detection kit (%)

[0184]

[0185]

[0186] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or basic characteristics. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0187] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A high-hook, interference-resistant HBP detection kit, characterized in that, Including reagents R1 and R2; The reagent R1 comprises the following components at the following concentrations: buffer solution: 20-60 mM / L; inorganic salt: 40-60 g / L; anti-interference agent: 10-20 g / L; polymerization promoter: 3-15 g / L; wherein the anti-interference agent is prepared by mixing 3-[3-(cholephthalamide propyl)dimethylamino]propanesulfonic acid inner salt and sodium dodecyl sulfate in a mass ratio of 1:3-1:

5. The reagent R2 comprises the following components at the following concentrations: latex microspheres 1: 0.5-1.2 mg / L; latex microspheres 2: 0.5-1.2 mg / L; activation system: 0.006-0.030 g / mL; buffer: 20-50 mM / L; HBP monoclonal antibody 1: 0.04-0.06 mg / mL; HBP monoclonal antibody 2: 0.04-0.06 mg / mL; blocking agent: 0.02-0.06 g / mL; Preservative: 0.8-1.2 g / L; stabilizer: 0.04-0.08 g / mL; wherein the particle size of latex microsphere 1 is 60-180 nm, and the particle size of latex microsphere 2 is 180-350 nm; the activation system includes one or both of EDC and NHS, wherein the concentration of EDC is 0.006-0.008 g / mL, and the concentration of NHS is 0.01-0.03 g / mL.

2. The HBP detection kit with high hooking capability and anti-interference as described in claim 1, characterized in that, Both HBP monoclonal antibody 1 and HBP monoclonal antibody 2 are mouse anti-human HBP antibodies, and their antigen recognition epitopes are different.

3. The HBP detection kit with high hooking capability and anti-interference as described in claim 1, characterized in that, The buffer solutions in reagents R1 and R2 are one or more of Tris buffer, borate-borax buffer, HEPES buffer, and MES buffer.

4. The HBP detection kit with high hooking capability and anti-interference as described in claim 1, characterized in that, The inorganic salt in reagent R1 is one or more of sodium chloride, calcium chloride, and magnesium chloride; the polymerization promoter is one or more of PEG6000, PEG8000, and PEG12000.

5. The HBP detection kit with high hooking capability and anti-interference as described in claim 1, characterized in that, The blocking agent in reagent R2 is one or more of BSA, casein, and ethanolamine; the preservative is one or more of Proclin 300, sodium azide, and thimerosal; and the stabilizer is one or more of trehalose, sucrose, casein, gelatin, and EDTA-2Na.

6. A method for preparing an HBP detection kit with high hook capability and anti-interference according to any one of claims 1 to 5, characterized in that, Includes the following steps: Step 1, preparation of reagent R1: Dissolve the buffer solution in water according to the component content of reagent R1 and adjust the pH value of reagent R1 to 7.0-8.0; then add inorganic salt, anti-interference agent and polymerization accelerator in sequence and stir evenly. After stirring evenly, reagent R1 is obtained. Step two, preparation of reagent R2; (1) Preparation of solution 1: Take latex microspheres 1 and add them to an appropriate amount of pure water. Place them on a shaker at 37°C and shake at 200 r / min for 5 min. Then add the prepared activation system solution and activate the latex for 20 min. Subsequently, a mixture of buffer solution and HBP monoclonal antibody 1 was added to a magnetic stirrer and coupled for 2 hours to obtain solution 1; (2) Preparation of solution 2: Replace latex microspheres 1 and HBP monoclonal antibody 1 in step (1) with latex microspheres 2 and HBP monoclonal antibody 2, and prepare solution 2 using the same preparation method as in step (1); (3) Take out the solutions 1 and 2 prepared above from the shaker, place them on the stirrer, add buffer solution, incubate at 37°C for 30 min, add blocking agent, and incubate for 1 h; finally add stabilizer and preservative, stabilize at 37°C overnight, and obtain antibody-loaded latex microsphere reagent 1 and antibody-loaded latex microsphere reagent 2 respectively. (4) The antibody-loaded latex microsphere reagent 1 and antibody-loaded latex microsphere reagent 2 prepared in step (3) are mixed in a certain proportion and mixed evenly to obtain reagent R2.

7. The method for preparing a high-hook, interference-resistant HBP detection kit according to claim 6, characterized in that, In step (4), the mass ratio between antibody-loaded latex microsphere reagent 1 and antibody-loaded latex microsphere reagent 2 is 5:1.

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