Chemiluminescence heparin binding protein kit and determination method thereof

Abstract

The invention discloses a chemiluminescence heparin binding protein kit and a determination method thereof. The kit comprises reagent components of a reagent R1, a reagent R2 and a reagent R3, as well as a matched calibrator, a matched quality control product, a matched pre-excitation liquid, a matched excitation liquid and a matched cleaning liquid, wherein the reagent R1 contains magnetic particles coated with streptavidin, and the reagent R2 and the reagent R3 are respectively monoclonal antibodies which are labeled by acridinium ester and biotin and are used for recognizing different epitopes of human HBP. The kit shows better sensitivity, wider 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 invention belongs to the technical field of immunological detection, and specifically relates to a chemiluminescent heparin-binding protein kit and a determination method thereof, and also relates to a heparin-binding protein determination kit using a chemiluminescent immunoassay and a determination method thereof. Background Art

[0002] Heparin-binding protein (HBP) was first isolated from neutrophil azurophilic granules by Shafer et al. in 1984. Due to its molecular weight of 37 kDa and its cationic antimicrobial properties, it was named CAP37 (Cationic Antimicrobial Protein 37). Subsequently, Gabay et al. named it azurocidin. Further studies revealed that its structure contains 18 arginine residues, which confer antimicrobial activity and heparin-binding capacity, ultimately leading to its designation as heparin-binding protein (HBP). In response to bacterial infection or inflammation, neutrophils rapidly release HBP extracellularly through degranulation, with elevated concentrations becoming detectable in the blood within 1-2 hours. HBP binds to heparan sulfate on the surface of vascular endothelial cells, activating the intracellular RhoA / ROCK signaling pathway, leading to cytoskeletal reorganization, tight junction disruption, and a significant increase in vascular permeability (manifesting as "capillary leak syndrome"). HBP can also stimulate monocytes to release pro-inflammatory cytokines (such as IL-6 and TNF-α), while simultaneously inducing neutrophil chemotaxis, forming a positive feedback loop that exacerbates systemic inflammatory responses. Furthermore, HBP can inhibit tissue factor pathway inhibitor (TFPI), promoting thrombin generation and exacerbating sepsis-related disseminated intravascular coagulation (DIC). Therefore, as HBP is rapidly released into the bloodstream, it intensifies the inflammatory response by inducing increased endothelial cell permeability and monocyte activation, playing a key role in pathological processes such as sepsis and septic shock, becoming a core biomarker for these critical illnesses.

[0003] In bacterial infections and sepsis, HBP rises significantly within 1-2 hours of infection. Its specificity is higher than that of viral or non-infectious inflammation, making it a key indicator for early warning of sepsis. As a complementary indicator for the early diagnosis of sepsis, HBP testing within 6 hours can guide antibiotic use and help reduce antibiotic overuse. Testing can also be performed on cerebrospinal fluid or urine to localize infection and aid in the diagnosis of meningitis and urinary tract infections. Studies have shown that HBP can be detected as early as 72 hours before the onset of sepsis, earlier than traditional markers such as C-reactive protein and procalcitonin. HBP has a short half-life (<1 hour), and its levels are positively correlated with infection severity, allowing for dynamic monitoring and real-time assessment of infection risk and control effectiveness. As an "early, specific, and dynamic" marker, HBP is recommended for rapid differential diagnosis of bacterial infections, early warning of sepsis and monitoring of efficacy, and guidance on appropriate antibiotic use to reduce drug resistance.

[0004] Regarding the detection and analysis of HBP in clinical samples from hospital patients, the HBP concentration in 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 advanced diseases such as multiple organ failure or refractory shock, the HBP concentration may even exceed 1000 ng / mL.

[0005] Currently, the most authoritative HBP detection method is an enzyme-linked immunosorbent assay (ELISA) kit from a diagnostic company. However, this method is time-consuming (1–2 hours), making it difficult to meet the "golden window" for early sepsis diagnosis. Imported diagnostic reagents are generally expensive. Domestic manufacturers also offer other methods for HBP detection, such as immunochromatography, latex-enhanced immunoturbidimetry, and magnetic microparticle chemiluminescence. However, most methods lack sensitivity and specificity, and their detection range falls short of the required range. Traditional methods are prone to false negatives in low-concentration samples, and the test throughput cannot meet the requirements for testing large numbers of samples in a short period of time. Furthermore, some methods are highly equipment-dependent and require specialized instruments, limiting their application in primary care hospitals.

[0006] CN112858696A and CN204882575U both disclose immunofluorescence chromatography kits for detecting HBP. While simple to operate, these kits lack high sensitivity and a narrow linear range, enabling only qualitative and semi-quantitative detection. The accuracy of the test results does not fully meet the clinical needs for quantitative testing. CN204882575U and CN108956978A also disclose latex-enhanced immunoturbidimetry and magnetic microparticle chemiluminescence kits for detecting HBP, respectively. Summary of the Invention

[0007] In response to the increasing performance requirements for test kits in actual clinical diagnosis in existing HBP detection technologies, the present invention provides a chemiluminescent heparin-binding protein (HBP) assay kit and its assay method, as well as a HBP assay kit and its assay method using a chemiluminescent immunoassay. These kits aim to provide medical professionals with a test kit that delivers high accuracy, a high degree of automation, and high efficiency, meeting the needs of testing serum and plasma samples. Furthermore, they offer research and technical personnel a preparation method and strategy for optimizing and improving the performance of HBP assay kits.

[0008] The technical solutions of the present invention are as follows: The present invention provides a chemiluminescent heparin-binding protein kit, also a heparin-binding protein assay kit using a chemiluminescent immunoassay. The kit comprises reagent components R1, R2, and R3, as well as matching calibrators, quality control products, a pre-excitation solution, an excitation solution, and a cleaning solution. Reagent R1 comprises streptavidin-coated magnetic microparticles, while reagents R2 and R3 are monoclonal antibodies that recognize different epitopes of human HBP, labeled with an acridinium ester and biotin, respectively. The present invention illustrates the complete preparation process for a chemiluminescent HBP assay kit. The assay kit exhibits good sensitivity and a wide detection range, can effectively reduce potential endogenous and exogenous interference in clinical samples from potentially interfering populations, and has high specificity.

[0009] The purpose of the present invention is achieved through the following technical solutions: A chemiluminescent heparin-binding protein kit, namely a heparin-binding protein (HBP) determination kit using a chemiluminescent immunoassay, comprises: reagent components reagent R1, reagent R2 and reagent R3 as well as matching calibrators, quality control products, pre-excitation solution, excitation solution and cleaning solution.

[0010] 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 in R1 diluent with a pH of 6.0~8.0, 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, and 0.5 g / L~2 g / L preservative; The components of the reagent R2 include: 0.5 mg / L to 8 mg / L acridinium ester HBP antibody 1 marker, 2 g / L to 80 g / L buffer salt in R2 diluent with a pH of 5.0 to 7.0, 5 g / L to 30 g / L buffer stabilizer, 0.5 g / L to 5 g / L chelating agent, 0.5 g / L to 5 g / L dispersant, 0.5 g / L to 5 g / L surfactant, 10 mg / L to 500 mg / L blocking agent, 2 g / L to 10 g / L protein protectant, and 0.5 g / L to 2 g / L preservative; The components of the reagent R3 include: 0.5 mg / L to 8 mg / L of biotin HBP antibody 2 marker, 2 g / L to 80 g / L of buffer salt in R3 diluent with a pH of 6.0 to 8.0, 5 g / L to 30 g / L of buffer stabilizer, 0.5 g / L to 5 g / L of chelating agent, 0.5 g / L to 5 g / L of surfactant, 2 g / L to 20 g / L of protein protective agent, and 0.5 g / L to 2 g / L of preservative; The calibrator comprises a matrix 1 of HBP at different concentrations, namely 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 control product comprises a matrix 2 of HBP at different concentrations, namely (40±4) ng / mL and (600±60) ng / mL; 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% hydrogen peroxide (w / v), 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.

[0011] Furthermore, the buffer salt is at least one of sodium dihydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, morpholineethanesulfonic acid (MES), hydroxyethylpiperazineethanesulfonic acid (HEPES), tris(hydroxymethyl)aminomethane (TRIS), and bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (BIS-TRIS); The buffer stabilizer is at least one of sodium chloride, potassium chloride, calcium chloride, and calcium sulfate; 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.

[0012] Furthermore, the chelating agent is at least one of disodium ethylenediaminetetraacetic acid (EDTA), dipotassium ethylenediaminetetraacetic acid (EDTA), sodium citrate, and sodium oxalate; 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 carbohydrate stabilizer is at least one of sucrose, trehalose, dextran, and mannose; 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 (MIT), and sodium benzoate.

[0013] The above-mentioned heparin-binding protein (HBP) assay kit using chemiluminescent immunoassay has a reagent sensitivity of ≤0.05 ng / mL for HBP detection; a linear range of 0.05 ng / mL to 2000 ng / mL; trueness and recovery within 100%±5%; intra-batch precision <3.5%, and inter-batch precision <5%; interference bias rate: bilirubin, triglycerides, hemoglobin, and rheumatoid factor (RF) are within the range of ±5%, and human anti-mouse antibody (HAMA) is within the range of ±6%; specificity for inflammatory infection markers: serum amyloid A, C-reactive protein, procalcitonin, interleukin-2, and interleukin-6 have basically no cross-reactivity.

[0014] Furthermore, the streptavidin magnetic beads described in reagent R1 are obtained by coating streptavidin on solid phase magnetic beads, and 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 (EDC·HCl) and 0.2 g / L~5 g / L of N-hydroxysuccinimide (NHS), 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 (streptavidin coating mass fraction 0.5%~5%) 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 store in a refrigerator at 2°C~8°C for later use; 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 sodium dihydrogen 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, and pH=7.4±0.4.

[0015] Furthermore, the acridinium ester HBP antibody 1 marker described in reagent R2 is obtained by coupling acridinium active ester to HBP antibody to obtain the acridinium ester HBP antibody 1 marker, and the preparation method includes the following steps: (1) Take 0.2 mg~2 mg of HBP antibody 1 in premix II, place it in a refrigerated centrifuge at 2℃~8℃ with an ultrafiltration tube, speed 2000rpm~6000rpm, 10min, 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 (antibody to acridinium ester molar ratio 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.

[0016] Furthermore, the biotin-HBP antibody 2 marker described in reagent R3 is obtained by coupling a biotin active ester to an HBP antibody to obtain a biotin-HBP antibody 2 marker, and the preparation method includes 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, 30min, 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 (antibody to biotin molar ratio 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, purify with a desalting column, collect the acridine-labeled antibody fraction, concentrate with an ultrafiltration tube, add preservation solution II, and store in a cryopreservation chamber at -20°C to -80°C for later use; 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 (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, and 0.5 g / L~2 g / L ProClean 300, with a pH of 5.0~7.0.

[0017] The present invention also provides a method for determining heparin binding protein (HBP) using a chemiluminescent immunoassay kit, wherein the reagent calibration comprises the following steps: S1. Prepare the pre-excitation solution, excitation solution, and cleaning solution on the fully automatic chemiluminescence immunoassay analyzer. After the instrument is primed with a peristaltic pump, place the heparin-binding protein (HBP) assay kit in the reagent compartment, maintain the instrument at 2°C to 8°C, load the heparin-binding protein (HBP) assay kit, and wait for 10 to 30 minutes. S2: After the instrument fully mixes the reagent R1 magnetic beads, it tests six HBP calibrators of different concentrations to obtain different luminescence values ​​(RLU). The calibrator concentration-luminescence value is weighted by four-parameter fitting to obtain the corresponding calibrated working curve; S3. The results of the calibrated reagents are presented in the form of concentration. Continue to test the concentration of the quality control samples and conduct other sample tests within the target value range.

[0018] Furthermore, the fully automatic chemiluminescence immunoassay analyzer described in S1 is selected from the F2000, F180, F120 of G Biotechnology Co., Ltd., SMART 6500 of I Company and Shine i9000 of J Biotechnology Co., Ltd., preferably the fully automatic chemiluminescence immunoassay analyzer F2000 produced by G Biotechnology Co., Ltd.

[0019] Furthermore, the present invention also provides a method for determining HBP using the chemiluminescent heparin-binding protein kit, which is also a method for determining HBP using a chemiluminescent immunoassay. The method employs a double-antibody sandwich reaction mode, wherein each sample is tested within 10 minutes at the fastest. The fully automatic chemiluminescent immunoassay analyzer continuously detects samples, with a detection throughput of up to 200 samples per hour. The method comprises the following steps: A1. Sample needle S draws 10 μL of sample at the sample application track position, and reagent needle A draws 50 μL of reagent R2 component at the corresponding project reagent position. These components are added to the reaction cup in sequence and incubated at 37°C for 5 minutes (acridinium ester-labeled antibody 1 captures HBP in the sample). A2. Reagent needle B is automatically perfused and cleaned. 50 μL of reagent components R1 and R3 are drawn from the corresponding reagent positions in the reagent compartment and added to the reaction cup for incubation (forming a double antibody sandwich product). A3. After the incubation is completed, the mixed solution in the reaction cup is magnetically separated. After the waste liquid is removed by the waste liquid needle C1, the sample needle D1 continues to add cleaning solution to the liquid surface to resuspend it; A4, pipette D2 continues to add 100 μL of pre-stimulation solution and pipette the liquid surface to mix. Pipet D3 continues to add 300 μL of stimulation solution and pipette the liquid surface to mix. A5. The reaction begins, and the acridinium ester marker in the reaction cup emits light. The photomultiplier tube detects the light signal and converts it into an electrical signal. The stronger the electrical signal, the stronger the luminescence, and the more HBP to be detected is captured, indicating that the sample contains more HBP, which is a positive feedback loop (acridinium ester reacts with hydrogen peroxide under alkaline conditions to release photons). A6. After the luminescence reaction is completed, the waste liquid needle C2 absorbs the suspension and discharges it into the waste liquid collection pipe. The sample cup gripper takes out the reaction cup and puts it into the waste bucket.

[0020] Compared with the prior art, the present invention has the following beneficial effects: 1. The heparin-binding protein (HBP) assay kit using chemiluminescent immunoassay described in the present invention uses a long-chain biotin coupling labeling process to reduce the interference of magnetic beads on antibodies and improve the sensitivity of the reagent, with the sensitivity not exceeding 0.05 ng / mL.

[0021] 2. The present invention discloses a heparin-binding protein (HBP) assay kit using a chemiluminescent immunoassay. The addition of a dispersant and amphoteric surfactants to reagent R2 significantly reduces exogenous interference from triglycerides, bilirubin, and hemoglobin. The addition of a blocking agent to reagent R2 significantly reduces the deviation rate from endogenous interference from rheumatoid factor (RF) and human anti-mouse antibodies (HAMA). The overall deviation for triglycerides, bilirubin, hemoglobin, and RF is within ±5%, and for HAMA is within ±6%.

[0022] 3. The heparin-binding protein (HBP) assay kit using chemiluminescence immunoassay described in the present invention shows that when related inflammatory infections occur, the related inflammatory factor indicators rise rapidly, and the specificity of the detection markers: serum amyloid A, C-reactive protein, procalcitonin, interleukin-2, and interleukin-6 have basically no obvious cross-reaction.

[0023] 4. The heparin-binding protein (HBP) assay kit using chemiluminescent immunoassay described in the present invention has good correlation with clinical samples using the control reagent enzyme-linked immunosorbent assay kit imported from the British company K Diagnostics, and is suitable for testing serum and plasma samples. The kit is compatible with various models of fully automatic chemiluminescent immunoassay analyzers, including the F2000, F180, and F120 from G Biotechnology Co., Ltd., the SMART 6500 from I Company, and the Shine i9000 from J Biotechnology Co., Ltd., meeting the needs of large-scale sample testing.

[0024] 5. The assay method of the heparin-binding protein (HBP) assay kit using chemiluminescent immunoassay described in the present invention adopts a suitable antibody labeling process to improve the linear range width, with the detection range being 0.05 ng / mL~2000 ng / mL; the accuracy of the spike recovery is improved to within 100%±5%; the intra-batch precision is <3.5%, and the inter-batch precision is <5%. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used together with the embodiments of the present invention to explain the present invention but do not constitute a limitation of the present invention.

[0026] Figure 1This is a schematic diagram of the HBP detection principle of a heparin binding protein assay kit using a chemiluminescent immunoassay according to an embodiment of the present invention; Figure 2 This is a graph showing the linear range of HBP detection in a heparin-binding protein assay kit using a chemiluminescent immunoassay as described in an embodiment of the present invention; Figure 3 This is a graph showing the correlation results between HBP detection in the assay method of a heparin-binding protein assay kit using chemiluminescent immunoassay as described in Example 1 of the present invention and the enzyme-linked immunosorbent assay kit of K Diagnostics Company. DETAILED DESCRIPTION

[0027] In order to facilitate the understanding of the technical content and technical effects of the present invention by relevant technical personnel, the present invention is further explained below in conjunction with specific embodiments. The described embodiments are only a part of the implementation form cases of the present invention, which are only preferred embodiments of the present invention and are not intended to limit the scope of protection claimed by the present invention. Based on the cases in the implementation mode, other embodiments obtained by those skilled in the art without making creative work are all within the scope of protection of the present invention.

[0028] The labeled HBP antibodies are all mouse anti-human HBP monoclonal antibodies with high immune reaction specificity, purchased from A Biotechnology Co., Ltd. and B Medical Technology Co., Ltd.; The markers biotin active ester and streptavidin were purchased from C Biochemical Technology Co., Ltd.; Magnetic particles were purchased from D Pharmaceutical Excipient Company; Acridinium ester was purchased from E Biochemical Technology Co., Ltd.; The serum matrix was purchased from F Biotechnology Co., Ltd.

[0029] Unless otherwise specified, the experimental methods in the following examples are all conventional methods. The experimental materials and kits in the following examples are all purchased from conventional suppliers in the market unless otherwise specified.

[0030] The results of the following examples were determined mainly using the fully automatic chemiluminescence immunoassay analyzer F2000 produced by G Biotechnology Co., Ltd.

[0031] Example 1 A heparin binding protein assay kit using chemiluminescent immunoassay, the HBP assay principle diagram is as follows Figure 1 As shown, it includes reagent components R1, R2 and R3, as well as matching calibrators, quality control products, pre-excitation solution, excitation solution and cleaning solution; In the process of preparing the solutions of each component, 2M hydrochloric acid and 2M sodium hydroxide were used to adjust the pH of the system.

[0032] Reagent components Reagent R1 component: Streptavidin magnetic beads in R1 diluent at a concentration of 0.35 g / L; The steps for preparing streptavidin magnetic beads are as follows: (1) Take 20 mg of carboxyl magnetic beads, remove the supernatant after magnetic separation, wash three times with premixed solution I (10.32 g / L sodium bicarbonate, 9.0 g / L sodium chloride, 0.5 g / L ProClean 300, pH = 8.5), and resuspend; (2) Continue to add 1.82 g / L of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and 0.96 g / L of N-hydroxysuccinimide (NHS), mix well, and place on a rotary mixer in the dark at room temperature for 2 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) Add 0.3 mg of streptavidin to the activated magnetic bead solution and continue mixing on a rotating mixer in the dark at 37°C for 5 h. (5) After the reaction, the mixed solution was magnetically separated and the supernatant was removed. 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 ProClean300, pH = 8.5) was added to resuspend the mixture and continued to be placed on a rotating mixer in the dark at 37 °C for 2 h. (6) After the reaction, remove the supernatant after magnetic separation, wash three times with preservation solution I (4.77 g / L morpholineethanesulfonic 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), resuspend, and store in a refrigerator at 2°C to 8°C for later use; The components of R1 diluent are: 1.36 g / L sodium dihydrogen phosphate, 4.22 g / L sodium dihydrogen 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; Components of reagent R2 include: Acridinium ester-labeled HBP antibody 1 at a concentration of 3.0 mg / L in R2 diluent; The steps for labeling HBP antibody with acridinium ester 1 are: (1) Take 0.5 mg of HBP antibody 1 and dissolve it in premix II (1.56 g / L sodium dihydrogen phosphate, 31.62 g / L sodium dihydrogen phosphate, 9.0 g / L sodium chloride, 0.5 g / L ProClean 300, pH = 8.0). Place the ultrafiltration tube in a refrigerated centrifuge at 2°C to 8°C, at a speed of 2000 rpm to 6000 rpm for 10 min, and separate and replace 3 times before transferring to the reaction bottle; (2) Continue to add 0.02 mg of acridine active ester (NSP-SA-NHS) dissolved in dimethyl sulfoxide, mix well, and place on a rotary mixer in the dark at room temperature for 5 hours; (3) After the reaction is completed, add blocking solution II (1.56 g / L sodium dihydrogen phosphate, 4.92 g / L sodium dihydrogen 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) and continue the reaction for 2 h; (4) After the termination reaction, the acridine-labeled antibody fraction was collected by desalting column purification, concentrated by ultrafiltration tube, and added to preservation solution II (9.73 g / L morpholineethanesulfonic 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). After aliquoting, it was frozen and stored at -20℃~-80℃ for later use.

[0033] The components of R2 diluent are: 9.73 g / L morpholineethanesulfonic acid, 9.0 g / L sodium chloride, 0.5 g / L disodium ethylenediaminetetraacetic acid, 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; Components of reagent R3 include: biotinylated HBP antibody 2 at a concentration of 2.5 mg / L in R3 diluent; The steps for biotin labeling HBP antibody 2 are: (1) Take 0.5 mg of HBP antibody 2 and mix it with premix II. Place the ultrafiltration tube in a refrigerated centrifuge at 2°C~8°C, at a speed of 2000 rpm~6000 rpm, for 10 min, separate and replace 3 times, and transfer to the reaction bottle; (2) Continue to add biotin active ester (Biotin-PEG 24 -NHS) 0.03 mg, mix well and place on a rotary mixer in the dark at room temperature for 5 h; (3) After the reaction is completed, add blocking solution II and continue the reaction for 2 hours; (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.

[0034] The components of R3 diluent are: 6.13 g / L tris(hydroxymethyl)aminomethane, 9.0 g / L sodium chloride, 0.5 g / L disodium EDTA, 0.5 g / L Tween-20, 5.0 g / L bovine serum albumin, 0.5 g / L ProClean 300, pH = 7.4; The components of the calibrator are divided into 6 concentration levels C0~C5: The concentrations of HBP in matrix 1 were 5.01 ng / mL, 19.97 ng / mL, 100.36 ng / mL, 502.16 ng / mL, and 1997.89 ng / mL, and the concentration was 0 ng / mL in matrix 1 without serum matrix; Matrix 1 component consists of: 4.83 g / L morpholineethanesulfonic 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.

[0035] The components of the quality control product are divided into two concentration levels Q1~Q2: The target concentrations of HBP in matrix 2 were 40 ng / mL and 600 ng / mL; Matrix 2 components are: 4.77 g / L morpholineethanesulfonic 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.

[0036] The components of the pre-excitation solution are: 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.

[0037] The components of the excitation solution are: 10g / L sodium hydroxide, 4.86g / L sodium carbonate, 5.32g / L decyltrimethylammonium chloride, pH>13.

[0038] The components of the washing solution are: 12.32 g / L sodium dihydrogen phosphate, 31.62 g / L sodium dihydrogen phosphate, 9 g / L sodium chloride, 2 g / L Tween-20, 1 g / L ProClean 300, pH=7.4.

[0039] Example 2 A method for determining heparin binding protein using a chemiluminescent immunoassay kit, wherein the reagent calibration comprises the following steps: S1. Prepare the pre-excitation solution, excitation solution, and cleaning solution on the fully automatic chemiluminescence immunoassay analyzer. After the instrument is primed with a peristaltic pump, place the heparin-binding protein (HBP) assay kit in the reagent compartment, maintain the instrument at 2°C to 8°C, load the heparin-binding protein (HBP) assay kit, and wait for 10 to 30 minutes. The fully automatic chemiluminescence immunoassay analyzer is model F2000 from G Biotechnology Co., Ltd. S2: After the instrument fully mixes the reagent R1 magnetic beads, it tests six HBP calibrators of different concentrations to obtain different luminescence values ​​(RLU). The calibrator concentration-luminescence value is weighted by four-parameter fitting to obtain the corresponding calibrated working curve; S3. The results of the calibrated reagents are presented in the form of concentration. Continue to test the concentration of the quality control samples and conduct other sample tests within the target value range.

[0040] Example 3 A method for determining heparin binding protein using a chemiluminescent immunoassay kit employs a double-antibody sandwich reaction mode. Each sample is tested within 10 minutes at the fastest. A fully automated chemiluminescent immunoassay analyzer continuously detects samples, with a detection throughput of up to 200 samples per hour. The method comprises the following steps: A1. Sample needle S draws 10 μL of sample at the sample application track position, and reagent needle A draws 50 μL of reagent R2 component at the corresponding project reagent position. These components are added to the reaction cup in sequence and incubated at 37°C for 5 minutes (acridinium ester-labeled antibody 1 captures HBP in the sample). A2. Reagent needle B is automatically perfused and cleaned. 50 μL of reagent components R1 and R3 are drawn from the corresponding reagent positions in the reagent compartment and added to the reaction cup for incubation (forming a double antibody sandwich product). A3. After the incubation is completed, the mixed solution in the reaction cup is magnetically separated. After the waste liquid is removed by the waste liquid needle C1, the sample needle D1 continues to add cleaning solution to the liquid surface to resuspend it; A4, pipette D2 continues to add 100 μL of pre-stimulation solution and pipette the liquid surface to mix. Pipet D3 continues to add 300 μL of stimulation solution and pipette the liquid surface to mix. A5. The reaction begins, and the acridinium ester marker in the reaction cup emits light. The photomultiplier tube detects the light signal and converts it into an electrical signal. The stronger the electrical signal, the stronger the luminescence, and the more HBP to be detected is captured, indicating that the sample contains more HBP, which is a positive feedback loop (acridinium ester reacts with hydrogen peroxide under alkaline conditions to release photons). A6. After the luminescence reaction is completed, the waste liquid needle C2 absorbs the suspension and discharges it into the waste liquid collection pipe. The sample cup gripper takes out the reaction cup and puts it into the waste bucket.

[0041] Comparative Example 1 A heparin-binding protein assay kit using chemiluminescent immunoassay is disclosed. The raw materials and preparation method thereof are different from those of Example 1 in that short-chain Biotin-PEG-NHS is used in the biotin labeling process. Other aspects are the same as those of Example 1.

[0042] Comparative Example 2 A heparin-binding protein assay kit using chemiluminescent immunoassay is disclosed. The raw materials and preparation method thereof are different from those of Example 1 in that short-chain Biotin-PEG4-NHS is used in the biotin labeling process. Other aspects are the same as those of Example 1.

[0043] Comparative Example 3 A heparin-binding protein assay kit using chemiluminescent immunoassay is disclosed. The raw materials and preparation method thereof are different from those of Example 1 in that short-chain Biotin-PEG8-NHS is used in the biotin labeling process. Other aspects are the same as those of Example 1.

[0044] Comparative Example 4 A heparin-binding protein assay kit using chemiluminescent immunoassay, wherein the raw materials and preparation method thereof are different from those of Example 1 in that the component reagent R2 does not contain the dispersant PVP-8000 and the zwitterionic surfactant lauryl betaine, and the rest are the same as in Example 1.

[0045] Comparative Example 5 A heparin-binding protein assay kit using chemiluminescent immunoassay, wherein the raw materials and preparation method thereof are different from those of Example 1 in that the component reagent R2 does not contain blocking agents, mouse IgG HBR-5 and mouse IgG HBR-7, and the rest is the same as in Example 1.

[0046] Comparative Example 6 A commercially available heparin-binding protein detection kit based on the chemiluminescence method using H reagent was purchased and used as a comparison reagent.

[0047] Experimental Example 1 Sensitivity: The zero-value sample C0 of the calibrator was measured 20 times, and the mean RLU value M0 and standard deviation SD of the luminescence value were calculated. The adjacent low-value sample C1 (concentration 5.01 ng / mL) of the test calibrator was measured 3 times, and the mean RLU value M1 of the luminescence value was calculated. According to the relationship between the concentration and RLU value of the two calibrators, the linear equation y=kx+b was regressed and fitted, and M0+2SD was substituted into the equation to obtain the sensitivity of the corresponding kit. As shown in Table 1: The sensitivity results of Comparative Examples 1 to Comparative Examples 3 were 0.16 ng / mL, 0.14 ng / mL, and 0.09 ng / mL, respectively. The sensitivity of Example 1 was 0.02 ng / mL, and the test results had the best sensitivity.

[0048] Table 1 Sensitivity evaluation

[0049] Experimental Example 2 Linear range: Sample C5 (concentration 1997.89 ng / mL) with a high linearity value was diluted and spiked. Each sample was tested three times, and the average concentration was taken. A linear regression equation was fitted between the actual test value and the theoretical diluted concentration, and the correlation r value was calculated.

[0050] Figure 2 This is a graph showing the linear range of HBP detection in a heparin binding protein assay kit using a chemiluminescent immunoassay as described in the examples; as shown in Table 2 and Figure 2 As shown, the linearity is within the range of 0.05 ng / mL to 2000 ng / mL, and the correlation coefficients r for Comparative Examples 1 to 3 are 0.9913, 0.9926, and 0.9955, respectively, and are all ≥ 0.9900. The correlation coefficient r for Example 1 is 0.9994, indicating the best linear correlation.

[0051] Table 2 Linear range evaluation

[0052] Experimental Example 3 Correctness evaluation: Known Sample A: Sample A with known concentrations of (150±15) ng / mL and (50±5) ng / mL, respectively, was added to low-value clinical sample C. The volume ratio of A to B was 1:9, ensuring that the added sample concentration was near the medically determined level. The samples were tested using the kit from Example 1, with each sample concentration measured three times. The recovery rate was calculated according to formula (1): (1) in: R — recovery rate; C s —The concentration of sample A is known; C0—test concentration of serum or plasma sample B; C — the concentration of sample B after adding sample A solution; V—volume of liquid A added; V0—volume of serum or plasma sample B; As can be seen from Table 3, the recovery R of Comparative Examples 1 to 3 are within the range of 100±15%, 100±15%, and 100±10%, respectively. The recovery R of Example 1 (96.4% and 99.3%) is within the range of 100±5%, and the recovery accuracy is the highest.

[0053] Table 3 Correctness evaluation

[0054] Experimental Example 4 Precision: The kit of Example 1 was used to test high and low value quality control products, clinical serum and plasma samples, each tested 10 times, and the intra-assay coefficient of variation (CV) was calculated according to formula (2): (2) in: CV — coefficient of variation; SD —standard deviation; M —The mean of the calculated results.

[0055] Continue to select the other two batches of reagent kits in Example 1 and test the quality control products in the same way. Calculate the mean value M of the three batches of reagent test samples (30 times) 1-3 and standard deviation SD 1-3 , calculate the inter-batch repeatability coefficient of variation CV. As can be seen from the table below, the intra-batch precision of the kit of Example 1 is ≤3.5%, and the inter-batch precision is ≤5%.

[0056] Table 4 Precision evaluation

[0057] Experimental Example 5 interference: Calibrator C2 (concentration 19.97 ng / mL) was prepared and divided equally into three groups of identical samples. A control group was supplemented with a blank sample, and the other two groups were experimental groups with different concentrations of interfering substances added to the samples. The final interfering substance concentrations in the different samples of the experimental groups were 0.2 g / L and 0.5 g / L bilirubin, 10 g / L and 30 g / L triglycerides, 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 using the kit of Example 1, and the concentration of each sample was measured three times. Each sample was tested three times, and the deviation from the unspiked control sample was calculated.

[0058] As can be seen from Table 5, the interference deviations in the test of Example 1 are relatively small; for Comparative Example 4, which does not add the dispersant PVP-8000 and the amphoteric surfactant lauryl betaine, there are obvious deviations for bilirubin and triglycerides; for Comparative Example 5, which does not add the blockers HBR-5 and HBR-7, there are obvious deviations for RF and HAMA; and for Comparative Example 6, after increasing the amount of interfering substances, the control reagents all have obvious deviations; Table 5 Interference evaluation

[0059] Experimental Example 6 Specificity: A serum-free calibrator, C2 (20 ng / mL), was prepared and aliquoted, with relevant inflammatory and infection markers spiked into the calibrator. The concentrations of procalcitonin, C-reactive protein, serum amyloid A, interleukin-2, and interleukin-6 in the spiked samples were 100 ng / L, 50,000 ng / L, 50,000 ng / L, 100 ng / L, and 100 ng / L, respectively. As shown in Table 6, Example 1 showed essentially no cross-reactivity, demonstrating high specificity.

[0060] Table 6 Specificity evaluation

[0061] Experimental Example 7 Dependencies: 80 clinical samples (serum and plasma) with different concentration ranges were selected and tested using the kit of Example 1 as an evaluation reagent (the test time for a single sample was no more than 10 minutes) and a control reagent enzyme-linked immunosorbent assay kit (the test time for a single sample was 1 to 2 hours) from K Diagnostics, an authoritative and representative imported commercial company for HBP detection.

[0062] Figure 3 This is a graph showing the correlation between HBP detection in the assay method of a heparin binding protein assay kit using chemiluminescent immunoassay as described in Example 1 and the enzyme-linked immunosorbent assay kit of K Diagnostics. The test results are shown in Table 7 and Figure 3 As shown, with the control reagent as the X-axis and Example 1 as the Y-axis, the linear regression equation is y = 0.973x + 4.4681, r = 0.9965, indicating that this method has good clinical correlation with other detection kits.

[0063] Table 7 Correlation evaluation

[0064] Results and Discussion 1. By comparing Example 1 with Comparative Examples 1 to 3, the heparin-binding protein assay kit using a chemiluminescent immunoassay method according to the present invention uses a long-chain biotin conjugation process for antibody labeling in reagent R3. Compared to the short-chain biotin conjugation process used in Comparative Examples 1 to 3, the labeling method of Example 1 increases the distance between the magnetic beads and the antibody, indirectly reducing the interference of the magnetic beads on the antibody, and improving the overall sensitivity, linearity, and accuracy of spike recovery of the reagent. Good intra-batch precision was demonstrated for different sample types, including quality control samples, serum samples, and plasma samples, and the inter-batch precision of quality control samples also met the general industry standard (≤10%).

[0065] 2. By comparing Example 1 with Comparative Examples 4 to 5, the addition of a dispersant, two amphoteric surfactants, and a blocker to the reagent R2 diluent formula of Example 1 significantly reduced exogenous interference from triglycerides, bilirubin, and hemoglobin and endogenous interference deviation from rheumatoid factor (RF) and human anti-mouse antibody (HAMA) heterophilic antibodies.

[0066] 3. By comparing Example 1 with Comparative Example 6, Example 1 has strong anti-interference ability. When related inflammatory infection occurs, the related inflammatory factor indicators rise rapidly. The specificity of the detection markers: serum amyloid protein A, C-reactive protein, procalcitonin, interleukin-2, and interleukin-6 have basically no obvious cross-reaction.

[0067] 4. In terms of clinical performance, this kit shows good correlation with K Diagnostics' enzyme-linked immunosorbent assay (ELISA) kits for clinical samples. Methodologically, this kit offers rapid testing speed and can meet the actual testing needs of hospitals (① The final results of diagnostic reagent tests are traceable, providing a basis for clinical diagnosis; ② Some foreign companies' diagnostic reagents are highly authoritative (see above) but also expensive. To save costs, most hospitals will use relatively low-priced domestic products with equivalent clinical results; ③ Most relevant patents are compared with this company's, and no specific comparison is made, only with domestic reagents).

Claims

1. A chemiluminescent heparin-binding protein kit, characterized in that: This is a heparin binding protein determination kit using chemiluminescence immunoassay, including: reagent components 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 reagent R2 include an acridinium ester HBP antibody 1 marker, wherein the acridinium ester HBP antibody 1 marker is acridine active ester coupled to the HBP antibody; The components of reagent R3 include biotin-HBP antibody 2 labeling substance, wherein the biotin-HBP antibody 2 labeling substance is obtained by coupling biotin active ester to the HBP antibody.

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 in R1 diluent with a pH of 6.0~8.0, 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, and 0.5 g / L~2 g / L preservative; The components of the reagent R2 include: 0.5 mg / L to 8 mg / L acridinium ester HBP antibody 1 marker, 2 g / L to 80 g / L buffer salt in R2 diluent with a pH of 5.0 to 7.0, 5 g / L to 30 g / L buffer stabilizer, 0.5 g / L to 5 g / L chelating agent, 0.5 g / L to 5 g / L dispersant, 0.5 g / L to 5 g / L surfactant, 10 mg / L to 500 mg / L blocking agent, 2 g / L to 10 g / L protein protectant, and 0.5 g / L to 2 g / L preservative; The components of the reagent R3 include: 0.5 mg / L to 8 mg / L of biotin HBP antibody 2 marker, 2 g / L to 80 g / L of buffer salt in R3 diluent with a pH of 6.0 to 8.0, 5 g / L to 30 g / L of buffer stabilizer, 0.5 g / L to 5 g / L of chelating agent, 0.5 g / L to 5 g / L of surfactant, 2 g / L to 20 g / L of protein protective agent, and 0.5 g / L to 2 g / L of preservative; The calibrator comprises a matrix 1 of HBP at different concentrations, namely 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-80 g / L of buffer salt, 5 g / L-30 g / L of buffer stabilizer, 5 g / L-30 g / L of carbohydrate stabilizer, 0% v / v-30% v / v of serum matrix, 2 g / L-20 g / L of protein protectant, and 0.5 g / L-2 g / L of preservative, with a pH of 6.0-8.

0. The control product comprises a matrix 2 of HBP at different concentrations, namely (40±4) ng / mL and (600±60) ng / mL; 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 buffer stabilizer is at least one of sodium chloride, potassium chloride, calcium chloride, and calcium sulfate; 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 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 blocking agent is at least one of mouse immunoglobulin, sheep immunoglobulin, bovine immunoglobulin, and immunoglobulins of the same species that recognize different epitopes; 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. The 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 acridinium active ester to HBP antibody, and the preparation method includes the following steps: (1) Take 0.2 mg~2 mg of HBP antibody 1 in premix II, place it in a refrigerated centrifuge at 2℃~8℃ with an ultrafiltration tube, speed 2000rpm~6000rpm, 10min, 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 to obtain a biotin-HBP antibody 2 marker. The preparation method includes 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, 30min, 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 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.

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.

10. The method for determining heparin binding protein using a chemiluminescence assay kit according to any one of claims 1 to 9, characterized in that: This is also a method for determining heparin binding protein using a chemiluminescent immunoassay kit. It uses a double-antibody sandwich reaction mode. Each sample can be tested within 10 minutes at the fastest. The fully automatic chemiluminescent immunoassay analyzer continuously detects samples, with a detection throughput of up to 200 samples per hour. The method includes the following steps: A1, sample needle S draws 10 μL of sample at the sample application track position, and reagent needle A draws 50 μL of reagent R2 component at the corresponding project reagent position. These components are added to the reaction cup in sequence and incubated at 37°C for 5 minutes. Acridinium ester-labeled antibody 1 captures HBP in the sample. A2, reagent needle B automatically perfuses and cleans, draws 50 μL of reagent R1 component and R3 component at the corresponding reagent position in the reagent compartment, and continues to add them to the reaction cup for incubation to form a double antibody sandwich product; A3. After the incubation is completed, the mixed solution in the reaction cup is magnetically separated. After the waste liquid is removed by the waste liquid needle C1, the sample needle D1 continues to add cleaning solution to the liquid surface to resuspend it; A4, pipette D2 continues to add 100 μL of pre-stimulation solution and pipette the liquid surface to mix. Pipet D3 continues to add 300 μL of stimulation solution and pipette the liquid surface to mix. A5. The reaction begins, and the acridinium ester marker in the reaction cup emits light. The light signal is detected by a photomultiplier tube and converted into an electrical signal. The stronger the detected electrical signal, the stronger the luminescence, and the more HBP to be detected is captured, indicating that the sample contains more HBP, which is a positive correlation feedback. A6. After the luminescence reaction is completed, the waste liquid needle C2 absorbs the suspension and discharges it into the waste liquid collection pipe. The sample cup gripper takes out the reaction cup and puts it into the waste bucket.

Images