Chemiluminescence method heparin binding protein kit and determination method thereof

Abstract

The application discloses a chemiluminescence method heparin binding protein kit and a determination method thereof. The kit comprises reagent components reagent R1, reagent R2 and reagent R3, and matched calibrators, quality control samples, pre-excitation liquid, excitation liquid and cleaning liquid. The reagent R1 contains magnetic microparticles coated with streptavidin, the reagent R2 and the reagent R3 are respectively acridinium ester and biotin-labeled monoclonal antibodies for recognizing different epitopes of human HBP. The kit has good sensitivity, a wide detection range and high specificity, and can effectively reduce potential endogenous and exogenous interference in clinical samples of potential interference people.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of immunological detection, and particularly relates to a chemiluminescence method heparin-binding protein kit and a determination method thereof, and also relates to a heparin-binding protein determination kit and a determination method thereof using a chemiluminescence immunoassay method. BACKGROUND

[0002] Heparin-binding protein (HBP) was first discovered by Shafer et al. from the azurophilic granules of neutrophils in 1984, and was named CAP37 (Cationic Antimicrobial Protein 37) due to its molecular weight of 37 kDa and cationic antimicrobial properties. Subsequently, Gabay et al. named it Azurocidin, and further studies found that its structure contains 18 arginine residues, which endow it with antimicrobial activity and heparin binding capacity, and finally it was named HBP. Under the stimulation of bacterial infection or inflammation, neutrophils rapidly release HBP to the extracellular space through degranulation, and the concentration in the blood can be detected to increase within 1-2 hours. HBP can bind to the surface of vascular endothelial cells with heparan sulfate, activate the intracellular RhoA / ROCK signaling pathway, cause cytoskeleton rearrangement and tight junction disruption, and significantly increase vascular permeability (manifesting as "capillary leak syndrome"). HBP can also stimulate monocytes to release pro-inflammatory factors (such as IL-6, TNF-α), and at the same time induce neutrophil chemotaxis, forming a positive feedback loop to exacerbate systemic inflammatory response; on the other hand, HBP can inhibit tissue factor pathway inhibitor (TFPI) to promote thrombin generation, and exacerbate sepsis-related disseminated intravascular coagulation (DIC). At this point, with the rapid release of HBP into the blood, through the mechanisms of inducing endothelial cell permeability increase and activating monocytes, etc., the inflammatory response is exacerbated, and HBP plays a key role in the pathological processes of sepsis, septic shock, etc., and becomes a core biomarker of critical illness such as sepsis and septic shock.

[0003] When bacterial infection and sepsis occur, HBP increases significantly within 1-2 hours after bacterial infection, and the specificity of the condition is higher than that of viral or non-infectious inflammation, which is a key indicator for early warning of sepsis. As a supplementary indicator for early diagnosis of sepsis, HBP detection within 6 hours can guide the use of antibiotics, which helps to reduce antibiotic abuse. The detection sample can also be cerebrospinal fluid or urine, which can be used for local infection positioning and assist in the diagnosis of meningitis and urinary tract infection. Studies have shown that HBP can be detected to increase 72 hours before sepsis occurs, which is earlier than traditional markers such as C-reactive protein and procalcitonin. HBP has a short half-life (<1 hour) and a positive correlation with the severity of infection, which can be dynamically monitored and used to assess infection risk and control effect in real time. As an "early-specific-dynamic" marker, HBP is recommended for rapid differential diagnosis of bacterial infection, early warning and efficacy monitoring of sepsis, guidance for rational use of antibiotics, and reduction of drug resistance.

[0004] For the detection and analysis of HBP in clinical samples of hospital patients, the HBP concentration of normal healthy people is below 10 ng / mL; however, when local infection or early inflammation occurs, the HBP concentration is in the range of 20 ng / mL to 30 ng / mL; in severe infection (such as septic shock) or complex postoperative complications, the HBP concentration is in the range of 100 ng / mL to 1000 ng / mL; in extreme cases of late conditions such as multiple organ failure or refractory shock, the HBP concentration can even exceed 1000 ng / mL.

[0005] Currently, the most authoritative representative for detecting HBP is the enzyme-linked immunoassay kit of the diagnostic company, but this method takes a long time (1h-2h) and is difficult to meet the "golden window period" of early diagnosis of sepsis. Currently, imported diagnostic reagents from abroad are generally expensive, and domestic manufacturers also have other methods for HBP detection, such as immunochromatography, latex-enhanced immunoturbidimetry, and magnetic micro-particle chemiluminescence. However, most of these detection methods have insufficient sensitivity and specificity, and the detection range does not meet the above requirements. Traditional methods are prone to false negatives in low-concentration samples, and the test throughput cannot meet the requirements of a large number of samples in a short period of time. In addition, some methods have high dependence on equipment and require special instruments, which limits their application in primary hospitals.

[0006] CN112858696A and CN204882575U both disclose an immunofluorescence chromatography method for detecting HBP kit, which is simple to operate but has insufficient sensitivity and a narrow linear range, and can only perform qualitative and semi-quantitative detection, so the accuracy of the test results cannot fully meet the needs of clinical quantitative test results. CN204882575U and CN108956978A also disclose latex-enhanced immunoturbidimetry and magnetic micro-particle chemiluminescence methods for detecting HBP kit, respectively. SUMMARY

[0007] In view of the performance requirements of the reagent kit in the actual clinical diagnosis, in order to meet the actual needs of the reagent kit with high sensitivity, wide linear range, good repeatability, strong anti-interference and specificity, high test efficiency, and the test of different sample types such as serum and plasma, the present application provides a chemiluminescence method heparin binding protein reagent kit and a determination method thereof, which is also a heparin binding protein determination reagent kit and a determination method thereof by using chemiluminescence immunoassay, aiming to provide a test result with good accuracy, high automation, high efficiency and meeting the needs of serum and plasma sample type test for medical care.

[0008] The technical scheme of the present application is as follows:

[0009] The present application provides a chemiluminescence method heparin binding protein reagent kit, which is also a heparin binding protein determination reagent kit by using chemiluminescence immunoassay, the reagent kit comprising: reagent components reagent R1, reagent R2 and reagent R3, and matched calibrant, quality control, pre-excitation solution, excitation solution and cleaning solution; wherein the reagent R1 contains magnetic microspheres coated with streptavidin, and the reagent R2 and the reagent R3 are respectively acridinium ester and biotin-labeled monoclonal antibody recognizing different epitopes of human HBP. The present application illustrates the complete preparation process of the HBP reagent kit determined by chemiluminescence immunoassay, and the determination reagent kit exhibits good sensitivity and wide detection range, can effectively reduce the potential endogenous and exogenous interference in the clinical samples of the potential interference population, and has high specificity.

[0010] The object of the present application is achieved by the following technical scheme:

[0011] A chemiluminescence method heparin binding protein reagent kit, which is a heparin binding protein (HBP) determination reagent kit by using chemiluminescence immunoassay, comprising: reagent components reagent R1, reagent R2 and reagent R3, and matched calibrant, quality control, pre-excitation solution, excitation solution and cleaning solution.

[0012] The components of the reagent R1 include: 0.1 g / L~1.0 g / L of streptavidin magnetic beads, 2 g / L~80 g / L of buffer salt, 5 g / L~30 g / L of buffer stabilizer, 0.5 g / L~3 g / L of dispersing agent, 0.5 g / L~5 g / L of surfactant, 2 g / L~20 g / L of protein protective agent, and 0.5 g / L~2 g / L of preservative in the R1 diluent with pH of 6.0~8.0;

[0013] The components of the reagent R2 include: 0.5 mg / L-8 mg / L acridinium ester HBP antibody 1 marker, 2 g / L-80 g / L buffer salt, 5 g / L-30 g / L buffer stabilizer, 0.5 g / L-5 g / L chelating agent, 0.5 g / L-5 g / L dispersing agent, 0.5 g / L-5 g / L surfactant, 10 mg / L-500 mg / L blocking agent, 2 g / L-10 g / L protein protective agent, 0.5 g / L-2 g / L preservative in R2 diluent with pH 5.0-7.0;

[0014] The components of the reagent R3 include: 0.5 mg / L-8 mg / L biotin HBP antibody 2 marker, 2 g / L-80 g / L buffer salt, 5 g / L-30 g / L buffer stabilizer, 0.5 g / L-5 g / L chelating agent, 0.5 g / L-5 g / L surfactant, 2 g / L-20 g / L protein protective agent, 0.5 g / L-2 g / L preservative in R3 diluent with pH 6.0-8.0;

[0015] The components of the calibrator are matrix 1 of HBP with different concentrations, which are 0 ng / mL, 5.01 ng / mL, 19.97 ng / mL, 100.36 ng / mL, 502.16 ng / mL and 1997.89 ng / mL respectively; 2 g / L-80 g / L buffer salt, 5 g / L-30 g / L buffer stabilizer, 5 g / L-30 g / L saccharide stabilizer, 0 %(v / v)-30 %(v / v) serum matrix, 2 g / L-20 g / L protein protective agent, 0.5 g / L-2 g / L preservative in matrix 1 with pH 6.0-8.0;

[0016] The components of the quality control are matrix 2 of HBP with different concentrations, which are (40±4) ng / mL, (600±60) ng / mL respectively; 2 g / L-80 g / L buffer salt, 5 g / L-30 g / L buffer stabilizer, 3 g / L-30 g / L saccharide stabilizer, 10 %(v / v)-30 %(v / v) serum matrix, 2 g / L-10 g / L protein protective agent, 0.5 g / L-2 g / L preservative in matrix 2 with pH 6.0-8.0;

[0017] The components of the pre-priming solution are 20 mM-100 mM nitric acid, 30 mM-200 mM hydrochloric acid, 0.1 %-0.5 % hydrogen peroxide (w / v), 0.1 g / L-10 g / L zinc chloride, 0.5 g / L-2 g / L sodium dodecyl sulfonate, pH<2;

[0018] The excitation liquid component includes: 4 g / L-20 g / L of sodium hydroxide, 1 g / L-10 g / L of sodium carbonate, 1 g / L-10 g / L of decyl trimethyl ammonium chloride, pH>13;

[0019] The component of the cleaning liquid includes: 1 g / L-15 g / L of sodium dihydrogen phosphate, 10 g / L-60 g / L of disodium hydrogen phosphate, 5 g / L-30 g / L of sodium chloride, 0.5 g / L-10 g / L of Tween-20, 0.5 g / L-2 g / L of ProClean 300, pH=7.4±0.4.

[0020] Further, the buffer salt is at least one of sodium dihydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, morpholine ethanesulfonic acid (MES), hydroxyethyl piperazine ethanesulfonic acid (HEPES), tris (hydroxymethyl) aminomethane (TRIS), bis (2-hydroxyethyl) amino-tris (hydroxymethyl) methane (BIS-TRIS);

[0021] The buffer stabilizer is at least one of sodium chloride, potassium chloride, calcium chloride, calcium sulfate;

[0022] The dispersing agent is at least one of polyethylene glycol PEG-2000, polyethylene glycol PEG-4000, polyethylene glycol PEG-6000, polyvinylpyrrolidone PVP-8000, polyvinylpyrrolidone PVP-10000, polyvinylpyrrolidone PVP-30000;

[0023] The surfactant is at least one of Tween-20, Tween-80, Triton x-100, lauryl betaine, 3-sulfopropyl hexadecyl dimethyl betaine.

[0024] Further, the chelating agent is at least one of ethylenediaminetetraacetic acid (EDTA) disodium, ethylenediaminetetraacetic acid (EDTA) dipotassium, sodium citrate, sodium oxalate;

[0025] The blocking agent is at least one of mouse immunoglobulin, sheep immunoglobulin, bovine immunoglobulin, and homologous immunoglobulin recognizing different epitopes;

[0026] The saccharide stabilizer is at least one of sucrose, trehalose, dextran, mannose;

[0027] The serum matrix is at least one of horse serum, goat serum, and newborn calf serum;

[0028] The protein protective agent is at least one of bovine serum albumin, human serum albumin, casein, sodium caseinate, and ovalbumin;

[0029] The preservative is at least one of ProClean 300, ProClean 950, methylisothiazolinone (MIT), sodium benzoate.

[0030] The HBP detection reagent has a sensitivity of ≤0.05 ng / mL, a linear range of 0.05 ng / mL-2000 ng / mL, a recovery within 100%±5%, an intra-batch precision of <3.5%, an inter-batch precision of <5%, a bias rate of bilirubin, triglyceride, hemoglobin and rheumatoid factor (RF) within ±5%, a bias rate of human anti-mouse antibody (HAMA) within ±6%, and no cross-reaction with serum amyloid A, C-reactive protein, procalcitonin, interleukin-2 and interleukin-6.

[0031] Further, the streptavidin magnetic beads of the reagent R1 are prepared by coating streptavidin on solid-phase magnetic beads, and the preparation method comprises the following steps:

[0032] (1) 5 mg-50 mg carboxyl magnetic beads are taken, the supernatant is removed after magnetic separation, and the magnetic beads are washed with premix solution I for 3 times and resuspended;

[0033] (2) 0.2 g / L-5 g / L 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (EDC·HCl) and 0.2 g / L-5 g / L N-hydroxysuccinimide (NHS) are continuously added, the mixture is uniformly mixed, and then placed in a rotary mixer for reaction at room temperature for 0.5 h-3 h in the dark;

[0034] (3) After the reaction is completed, the supernatant is removed after magnetic separation of the mixed solution, the magnetic beads are washed with premix solution I for 3 times and resuspended;

[0035] (4) 0.025 mg-2.50 mg (streptavidin coating mass fraction 0.5%-5%) of streptavidin is continuously added to the activated magnetic bead solution, and the mixture is uniformly mixed and reacted at 37°C for 2 h-10 h in the dark in a rotary mixer;

[0036] (5) After the reaction is completed, the supernatant is removed after magnetic separation of the mixed solution, blocking solution I is added for resuspension, and the mixture is uniformly mixed and reacted at 37°C for 0.5 h-3 h in the dark in a rotary mixer;

[0037] (6) After the reaction is completed, the supernatant is removed after magnetic separation, the magnetic beads are washed with storage solution I for 3 times and resuspended, and then stored at 2°C-8°C for standby;

[0038] The premix liquid I component includes 5.0 g / L-50 g / L sodium bicarbonate, 5 g / L-30 g / L sodium chloride, 0.5 g / L-2 g / L ProClean 300, and the pH is 7.0-9.0; the blocking liquid I component includes 5.0 g / L-50 g / L sodium bicarbonate, 5 g / L-30 g / L sodium chloride, 1 g / L-20 g / L lysine, 2 g / L-20 g / L casein, 0.5 g / L-2 g / L Tween-20, 0.5 g / L-2 g / L ProClean 300, and the pH is 7.0-9.0;

[0039] The storage liquid I component includes 1 g / L-15 g / L sodium dihydrogen phosphate, 10 g / L-60 g / L disodium hydrogen phosphate, 5 g / L-30 g / L sodium chloride, 2 g / L-50 g / L fish gelatin, 1 g / L-10 g / L bovine serum albumin, 0.5 g / L-2 g / L ProClean 300, and the pH is 7.4±0.4.

[0040] Further, the acridinium ester HBP antibody 1 label of reagent R2 is acridinium active ester coupled on HBP antibody to obtain acridinium ester HBP antibody 1 label, and the preparation method comprises the following steps:

[0041] (1) 0.2 mg-2 mg HBP antibody 1 is taken in the premix liquid II, an ultrafiltration tube is placed in a 2°C-8°C refrigerator centrifuge, the rotation speed is 2000 rpm-6000 rpm, 10 min, and the separation is replaced for 3 times, and then transferred to a reaction bottle;

[0042] (2) 0.003 mg-0.3 mg of acridinium active ester dissolved in dimethyl sulfoxide (antibody and acridinium ester molar ratio 1:3-1:30) is continuously added, mixed uniformly, and then placed in a rotary mixer in a light-proof room temperature mixing instrument for 2 h-10 h;

[0043] (3) after the reaction is completed, the blocking liquid II is added and reacted for 0.5 h-3 h;

[0044] (4) after the reaction is stopped, a desalination column is purified, the acridinium labeled antibody component is collected, concentrated by an ultrafiltration tube, and then the storage liquid II is added, and after being divided, it is stored under the condition of-20°C--80°C for standby use.

[0045] Further, the biotin HBP antibody 2 label of reagent R3 is biotin active ester coupled on HBP antibody to obtain biotin HBP antibody 2 label, and the preparation method comprises the following steps:

[0046] (1) take 0.2 mg~2mg HBP antibody 2 in premix solution II, use ultrafiltration tube to be placed in 2℃~8℃ refrigerator centrifuge, rotation speed 2000 rpm~6000 rpm, 30 min, separate displacement 3 times, transfer to reaction bottle;

[0047] (2) continue to add biotin active ester dissolved in dimethyl sulfoxide 0.006~0.6mg (antibody and biotin molar ratio 1:3~1:30), mix well and place in rotary mixer for 2h~10h reaction at room temperature in the dark;

[0048] (3) after the reaction, add blocking solution II and continue to react for 0.5h~3h;

[0049] (4) after the reaction is stopped, desalination column purification, collect acridine labeled antibody components, concentrate with ultrafiltration tube, add storage solution II, after dispensing, freeze at-20℃~-80℃ for standby;

[0050] Among them, the premix solution II component includes 1 g / L~15 g / L sodium dihydrogen phosphate, 10 g / L~60 g / L disodium hydrogen phosphate, 5 g / L~30 g / L sodium chloride, 0.5 g / L~2 g / L ProClean 300, pH 7.0~8.5;

[0051] The blocking solution II component includes 1 g / L~15 g / L sodium dihydrogen phosphate, 10 g / L~60 g / L disodium hydrogen phosphate, 5 g / L~30 g / L sodium chloride, 0.5 g / L~10 g / L glycine, 2 g / L~20 g / L casein, 0.5 g / L~2 g / L Tween-20, 0.5 g / L~2 g / L ProClean 300, pH 7.0~8.5;

[0052] The storage solution II component includes 4 g / L~40 g / L morpholine ethanesulfonic acid, 3 g / L~30 g / L tris-hydroxymethyl aminomethane (TRIS), 5 g / L~30 g / L sodium chloride, 2%~30% (v / v) glycerol, 2 g / L~20 g / L human serum albumin, 0.5 g / L~2 g / L Tween-20, 0.5 g / L~2 g / L ProClean 300, pH 5.0~7.0.

[0053] The application also provides a determination method of a heparin binding protein (HBP) determination kit using a chemiluminescence immunoassay method, wherein the reagent calibration comprises the following steps:

[0054] S1, the full-automatic chemiluminescence immunoassay analyzer is equipped with pre-excitation liquid, excitation liquid and cleaning liquid; after the instrument is perfused under the action of the peristaltic pump, the heparin binding protein (HBP) determination kit is placed in the reagent bin, and is stored on board at 2-8 DEG C; the heparin binding protein (HBP) determination kit is loaded, and standby is 10-30 minutes;

[0055] S2, after the reagent R1 magnetic bead is fully mixed in the instrument, the HBP calibrant of 6 different concentrations is tested to obtain different luminescence values (RLU), and the concentration-luminescence value after weighting is fitted to obtain the working curve after calibration;

[0056] S3, the determination result of the calibrated reagent is presented in the form of concentration, the quality control sample concentration is continuously tested, and other samples are tested within the target value range.

[0057] Further, the full-automatic chemiluminescence immunoassay analyzer in S1 is selected from F2000, F180, F120 of G Biotech Co., Ltd., SMART 6500 of I company and Shine i9000 of J Biotech Co., Ltd., and preferably the full-automatic chemiluminescence immunoassay analyzer F2000 produced by G Biotech Co., Ltd.

[0058] Further, the application also provides a determination method of the above-mentioned heparin binding protein (HBP) determination kit by chemiluminescence method, which is also a determination method of the heparin binding protein (HBP) determination kit by chemiluminescence immunoassay method, adopts a reaction mode of double-antibody sandwich method, and each sample test is completed within 10 minutes at the fastest, the full-automatic chemiluminescence immunoassay analyzer continuously detects samples, and the detection throughput reaches 200 samples per hour, and the method comprises the following steps:

[0059] A1, the sample needle S absorbs 10 muL of sample at the application sample track position, the reagent needle A absorbs 50 muL of reagent R2 component at the corresponding project reagent bin position, and is added into the reaction cup in sequence, and is incubated at 37 DEG C for 5 minutes (acridinone-labeled antibody 1 captures HBP in the sample);

[0060] A2, the reagent needle B is automatically perfused, 50 muL of reagent R1 component and R3 component are absorbed at the corresponding reagent position of the reagent bin, and are continuously added into the reaction cup for incubation (to form a double-antibody sandwich product);

[0061] A3, after the incubation is completed, the mixed liquid in the reaction cup is subjected to magnetic separation, the supernatant is absorbed by the waste liquid needle C1, and then the waste liquid is discharged, and the sample needle D1 continues to add cleaning liquid to resuspend the liquid surface;

[0062] A4, the sample needle D2 continues to add 100 muL of pre-excitation liquid to mix the liquid surface, and the sample needle D3 continues to add 300 muL of excitation liquid to mix the liquid surface.

[0063] A5、reaction starts, acridinium ester label in the reaction cup emits light, light signal is detected by photomultiplier tube and converted into electric signal, the stronger the detected electric signal, the stronger the light emission, and the more the captured HBP to be detected, indicating a positive correlation feedback (acridinium ester reacts with hydrogen peroxide under alkaline conditions to release photons) that the sample contains more HBP;

[0064] A6、after the light-emitting reaction ends, the waste liquid needle C2 sucks the suspension, and is discharged into the waste liquid collection pipeline; the sample cup gripper takes out the reaction cup and puts it into the waste barrel.

[0065] Compared with the prior art, the present application has the following beneficial effects:

[0066] 1、The heparin-binding protein (HBP) assay kit for application of a chemiluminescence immunoassay method, selects a long-chain biotin coupling labeling process, reduces the interference of magnetic beads on antibodies, and improves the sensitivity of reagents, and the sensitivity is not more than 0.05 ng / mL.

[0067] 2、The heparin-binding protein (HBP) assay kit for application of a chemiluminescence immunoassay method, the addition of a dispersing agent, two kinds of surfactants with dual properties in the reagent R2 formula significantly reduces the exogenous interference of triglycerides, bilirubin and hemoglobin; the addition of a blocking agent in the reagent R2 formula significantly reduces the endogenous interference deviation rate of heterophilic antibodies such as rheumatoid factor (RF) and human anti-mouse antibody (HAMA). The combined deviation of triglycerides, bilirubin, hemoglobin and RF is within ±5%, and the deviation of HAMA is within ±6%.

[0068] 3、The heparin-binding protein (HBP) assay kit for application of a chemiluminescence immunoassay method, when relevant inflammation occurs, relevant inflammatory factor indexes rapidly rise, and the specificity of the detection marker is basically free of obvious cross-reaction.

[0069] 4、The heparin-binding protein (HBP) assay kit for application of a chemiluminescence immunoassay method has good correlation with the imported commercial K diagnostic company in the United Kingdom control reagent enzyme-linked immunoassay kit for clinical samples, is suitable for testing of serum and plasma samples; the kit is suitable for different models of full-automatic chemiluminescence immunoassay analyzers, involves F2000, F180 and F120 of G Biotech Co., Ltd., SMART 6500 of I company and Shine i9000 of J Biotech Co., Ltd., and can meet the needs of large-scale sample testing.

[0070] 5. The determination method of the heparin binding protein (HBP) determination kit for application of the chemiluminescence immunoassay method, adopts a suitable antibody labeling process, improves the linear range width, and the detection range is 0.05 ng / mL-2000 ng / mL; the accuracy recovery is less than 100%±5%; the within-batch precision is less than 3.5%, and the between-batch precision is less than 5%. BRIEF DESCRIPTION OF DRAWINGS

[0071] The accompanying drawings are included to provide a further understanding of the application, and constitute a part of the specification, which together with the embodiments of the application serve to explain the application, and do not constitute a limitation on the application.

[0072] Figure 1 The HBP detection principle diagram of the heparin binding protein determination kit for application of the chemiluminescence immunoassay method according to the embodiment of the application;

[0073] Figure 2 The HBP detection linear range result diagram in the determination method of the heparin binding protein determination kit for application of the chemiluminescence immunoassay method according to the embodiment of the application;

[0074] Figure 3 The HBP detection correlation result diagram in the determination method of the heparin binding protein determination kit for application of the chemiluminescence immunoassay method according to the embodiment 1 of the application and the enzyme-linked immunoassay kit of K diagnostic company. DETAILED DESCRIPTION

[0075] In order to facilitate the understanding of the technical content of the application and the achieved technical effects by the related technical personnel, the application is further described below in combination with specific embodiments. The described embodiments are only a part of the implementation form cases of the application, and are only the preferred embodiments of the application, and do not limit the scope of the application. Based on the cases in the embodiments, other embodiments obtained by the skilled in the art without creative labor are within the protection scope of the application.

[0076] The labeled HBP antibody is a mouse anti-human HBP monoclonal antibody with high immunoreaction specificity, which is purchased from A biotechnology Co., Ltd. and B medical technology Co., Ltd.;

[0077] The label biotin active ester and streptavidin are purchased from C biochemical technology Co., Ltd.

[0078] The magnetic microparticle is purchased from D pharmaceutical auxiliary material Co., Ltd.

[0079] The acridinium ester is purchased from E biochemical technology Co., Ltd.

[0080] The serum matrix is purchased from F biotechnology Co., Ltd.

[0081] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials and kits in the following examples are commercially available from conventional suppliers unless otherwise specified.

[0082] The determination of the results of the following examples is mainly carried out by using the full-automatic chemiluminescence immunoassay analyzer F2000 of G Biotech Co., Ltd.

[0083] Example 1

[0084] A heparin-binding protein assay kit using chemiluminescence immunoassay method, the HBP detection principle diagram is as shown in Figure 1 The kit includes reagent components reagent R1, reagent R2 and reagent R3, as well as matched calibrators, quality control products, pre-activation solution, activation solution and cleaning solution.

[0085] In the preparation process of each component solution, 2M hydrochloric acid and 2M sodium hydroxide are respectively used to adjust the pH of the system.

[0086] The reagent component reagent R1 component: the concentration of streptavidin magnetic beads in R1 diluent is 0.35g / L.

[0087] The preparation steps of streptavidin magnetic beads are as follows:

[0088] (1) Take 20mg carboxyl magnetic beads, remove the supernatant after magnetic separation, and wash with pre-mixed solution I (10.32g / L sodium bicarbonate, 9.0g / L sodium chloride, 0.5g / L ProClean 300, pH=8.5) for 3 times, and resuspend;

[0089] (2) Continue to add 1.82g / L of 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (EDC·HCl) and 0.96g / L of N-hydroxysuccinimide (NHS), mix well, and then place in a rotary mixer for 2h of reaction at room temperature in the dark;

[0090] (3) After the reaction is completed, the mixed solution is magnetically separated to remove the supernatant, and then washed with pre-mixed solution I for 3 times, and resuspended;

[0091] (4) Continue to add 0.3mg of streptavidin to the activated magnetic bead solution, and continue to place in a rotary mixer for 5h of reaction at 37℃ in the dark;

[0092] (5) After the reaction is completed, the supernatant is removed after magnetic separation of the mixed solution, a blocking solution I (10.32 g / L sodium bicarbonate, 9.0 g / L sodium chloride, 4.86 g / L lysine, 10 g / L casein, 0.5 g / L Tween-20, 0.5 g / L ProClean 300, pH = 8.5) is added for resuspension, and the mixture is uniformly mixed on a rotary mixer at 37°C for 2 h in the dark;

[0093] (6) After the reaction is completed, the supernatant is removed after magnetic separation, and a storage solution I (4.77 g / L morpholinoethanesulfonic acid, 3.12 g / L tris(hydroxymethyl)aminomethane (TRIS), 9.0 g / L sodium chloride, 10 g / L fish gelatin, 5 g / L bovine serum albumin, 0.5 g / L ProClean 300, pH = 7.2) is used for washing 3 times, resuspension, and cold storage at 2°C to 8°C for standby;

[0094] The R1 dilution solution comprises:

[0095] 1.36 g / L sodium dihydrogen phosphate, 4.22 g / L disodium hydrogen phosphate, 9.0 g / L sodium chloride, 1.0 g / L polyethylene glycol (PEG-2000), 1.0 g / L Tween-20, 5.0 g / L bovine serum albumin, 0.5 g / L ProClean 300, pH = 7.2;

[0096] The component reagent R2 comprises: the concentration of acridinium ester-labeled HBP antibody 1 in the R2 dilution solution is 3.0 mg / L;

[0097] The acridinium ester-labeled HBP antibody 1 procedure comprises:

[0098] (1) 0.5 mg of HBP antibody 1 is taken into an ultrafiltration tube in a premix solution II (1.56 g / L sodium dihydrogen phosphate, 31.62 g / L disodium hydrogen phosphate, 9.0 g / L sodium chloride, 0.5 g / L ProClean 300, pH = 8.0), and the ultrafiltration tube is placed in a centrifuge for cold storage at 2°C to 8°C, at a speed of 2,000 rpm to 6,000 rpm for 10 min, and is separated and replaced for 3 times, and is transferred to a reaction bottle;

[0099] (2) 0.02 mg of acridine active ester (NSP-SA-NHS) dissolved in dimethyl sulfoxide is continuously added, the mixture is uniformly mixed, and the mixture is uniformly mixed on a rotary mixer at room temperature for 5 h in the dark;

[0100] (3) After the reaction is completed, a blocking solution II (1.56 g / L sodium dihydrogen phosphate, 4.92 g / L disodium hydrogen phosphate, 9.0 g / L sodium chloride, 1.97 g / L glycine, 10 g / L casein, 0.5 g / L Tween-20, 0.5 g / L ProClean 300, pH = 8.0) is added for continuous reaction for 2 h;

[0101] (4) After the reaction is terminated, the reaction solution is purified by a desalting column, and the acridinium-labeled antibody component is collected, concentrated by an ultrafiltration tube, and then added with storage solution II (9.73 g / L morpholinoethanesulfonic acid, 9.0 g / L sodium chloride, 10% glycerol, 5.0 g / L human serum albumin, 0.5 g / L Tween-20, 0.5 g / L ProClean 300, pH = 6.5), and stored at -20°C to -80°C after being divided into aliquots.

[0102] The R2 diluent component is:

[0103] 9.73 g / L morpholinoethanesulfonic acid, 9.0 g / L sodium chloride, 0.5 g / L disodium ethylenediaminetetraacetate, 0.5 g / L Tween-20, 1 g / L lauryl betaine, 0.5 g / L polyvinylpyrrolidone (PVP-8000), 100 mg / L mouse IgG (HBR-5), 100 mg / L mouse IgG (HBR-7), 5 g / L bovine serum albumin, 0.5 g / L ProClean 300, pH = 6.0;

[0104] The R3 reagent component includes: the concentration of biotin-labeled HBP antibody 2 in the R3 diluent is 2.5 mg / L.

[0105] The biotin-labeled HBP antibody 2 procedure is:

[0106] (1) 0.5 mg of HBP antibody 2 is taken in premix solution II, and an ultrafiltration tube is placed in a 2°C to 8°C refrigerated centrifuge at a speed of 2,000 rpm to 6,000 rpm for 10 min, and is separated and replaced for three times, and is transferred to a reaction bottle;

[0107] (2) 0.03 mg of biotin active ester (Biotin-PEG 24 -NHS) dissolved in dimethyl sulfoxide is continuously added, and after mixing, it is placed in a rotary mixer for mixing at room temperature for 5 h in the dark;

[0108] (3) After the reaction is terminated, blocking solution II is added for continuous reaction for 2 h;

[0109] (4) After the reaction is terminated, the reaction solution is purified by a desalting column, and the acridinium-labeled antibody component is collected, concentrated by an ultrafiltration tube, and then added with storage solution II (9.73 g / L morpholinoethanesulfonic acid, 9.0 g / L sodium chloride, 10% glycerol, 5.0 g / L human serum albumin, 0.5 g / L Tween-20, 0.5 g / L ProClean 300, pH = 6.5), and stored at -20°C to -80°C after being divided into aliquots.

[0110] The R3 diluent component is:

[0111] 6.13 g / L tris-hydroxymethyl aminomethane, 9.0 g / L sodium chloride, 0.5 g / L disodium ethylenediaminetetraacetate, 0.5 g / L Tween-20, 5.0 g / L bovine serum albumin, 0.5 g / L ProClean 300, pH = 7.4;

[0112] The components of the calibrator are 6 concentration levels C0~C5:

[0113] The concentrations of HBP in matrix 1 are 5.01 ng / mL, 19.97 ng / mL, 100.36 ng / mL, 502.16 ng / mL and 1997.89 ng / mL, and the concentration of 0 ng / mL is the matrix 1 without serum matrix;

[0114] The components of matrix 1 are:

[0115] 4.83 g / L methanesulfonic acid, 3.08 g / L tris(hydroxymethyl)aminomethane (TRIS), 9.0 g / L sodium chloride, 10 g / L sucrose, 10% newborn calf serum, 5.0 g / L human serum albumin, 0.5 g / L ProClean 300, pH = 7.2.

[0116] The components of the quality control are 2 concentration levels Q1~Q2:

[0117] The target concentrations of HBP in matrix 2 are 40 ng / mL and 600 ng / mL;

[0118] The components of matrix 2 are:

[0119] 4.77 g / L methanesulfonic acid, 3.12 g / L tris(hydroxymethyl)aminomethane (TRIS), 9.0 g / L sodium chloride, 15 g / L trehalose, 20% newborn calf serum, 2.0 g / L human serum albumin, 0.5 g / L ProClean 300, pH = 7.2.

[0120] The components of the pre-priming solution are:

[0121] 3 mM nitric acid, 100 mM hydrochloric acid, 0.25% hydrogen peroxide, 0.48 g / L zinc chloride, 0.5 g / L sodium dodecyl sulfate, pH < 2.

[0122] The components of the priming solution are:

[0123] 10 g / L sodium hydroxide, 4.86 g / L sodium carbonate, 5.32 g / L decyltrimethylammonium chloride, pH > 13.

[0124] The components of the washing solution are: 12.32 g / L sodium dihydrogen phosphate, 31.62 g / L disodium hydrogen phosphate, 9 g / L sodium chloride, 2 g / L Tween-20, 1 g / L ProClean 300, pH = 7.4.

[0125] Example 2

[0126] The application discloses a determination method of a heparin-binding protein determination kit applying a chemiluminescence immunoassay method, wherein reagent calibration comprises the following steps:

[0127] S1, a pre-excitation liquid, an excitation liquid and a cleaning liquid are equipped on a full-automatic chemiluminescence immunoassay instrument; after the instrument is perfused under the action of a peristaltic pump, the heparin-binding protein (HBP) determination kit is placed in a reagent bin position, and is stored on the instrument at 2-8 DEG C; the heparin-binding protein (HBP) determination kit is loaded, and standby is performed for 10-30 minutes;

[0128] The full-automatic chemiluminescence immunoassay instrument is selected from F2000 of G Biotech Co., Ltd.

[0129] S2, after the reagent R1 magnetic beads are fully mixed in the instrument, the HBP calibrators of 6 different concentrations are tested to obtain different luminescence values (RLU), and the working curve after calibration is obtained by four-parameter fitting of the concentration-luminescence value after weighting;

[0130] S3, the determination result of the calibrated reagent is presented in the form of concentration, the quality control sample concentration is continuously tested, and other samples are tested within the target value range.

[0131] Embodiment 3

[0132] A determination method of a heparin-binding protein determination kit applying a chemiluminescence immunoassay method adopts a double-antibody sandwich method reaction mode, each sample test is completed within 10 minutes at the fastest, a full-automatic chemiluminescence immunoassay instrument continuously detects samples, and the detection throughput is as high as 200 samples per hour, and the method comprises the following steps:

[0133] A1, a sample needle S sucks 10 muL of a sample at an application sample track position, a reagent needle A sucks 50 muL of a reagent R2 component at a corresponding project reagent bin position, and is sequentially added into a reaction cup, and is incubated at 37 DEG C for 5 minutes (acridin ester labeled antibody 1 captures HBP in the sample);

[0134] A2, a reagent needle B is automatically perfused and cleaned, 50 muL of a reagent R1 component and a reagent R3 component are sucked at a corresponding reagent position of a reagent bin, and are continuously added into the reaction cup for incubation (to form a double-antibody sandwich product);

[0135] A3, after the incubation is completed, the mixed liquid in the reaction cup is subjected to magnetic separation, a waste liquid needle C1 sucks the supernatant to remove waste liquid, and a sample adding needle D1 continuously adds cleaning liquid to resuspend the liquid surface;

[0136] A4, a sample adding needle D2 continuously adds 100 muL of pre-excitation liquid to mix the liquid surface, and a sample adding needle D3 continuously adds 300 muL of excitation liquid to mix the liquid surface;

[0137] A5, after the reaction starts, the acridinium ester label in the reaction cup emits light, and the light signal is detected by the photomultiplier tube to convert into an electric signal. The stronger the detected electric signal, the stronger the light emission, and the more HBP captured for detection, indicating a positive correlation feedback (acridinium ester reacts with hydrogen peroxide under alkaline conditions to release photons);

[0138] A6, after the light-emitting reaction is over, the waste liquid needle C2 sucks the suspension liquid and discharges into the waste liquid collection pipeline, and the sample cup gripper takes out the reaction cup and puts it into the waste barrel.

[0139] Comparative Example 1

[0140] A kind of heparin binding protein determination kit for application chemical luminescence immunoassay, its raw materials and preparation method are different from example 1 in that short chain Biotin-PEG-NHS is used in biotin labeling process, and the rest is the same as example 1.

[0141] Comparative Example 2

[0142] A kind of heparin binding protein determination kit for application chemical luminescence immunoassay, its raw materials and preparation method are different from example 1 in that short chain Biotin-PEG4-NHS is used in biotin labeling process, and the rest is the same as example 1.

[0143] Comparative Example 3

[0144] A kind of heparin binding protein determination kit for application chemical luminescence immunoassay, its raw materials and preparation method are different from example 1 in that short chain Biotin-PEG8-NHS is used in biotin labeling process, and the rest is the same as example 1.

[0145] Comparative Example 4

[0146] A kind of heparin binding protein determination kit for application chemical luminescence immunoassay, its raw materials and preparation method are different from example 1 in that the component reagent R2 does not add dispersant PVP-8000 and amphoteric surfactant lauryl betaine, and the rest is the same as example 1.

[0147] Comparative Example 5

[0148] A kind of heparin binding protein determination kit for application chemical luminescence immunoassay, its raw materials and preparation method are different from example 1 in that the component reagent R2 does not add blocking agent mouse IgG HBR-5 and mouse IgG HBR-7, and the rest is the same as example 1.

[0149] Comparative Example 6

[0150] The heparin binding protein detection kit based on the H reagent on the market based on chemical luminescence method is purchased as a comparative reagent.

[0151] Experimental Example 1

[0152] Sensitivity:

[0153] The zero-value sample C0 of the calibrator was repeatedly measured for 20 times, and the average luminescence value RLU M0 and the standard deviation SD were calculated. The immediately lower sample C1 (concentration 5.01 ng / mL) of the calibrator was repeatedly measured for 3 times, and the average luminescence value RLU M1 was calculated. According to the regression fitting linear equation y=kx+b of the concentration and luminescence value RLU of the two calibrators, the sensitivity of the kit was obtained by substituting M0+2SD into the equation. As shown in Table 1, the sensitivity results of Comparative Examples 1-3 were 0.16 ng / mL, 0.14 ng / mL, and 0.09 ng / mL, respectively, and the sensitivity of Example 1 was 0.02 ng / mL, and the test result was the best.

[0154] Table 1 Sensitivity Evaluation

[0155]

[0156] Experimental Example 2

[0157] Linear Range:

[0158] The linear high-value sample C5 (concentration 1997.89 ng / mL) was diluted by addition; each sample was tested for 3 times, and the average concentration was taken. The actual value of the test was analyzed by regression fitting linear equation with the theoretical concentration after dilution, and the correlation r value was calculated.

[0159] Figure 2 The HBP detection linear range result graph in the determination method of the heparin binding protein determination kit according to the application was shown in Table 2 and Figure 2 As shown in Table 2 and

[0160] Table 2 Linear Range Evaluation

[0161]

[0162] Experimental Example 3

[0163] Accuracy Evaluation:

[0164] Known sample A: known sample A concentrations (150 ± 15) ng / mL and (50 ± 5) ng / mL, respectively, corresponding to the ratio of A to B added volume of 1:9 when added to low value clinical sample C, so that the added sample concentration is near the medical decision level. Test the sample with the kit of Example 1, each sample concentration is tested 3 times, and the recovery rate is calculated according to formula (1):

[0165] (1)

[0166] Wherein:

[0167] R — recovery rate;

[0168] C s — concentration of known sample A;

[0169] C0— test concentration of serum or plasma sample B;

[0170] C — detection concentration of sample B after adding sample A liquid;

[0171] V — volume of A liquid added;

[0172] V0— volume of serum or plasma sample B;

[0173] As can be seen from Table 3, the addition recovery R of Comparative Example 1~Comparative Example 3 is in the range of 100 ± 15%, 100 ± 15%, and 100 ± 10%, respectively. The addition recovery R of Example 1 (96.4% and 99.3%) is in the range of 100 ± 5%, and the addition recovery accuracy is the highest.

[0174] Table 3 accuracy evaluation

[0175]

[0176] Experimental Example 4

[0177] Precision:

[0178] Test the high and low value quality control samples, clinical serum and plasma samples with the kit of Example 1, each test 10 times, and calculate the within-run coefficient of variation CV according to formula (2):

[0179] (2)

[0180] Wherein:

[0181] CV — coefficient of variation;

[0182] SD — standard deviation;

[0183] M — mean value of calculation results.

[0184] The quality control samples were also tested with the kits of Example 1, and the mean M of 30 tests of the three batches of reagents was calculated 1-3 and the standard deviation SD 1-3 The coefficient of variation CV of batch repeatability was calculated. As shown in the following table, the intra-batch precision of the kit of Example 1 was ≤3.5%, and the inter-batch precision was ≤5%.

[0185] Table 4 Precision evaluation

[0186]

[0187] Experimental Example 5

[0188] Interference:

[0189] The calibration sample C2 (concentration 19.97 ng / mL) was prepared and divided into three groups of identical samples. The control group was added with a blank sample, and the other two groups were added with different concentrations of interferents, so that the final concentrations of the interferents in the samples of the experimental groups were 0.2 g / L and 0.5 g / L bilirubin, 10 g / L and 30 g / L triglyceride, 4 g / L and 10 g / L hemoglobin, 0.5 KIU / mL and 2 KIU / mL rheumatoid factor RF, and 0.05 mg / L and 0.2 mg / L human anti-mouse antibody HAMA. The samples were tested with the kit of Example 1, and each sample was tested 3 times, and the deviation from the control sample without addition was calculated.

[0190] As shown in Table 5, the deviations of the kit of Example 1 were relatively small; the comparative example 4 without the addition of the dispersing agent PVP-8000 and the amphoteric surfactant lauryl betaine had obvious deviations for bilirubin and triglyceride; the comparative example 5 without the addition of the blocking agents HBR-5 and HBR-7 had obvious deviations for RF and HAMA; the control reagent of comparative example 6 had obvious deviations after the addition of large amounts of interferents;

[0191] Table 5 Interference evaluation

[0192]

[0193] Experimental Example 6

[0194] Specificity:

[0195] The serum matrix-free calibration sample C2 (20 ng / mL) was reconstituted and aliquoted, and then the relevant inflammatory infection markers were added. The concentrations of procalcitonin, C-reactive protein, serum amyloid A, interleukin-2 and interleukin-6 in different added samples were 100 ng / L, 50000 ng / L, 50000 ng / L, 100 ng / L and 100 ng / L, respectively. As can be seen from Table 6, Example 1 has substantially no cross-reaction and exhibits high specificity.

[0196] Table 6 Specificity evaluation

[0197]

[0198] Experimental Example 7

[0199] Correlation:

[0200] Eighty clinical samples (serum and plasma) of different concentration ranges were selected, and the reagent kit of Example 1 was selected as the evaluation reagent (the time for testing a single sample was not more than 10 min), and the imported commercial K diagnosis company's enzyme-linked immunoassay kit (the time for testing a single sample was 1 h~2 h) which had the authority representative for detecting HBP was used as the control reagent to detect these samples.

[0201] Figure 3 The results of the correlation between the HBP detection in the determination method of the heparin-binding protein determination kit described in Example 1 and the enzyme-linked immunoassay kit of the K diagnosis company are shown in Table 7 and Figure 3 The linear regression equation is y = 0.973x + 4.4681, r = 0.9965, indicating that the method has good clinical correlation with other methods for detecting reagent kits.

[0202] Table 7 Correlation evaluation

[0203]

[0204] Results and discussion:

[0205] 1. By comparing Example 1 and Comparative Examples 1-3, the heparin-binding protein assay kit for application of chemiluminescent immunoassay in the present application, the reagent R3 labeled antibody is selected by long-chain biotin coupling labeling process. Compared with the short-chain biotin coupling process of Comparative Examples 1-3, the labeling method of Example 1 increases the distance between the magnetic beads and the antibody, indirectly reduces the interference of the magnetic beads on the antibody, and improves the overall sensitivity, linearity and accuracy of the reagent. For different types of samples including quality control products, serum samples and plasma samples, it shows good within-batch precision, and the between-batch precision of quality control products also meets the general line standard (≤10%).

[0206] 2. By comparing Example 1 and Comparative Examples 4-5, the addition of dispersants, two kinds of surfactants and blocking agents in the reagent R2 diluent formula of Example 1 significantly reduces the exogenous interference of triglycerides, bilirubin and hemoglobin, and the endogenous interference of rheumatoid factor (RF) and human anti-mouse antibody (HAMA) heterophilic antibodies.

[0207] 3. By comparing Example 1 and Comparative Example 6, Example 1 has strong anti-interference ability. When relevant inflammatory infection occurs, the relevant inflammatory factor index rises rapidly, and the specificity of the detection marker: serum amyloid A, C-reactive protein, procalcitonin, interleukin-2 and interleukin-6 has no obvious cross reaction.

[0208] 4. In terms of clinical performance, the reagent kit and the enzyme-linked immunoassay kit of K Diagnosis Company have good correlation in clinical sample results. In terms of methodology, the reagent kit test speed is fast and can meet the actual hospital detection needs (① The final result of the diagnostic reagent test has traceability, which provides a basis for clinical diagnosis; ② The reagent diagnostic products of some foreign companies are highly authoritative (see the description above), and the price is high. Most hospitals will use clinically equivalent domestic relatively low-priced products to save costs; ③ Most related patents will be compared with this company for correlation, and will not be deliberately compared with some domestic reagents).

Claims

1. A chemiluminescent heparin-binding protein kit, characterized in that: This is a heparin-binding protein determination kit using chemiluminescent immunoassay, including: reagent R1, reagent R2 and reagent R3, as well as matching calibrators, quality control products, pre-excitation solution, excitation solution and cleaning solution; The components of reagent R1 include streptavidin magnetic beads; The components of the reagent R2 include: 0.5 mg / L~8 mg / L acridinium ester HBP antibody 1 marker, 2 g / L~80 g / L buffer salt, 5 g / L~30 g / L buffer stabilizer, 0.5 g / L~5 g / L chelating agent, 0.5 g / L~5 g / L dispersant, 0.5 g / L~5 g / L surfactant, 10 mg / L~500 mg / L blocking agent, 2 g / L~10 g / L protein protectant, 0.5 g / L~2 g / L preservative, and R2 diluent with a pH of 5.0~7.

0. The acridinium ester HBP antibody 1 marker is acridine active ester coupled to the HBP antibody; The dispersant is at least one of polyethylene glycol PEG-2000, polyethylene glycol PEG-4000, polyethylene glycol PEG-6000, polyvinyl pyrrolidone PVP-8000, polyvinyl pyrrolidone PVP-10000, and polyvinyl pyrrolidone PVP-30000; The surfactant is at least one of Tween-20, Tween-80, Triton X-100, lauryl betaine, and 3-sulfopropyl hexadecyl dimethyl betaine; The blocking agent is at least one of mouse immunoglobulin, sheep immunoglobulin, bovine immunoglobulin, and immunoglobulins of the same species that recognize different epitopes; The components of reagent R3 include biotin HBP antibody 2 marker, wherein the biotin HBP antibody 2 marker is a biotin active ester coupled to the HBP antibody, wherein the biotin active ester is Biotin-PEG 24 -NHS; The buffer stabilizer is at least one of sodium chloride, potassium chloride, calcium chloride and calcium sulfate.

2. A chemiluminescent heparin-binding protein kit according to claim 1, characterized in that: The components of the reagent R1 include: 0.1 g / L~1.0 g / L streptavidin magnetic beads, 2 g / L~80 g / L buffer salt, 5 g / L~30 g / L buffer stabilizer, 0.5 g / L~3 g / L dispersant, 0.5 g / L~5 g / L surfactant, 2 g / L~20 g / L protein protectant, 0.5 g / L~2 g / L preservative, and R1 diluent with a pH of 6.0~8.0; The components of the reagent R3 include: 0.5 mg / L~8 mg / L biotin HBP antibody 2 marker, 2 g / L~80 g / L buffer salt, 5 g / L~30 g / L buffer stabilizer, 0.5 g / L~5 g / L chelating agent, 0.5 g / L~5 g / L surfactant, 2 g / L~20 g / L protein protective agent, 0.5 g / L~2 g / L preservative, and R3 diluent with a pH of 6.0~8.0; The calibrator comprises a matrix 1 containing different concentrations of HBP, the concentrations of which are 0 ng / mL, 5.01 ng / mL, 19.97 ng / mL, 100.36 ng / mL, 502.16 ng / mL, and 1997.89 ng / mL; the matrix 1 comprises 2 g / L to 80 g / L of buffer salt, 5 g / L to 30 g / L of buffer stabilizer, 5 g / L to 30 g / L of carbohydrate stabilizer, 0% v / v to 30% v / v of serum matrix, 2 g / L to 20 g / L of protein protectant, and 0.5 g / L to 2 g / L of preservative, with a pH value of 6.0 to 8.0; The quality control product comprises a matrix 2 containing different concentrations of HBP, the concentrations of which are 40±4 ng / mL and 600±60 ng / mL respectively; the matrix 2 contains 2 g / L~80 g / L of buffer salt, 5 g / L~30 g / L of buffer stabilizer, 3 g / L~30 g / L of carbohydrate stabilizer, 10% v / v~30% v / v of serum matrix, 2 g / L~10 g / L of protein protectant, and 0.5 g / L~2 g / L of preservative, with a pH value of 6.0~8.0; The pre-excitation solution comprises 20 mM to 100 mM nitric acid, 30 mM to 200 mM hydrochloric acid, 0.1% to 0.5% w / v hydrogen peroxide, 0.1 g / L to 10 g / L zinc chloride, and 0.5 g / L to 2 g / L sodium dodecyl sulfate, with a pH of <2. The exciting solution comprises: 4 g / L to 20 g / L sodium hydroxide, 1 g / L to 10 g / L sodium carbonate, and 1 g / L to 10 g / L decyltrimethylammonium chloride, with a pH value greater than 13; The cleaning solution comprises: 1 g / L to 15 g / L sodium dihydrogen phosphate, 10 g / L to 60 g / L sodium dihydrogen phosphate, 5 g / L to 30 g / L sodium chloride, 0.5 g / L to 10 g / L Tween-20, 0.5 g / L to 2 g / L ProClean 300, and a pH of 7.4±0.

4.

3. A chemiluminescent heparin-binding protein kit according to claim 2, characterized in that: The buffer salt is at least one of sodium dihydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, morpholineethanesulfonic acid, hydroxyethylpiperazineethanesulfonic acid, tris(hydroxymethyl)aminomethane, and bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane; The carbohydrate stabilizer is at least one of sucrose, trehalose, dextran and mannose.

4. A chemiluminescent heparin-binding protein kit according to claim 2, characterized in that: The chelating agent is at least one of disodium EDTA, dipotassium EDTA, sodium citrate, and sodium oxalate; The serum matrix is ​​at least one of horse serum, goat serum, and newborn calf serum; The protein protective agent is at least one of bovine serum albumin, human serum albumin, casein, sodium caseinate, and ovalbumin; The preservative is at least one of ProClean 300, ProClean 950, methylisothiazolinone, and sodium benzoate.

5. A chemiluminescent heparin-binding protein kit according to claim 2, characterized in that: The streptavidin magnetic beads described in reagent R1 are obtained by coating streptavidin on solid phase magnetic beads. The preparation method includes the following steps: (1) Take 5 mg to 50 mg of carboxyl magnetic beads, remove the supernatant after magnetic separation, wash three times with premixed solution I, and resuspend; (2) Continue to add 0.2 g / L~5 g / L of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.2 g / L~5 g / L of N-hydroxysuccinimide, mix well, and place on a rotary mixer in the dark at room temperature for 0.5 h~3 h; (3) After the reaction is completed, the mixed solution is magnetically separated and the supernatant is removed. The mixture is washed three times with premixed solution I and resuspended. (4) Continue to add 0.025 mg~2.50 mg of streptavidin to the activated magnetic bead solution, continue to place it on a rotating mixer in the dark at 37°C and mix for 2 h~10 h; (5) After the reaction is completed, the mixed solution is magnetically separated and the supernatant is removed. Blocking solution I is added to resuspend the mixture and the mixture is placed on a rotating mixer in the dark at 37°C for 0.5 h to 3 h. (6) After the reaction is completed, remove the supernatant after magnetic separation, wash three times with preservation solution I, resuspend, and refrigerate at 2℃~8℃ for future use.

6. A chemiluminescent heparin-binding protein kit according to claim 5, characterized in that: The premix I component includes 5.0 g / L~50 g / L sodium bicarbonate, 5 g / L~30 g / L sodium chloride, and 0.5 g / L~2 g / L ProClean 300, with a pH of 7.0~9.0; the blocking solution I component includes 5.0 g / L~50 g / L sodium bicarbonate, 5 g / L~30 g / L sodium chloride, 1 g / L~20 g / L lysine, 2 g / L~20 g / L casein, 0.5 g / L~2 g / L Tween-20, and 0.5 g / L~2 g / L ProClean 300, with a pH of 7.0~9.0; The components of the preservation solution I include 1 g / L~15 g / L sodium dihydrogen phosphate, 10 g / L~60 g / L disodium hydrogen phosphate, 5 g / L~30 g / L sodium chloride, 2 g / L~50 g / L fish gelatin, 1 g / L~10 g / L bovine serum albumin, 0.5 g / L~2g / L ProClean 300, pH=7.4±0.

4.

7. A chemiluminescent heparin-binding protein kit according to claim 2, characterized in that: The acridinium ester HBP antibody 1 marker described in reagent R2 is obtained by coupling an acridinium active ester to an HBP antibody. The preparation method comprises the following steps: (1) Take 0.2 mg~2 mg of HBP antibody 1 in premixed solution II, place it in a refrigerated centrifuge at 2℃~8℃ with an ultrafiltration tube, speed 2000rpm~6000rpm, for 10 minutes, separate and replace 3 times, and transfer to the reaction bottle; (2) Continue to add 0.003 mg~0.3 mg of acridinium active ester dissolved in dimethyl sulfoxide, with the molar ratio of antibody to acridinium ester being 1:3~1:30, mix well, and place on a rotary mixer in the dark at room temperature for 2h~10h; (3) After the reaction is completed, add blocking solution II and continue the reaction for 0.5h~3h; (4) After the reaction is terminated, the acridine-labeled antibody component is purified by desalting column, concentrated by ultrafiltration tube, and added to preservation solution II. After packaging, it is frozen and stored at -20℃~-80℃ for future use.

8. A chemiluminescent heparin-binding protein kit according to claim 2, characterized in that: The biotin-HBP antibody 2 marker described in reagent R3 is obtained by coupling a biotin active ester to an HBP antibody, and the preparation method comprises the following steps: (1) Take 0.2 mg~2 mg of HBP antibody 2 in premix II, place it in a 2℃~8℃ refrigerated centrifuge with an ultrafiltration tube, speed 2000 rpm~6000 rpm, 30 min, separate and replace 3 times, and transfer to the reaction bottle; (2) Continue to add 0.006~0.6mg of biotin active ester dissolved in dimethyl sulfoxide, with the molar ratio of antibody to biotin being 1:3~1:30, mix well, and place on a rotating mixer in the dark at room temperature for 2h~10h; (3) After the reaction is completed, add blocking solution II and continue the reaction for 0.5h~3h; (4) After the reaction is terminated, the biotin-labeled antibody components are collected by desalting column purification, concentrated by ultrafiltration tube, and added to preservation solution II. After packaging, they are frozen and stored at -20℃~-80℃ for future use.

9. A chemiluminescent heparin-binding protein kit according to claim 7 or 8, characterized in that: in, The premix II component includes 1 g / L~15 g / L sodium dihydrogen phosphate, 10 g / L~60 g / L disodium hydrogen phosphate, 5 g / L~30 g / L sodium chloride, 0.5 g / L~2 g / L ProClean 300, and a pH of 7.0~8.5; The blocking solution II component includes 1 g / L~15 g / L sodium dihydrogen phosphate, 10 g / L~60 g / L sodium dihydrogen phosphate, 5 g / L~30 g / L sodium chloride, 0.5 g / L~10 g / L glycine, 2 g / L~20 g / L casein, 0.5 g / L~2 g / L Tween-20, and 0.5 g / L~2 g / L ProClean 300, with a pH of 7.0~8.5; The components of the preservation solution II include 4 g / L~40 g / L morpholineethanesulfonic acid, 3 g / L~30 g / L tris(hydroxymethyl)aminomethane, 5 g / L~30 g / L sodium chloride, 2%~30% v / v glycerol, 2 g / L~20 g / L human serum albumin, 0.5 g / L~2 g / L Tween-20, and 0.5 g / L~2 g / L ProClean 300, with a pH of 5.0~7.0.

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