Heparin binding protein antibody, detection kit and application thereof

Abstract

The present invention relates to the field of biomedicine technology, and provides heparin binding protein antibodies, detection kits and applications thereof. The present invention provides two anti-HBP antibodies, which have high sensitivity and good specificity and can be used as HBP monoclonal antibody pairs. Based on the above antibodies, through appropriate reagent configuration, the present invention also provides an HBP detection kit with good performance, high accuracy, high sensitivity, wide linear range, high precision, good repeatability and strong applicability.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a heparin binding protein antibody, a detection kit and applications thereof. Background Art

[0002] Heparin-binding protein (HBP), also known as Azurocidin (AZU) and cationic antimicrobial protein of 3700 (CAP37), is a multifunctional single-chain glycoprotein that has the functions of changing endothelial cell permeability, antimicrobial activity, chemotaxis and regulating cell apoptosis. It is an important granule protein secreted by polymorphonuclear leukocytes (Linder A, Christensson B, Herwald H, et al. "Heparin-binding protein: an early marker of circulatory failure in sepsis." Clin Infect Dis, 2009,49:1044–50. 10.1086 / 605563.). HBP is a single-chain glycoprotein formed by removing 26 amino acid residues from the N-terminus and 3 amino acid residues from the C-terminus of a 251-amino acid precursor. Mature HBP consists of the remaining 222 amino acid residues and has a relative molecular mass of about 24,000. Amino acid sequence alignment shows that it has about 45% homology with human neutrophil elastase, about 42% homology with proteinase 3, and about 32% homology with cathepsin G in human neutrophils.

[0003] HBP is a circulating protein complex formed by Streptococcus M protein and fibrinogen. After the body activates neutrophils, when neutrophils adhere to vascular endothelial cells, HBP in neutrophil secretory granules and azurophilic granules is rapidly released into the blood in large quantities, inducing endothelial cell cytoskeleton rearrangement, leading to vascular leakage and edema formation. In addition, HBP also has the function of regulating monocytes and macrophages, further amplifying the inflammatory response and enhancing the body's immune response to infection. As neutrophils infiltrate tissues, HBP is continuously released, leading to tissue damage and organ dysfunction. HBP can change vascular permeability by binding to vascular epithelial cells, inducing plasma leakage and inflammatory response. Heparin-binding protein can also chemotactic and activate monocytes and macrophages to respond to bacterial infections and participate in regulating immune responses (Lingyun Zuo, Xiaoyun Li, LuhaoWang, et al. "Heparin-binding protein as a biomarker for the diagnosis of sepsis in the intensive care unit: a retrospective cross-sectional study in China." BMJ Open, 2024,Jun 10;14(6):e078687.).

[0004] Sepsis is a systemic infectious inflammatory response, a life-threatening organ dysfunction caused by an imbalance in the host's response to infection. Sepsis can cause symptoms such as changes in body temperature, increased heart rate, and rapid breathing, leading to systemic coagulation disorders and imbalances in immune inflammatory responses, inducing organ failure, and in severe cases, septic shock, known as septic shock, which is the main cause of death in sepsis patients. Many studies have shown that the concentration of HBP in plasma has a strong correlation with sepsis and septic shock, and can effectively predict the occurrence of sepsis and septic shock, making it an ideal clinical diagnostic indicator. In the "Guidelines for the Diagnosis and Treatment of Severe Sepsis / Septic Shock in China (2014)" (Chinese Society of Critical Care Medicine, Chinese Medical Association. "Guidelines for the Diagnosis and Treatment of Severe Sepsis / Septic Shock in China (2014)." Chinese Journal of Critical Care Medicine, 2015,27(6):401-426.) and "2020 Chinese experts' consensus on early prevention and interruption of sepsis in emergency medicine" (Wang Zhong, Wei Jie, et al. "Chinese experts' consensus on early prevention and interruption of sepsis in emergency medicine." Journal of Clinical Emergency Medicine, 2020,21(7):217-529.) HBP has been identified as an early diagnostic indicator for severe sepsis / septic shock.

[0005] The representative product of early HBP detection is the enzyme-linked immunosorbent assay kit of Axis-Shield Company in the UK, which uses the more traditional enzyme-linked immunosorbent assay (ELISA). In actual operation, this method has the defects of being time-consuming (generally 4 to 5 hours), inconvenient to use (many manual operation steps, inflexible detection of test personnel), narrow detection range (5.9-200.0ng / mL), and difficult to accurately quantify, so there are always great limitations. Subsequent methods developed include colloidal gold method and immunofluorescence chromatography. Although both can be detected quickly, they can only perform qualitative or semi-quantitative detection, and the accuracy is low, and they cannot provide accurate clinical judgment basis. At the same time, due to the limitations of the method itself, due to the use of a heterogeneous reaction system, the precision of the detection is poor, and the interference cannot be removed from the operating procedure, so the accuracy of the test cannot be guaranteed. The immunoturbidimetric method based on latex microspheres for detecting HBP is also greatly interfered by turbid substances such as lipids that may exist in the sample.

[0006] Chemiluminescence has the advantages of high sensitivity, strong precision, wide linear range and low susceptibility to interference, and is widely used in the detection of clinical disease indicators. The main determinant of the performance of HBP detection reagents currently lies in the functional characteristics of HBP binding antibodies. Good antibody pairing can effectively improve the diagnostic efficiency of diseases. The number of diagnostic reagents using chemiluminescence for HBP detection on the market is still limited, and there are certain deficiencies in sensitivity, linear range and precision. Summary of the invention

[0007] The present invention provides a heparin binding protein (HBP) antibody and a detection kit to solve the problems of low sensitivity, narrow linear range, poor precision and the like of the existing HBP detection antibodies, and to improve the performance of HBP antibody raw materials and HBP antigen detection reagent products.

[0008] In a first aspect, the present invention provides a heparin-binding protein antibody or an antigen-binding fragment thereof, comprising:

[0009] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2; or,

[0010] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:4.

[0011] Optionally, the antibody or antigen-binding fragment thereof can recognize and / or specifically bind to an epitope of a heparin-binding protein as shown in SEQ ID NO:5 or SEQ ID NO:6.

[0012] Optionally, the antibody is a monoclonal antibody; the antigen-binding fragment of the antibody is selected from F(ab')2, F(ab)2, Fab', Fab, Fv or scFv.

[0013] The second aspect of the present invention provides a nucleic acid encoding the above-mentioned heparin binding protein antibody or antigen-binding fragment thereof.

[0014] The third aspect of the present invention provides an expression vector comprising the above nucleic acid.

[0015] A fourth aspect of the present invention provides a transgenic cell line or a recombinant bacterium comprising the above nucleic acid or the above expression vector.

[0016] The fifth aspect of the present invention provides an immunoconjugate, comprising an antibody portion and a coupling portion coupled to the antibody portion, wherein the antibody portion comprises the above-mentioned heparin-binding protein antibody or its antigen-binding fragment, and the coupling portion is selected from a fluorescent substance, a chemiluminescent substance, a colored substance, an enzyme or a combination thereof.

[0017] In a sixth aspect of the present invention, a heparin-binding protein detection kit is provided, which comprises a first antibody and / or a second antibody; the first antibody comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 1 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 2; the second antibody comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 3 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 4.

[0018] Optionally, the kit is used for detecting heparin binding protein by immunochemiluminescence.

[0019] Optionally, the kit comprises a first antibody and a second antibody used in pair; the first antibody serves as a capture antibody and the second antibody serves as a detection antibody, or the first antibody serves as a capture antibody and the second antibody serves as a detection antibody.

[0020] Optionally, the kit includes magnetic particles coated with specific HBP capture antibodies, specific HBP detection antibodies labeled with luminescent markers, substrates and stimulating solutions compatible with the luminescent markers, and quality control products and calibrators of HBP antigen series.

[0021] In a seventh aspect, the present invention provides use of the above-mentioned heparin-binding protein antibody or antigen-binding fragment thereof in the preparation of a sepsis diagnostic reagent or kit.

[0022] The present invention obtains HBP monoclonal antibodies and monoclonal antibody pairs with high sensitivity and good specificity through immunization of recombinant HBP antigens and screening of monoclonal antibodies. Furthermore, through appropriate reagent configuration, an HBP detection kit with good performance is obtained. The HBP detection kit of the present invention has high reagent accuracy, high sensitivity, wide linear range, and high correlation; the reagent has high precision, good repeatability, and strong applicability. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the SDS-PAGE diagram of the recombinant HBP antigen protein. In the figure, the lanes from left to right are 1. protein marker, 2. stock solution before purification, 3. flow-through solution after purification that is not bound to the chromatography column, 4. protein components eluted with 20mM imidazole solution, 5. protein components eluted with 80mM imidazole solution, and 6. protein components eluted with 500mM imidazole solution.

[0024] Figure 2 This is the test result analysis curve of the linear range of the HBP kit in Example 6. DETAILED DESCRIPTION

[0025] The present invention provides an antibody or an antigen-binding fragment thereof that can specifically bind to heparin-binding protein (HBP). The heparin-binding protein or the abbreviation HBP described in the present invention have the same meaning. The present invention obtains a recombinant human heparin-binding protein with biological activity by constructing a human heparin-binding protein expression gene and by recombinant expression; two antibodies are selected by autonomous animal immunization and monoclonal cell line screening. The two antibodies can not only be used as anti-HBP antibodies respectively, but also can be paired as monoclonal antibodies to bind to recombinant human heparin-binding protein with high sensitivity and strong specificity by a double antibody sandwich method. That is, the present invention also provides an anti-human heparin-binding protein monoclonal antibody or an antigen-binding fragment thereof for use in detecting human heparin-binding protein, and for use in preparing a sepsis diagnostic reagent or kit.

[0026] In some embodiments, the antibody comprises all or part of the constant region of the antibody. In some embodiments, the constant region is selected from the following types, including: IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3 or IgG4) and IgM, etc. The present invention also discloses an antigen binding fragment that can specifically bind to the HBP antibody. Examples of antigen binding fragments include F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc.

[0027] For monoclonal antibodies of the present invention, any available or known method in the art can be used to obtain monoclonal antibodies, including expression in any eukaryotic, prokaryotic or phage system. Monoclonal antibodies for the present invention can be prepared using a wide variety of techniques known in the art, including hybridoma technology, recombinant technology or phage display technology, etc.

[0028] Products in other aspects of the present invention also include intermediates in the process of preparing antibodies, including expression vectors, transgenic cell lines or recombinant bacteria, or compositions containing the above substances. Specifically, the expression vector may be a recombinant expression vector such as a eukaryotic expression vector, and the transgenic cell line or recombinant bacteria may also include eukaryotic cells or prokaryotic cells, such as Escherichia coli, yeast or animal cells (e.g. mammalian cells, such as mouse cells, human cells, etc.) and cell lines.

[0029] The present invention also provides a HBP detection kit or detection reagent, which comprises the antibody or antigen-binding fragment thereof of the present invention. In some embodiments, the kit further comprises a solid phase substrate for the antibody or antigen-binding fragment thereof to attach, and the solid phase substrate includes but is not limited to microplates, magnetic particles, filter paper for immunochromatography, polymers such as polystyrene, glass filters and other insoluble carriers. In some embodiments, the kit further comprises some other components, including but not limited to enzymes for labeling, corresponding substrates, radioactive isotopes, fluorescent substances, colored substances, buffers, etc.

[0030] HBP is detected using the above-mentioned kit or detection reagent. In some embodiments, the detection method includes: combining the antibody or antigen-binding fragment of the present invention with the HBP antigen in the sample to be tested to form an antibody-antigen or antibody fragment-antigen complex; then, detecting the sample complex to determine whether the target antigen is present in the sample.

[0031] Specifically, in some embodiments, the detection reagent or kit comprises the antibody or antigen-binding fragment thereof of the present invention (as the first antibody), and a detectable, labeled second antibody. The specific detection method comprises: adsorbing the first antibody onto a solid support; then adding a sample to be tested that may contain HBP to the support; adding a second antibody with a marker to the support; detecting the presence of the marker to determine whether HBP exists. In addition, the specific detection method may also be a known detection method such as enzyme immunoassay, immunochemiluminescence detection, radioimmunoassay, fluorescent immunoassay, immunochromatography, competitive method or sandwich method.

[0032] In a more preferred embodiment, the present invention provides an immunochemiluminescence HBP detection kit prepared using the above-mentioned anti-human heparin binding protein monoclonal antibody; the human heparin binding protein detection kit (immunochemiluminescence method) provided by the present invention has the characteristics of higher accuracy and sensitivity, wider linear range and considerable precision.

[0033] Specifically, the HBP detection kit of the present invention has the following advantages:

[0034] 1. The reagent has high accuracy, and the relative deviation of the test results of samples of each concentration does not exceed 5%;

[0035] 2. The reagent has high sensitivity, a wide linear range, and a high degree of correlation. The wide linear range can accommodate sample testing needs in more situations. The lower limit of the linear range (5.9ng / mL) is much lower than the reference value (11.4 ng / mL), avoiding false positive misjudgments; the upper limit of the linear range (500ng / mL) is better than the upper limit of the linear range of similar products (300ng / mL), which can keep the results accurate when testing high-concentration samples without dilution. At the same time, within this range, the linear correlation coefficient r of the reagent is as high as 0.9998, which ensures the accuracy of the full range of test results and can accurately monitor the changes and fluctuations of patients' indicators;

[0036] 3. The reagents have high precision and good repeatability. The CV of both indoor and inter-laboratory precision is less than 3%, which can provide a reliable traceability basis for the test data;

[0037] 4. The reagent has good applicability, which means it is suitable for both high-speed fully automatic chemiluminescence workstations and small chemiluminescence analyzers. It can meet a variety of different hospital application scenarios including outpatient clinics, emergency departments and community hospitals, while maintaining stable detection performance.

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

[0039] Unless otherwise specified, the experimental materials used in the examples of the present invention can be obtained from commercial channels, and the experimental operation methods, unless otherwise specified, are performed using means known in the art.

[0040] Example 1: Recombinant expression of HBP antigen

[0041] According to the heparin binding protein sequence information (NP_001691.1) published by the National Center for Biotechnology Information (NCBI), the full-length sequence was selected and cloned into the pCDNA3.4 eukaryotic expression vector. The resulting plasmid was transfected into HEK293 cells (human embryonic kidney cells 293) using PEI transfection reagent (polyethylenimine). After culturing for 7 days in a constant temperature shaker at 37°C, 8% CO2 and an appropriate speed, the cell supernatant was collected by centrifugation and purified by nickel medium affinity chromatography to obtain the recombinant expressed HBP protein, which was approximately 27.6 kDa ( Figure 1 ).

[0042] Example 2: Immunization of animals with recombinant HBP protein

[0043] The recombinant HBP antigen prepared in Example 1 was diluted to 1 mg / mL with 20 mM PBS pH 7.4 solution (phosphate buffer) and mixed with Freund's adjuvant in a 1:1 ratio for emulsification. The emulsified recombinant HBP antigen was intraperitoneally injected into 6- to 8-week-old Balb / c mice, with 100 μg per mouse. Freund's complete adjuvant was used for the initial immunization and Freund's incomplete adjuvant was used for the booster immunization. After three injections, tail vein blood was collected to determine the titer, and cells with a titer of more than 1 million were selected for cell fusion. Spleen direct immunization was performed 72 hours before fusion, with 30 μg of HBP antigen injected.

[0044] Example 3: Screening of monoclonal antibodies

[0045] (1) Fusion cells: The mice to be fused were killed by cervical dislocation, and their spleens were taken out and ground and filtered to make spleen cell suspension, which was then transferred to a 50 mL centrifuge tube. The cells were resuspended in serum-free RPMI1640 medium and washed three times, each time at 15,000 rpm for 5 min. The myeloma cells were resuspended in serum-free RPMI1640 medium and washed three times, each time at 15,000 rpm for 5 min. The spleen cell suspension and myeloma cell suspension were mixed, centrifuged at 1,500 rpm for 5 min, the supernatant was discarded, and the bottom of the tube was flicked to disperse the cells. At the same time, 1 mL of PEG1450 pre-warmed to 37°C was slowly added. After adding within 60 seconds, the mixture was mixed by pipetting for 30 seconds, and then 40 mL of RPMI 1640 medium pre-warmed to 37°C was immediately added to terminate the fusion reaction, and the cells were transferred to RPMI 1640 HAT cell screening medium. After mixing, the cells were plated on a 96-well cell culture plate. After 7 days, the RPMI 1640 cell culture medium was replaced, and after 3 days, part of the supernatant was aspirated for subsequent operations.

[0046] (2) Supernatant detection: The recombinantly expressed HBP protein was coated in a polystyrene 96-well ELISA plate at a total amount of 50 ng / well. The cell supernatant obtained in Example 3 (1) was added to the coated 96-well plate. After incubation and washing, the plate was incubated with HRP-labeled goat anti-mouse secondary antibody, reaction substrate and stop solution in sequence. The cell wells corresponding to the high-value positive wells were selected for subcloning. After five subclones, 11 hybridoma monoclonal cells with good reactivity to the recombinant HBP protein (OD value ≥ 3.0) were finally selected.

[0047] Example 4: Pairing screening of monoclonal antibodies

[0048] (1) HRP-labeled antibody: Dissolve 30 mg HRP dry powder in 1.5 mL pure water, add 1.2 mL 25 mg / mL NaIO4 aqueous solution, mix well, and react at 4°C for 30 min. Remove and add 0.2 mL ethylene glycol solution, leave at room temperature for 30 min to complete HRP activation, and store at 4°C for later use. Dilute the antibody to 2 mg / mL with 20 mM carbonate buffer pH 9.6, mix 2 mL of the antibody to be labeled with the activated HRP labeling solution, and dialyze overnight in 20 mM carbonate buffer pH 9.6. Remove and add 0.2 mL 2 mg / mL NaBH4 solution, and react at 4°C for 2 hours. Add an equal volume of saturated ammonium sulfate solution, leave at 4°C for 30 min, centrifuge, resuspend the precipitate with 1 mL 20 mM PBS pH 7.4 solution, add an equal volume of glycerol, mix well, and store.

[0049] (2) Paired detection: Use 20mM PB pH7.4 solution to dilute the anti-HBP antibody to 1μg / mL, 100μL / well is coated on a polystyrene 96-well ELISA plate, and incubate at 37℃ for 2 hours. Wash the 96-well plate 5 times with PBST, add blocking solution for blocking, and discard the blocking solution after blocking. Add 100μL / well of recombinant HBP protein diluted to 10ng / mL to the 96-well plate, incubate at 37℃ for 1 hour, wash the 96-well plate 5 times with PBST, add HRP-labeled anti-HBP antibody, incubate at 37℃ for 30min, wash the 96-well plate 5 times with PBST, and then incubate the reaction substrate and stop solution in turn, and place it in an ELISA reader for reading. All 11 antibodies are paired in pairs as coating antibodies and labeled antibodies. After testing, the monoclonal antibody pair with the highest sensitivity and best specificity is selected, that is, monoclonal antibodies HBP-10# and HBP-19#.

[0050] Example 5: Sequence determination and binding epitope analysis of monoclonal antibodies

[0051] (1) Antibody sequence determination: Nanjing GenScript Biotech Co., Ltd. was commissioned to perform de-novo protein sequencing on two monoclonal antibodies HBP-10# and HBP-19#. After spectral analysis, the amino acid sequences of the heavy chain and light chain variable regions of the two monoclonal antibodies HBP-10# and HBP-19# were obtained. The specific amino acid sequences are shown in Table 1. Both antibodies are mouse antibody IgG1.

[0052] Table 1 Amino acid sequences of the variable regions of monoclonal antibodies

[0053]

[0054] (2) Antibody binding epitope analysis: Nanjing GenScript Biotech Co., Ltd. was commissioned to analyze the binding epitopes of two monoclonal antibodies HBP-10# and HBP-19#. The binding epitopes of both antibodies are linear epitopes, and the relevant information is shown in Table 2. Among them, the binding epitope of monoclonal antibody HBP-10# is the amino acid sequence GSQRSGGRLS (Gly-Ser-Gln-Arg-Ser-Gly-Gly-Arg-Leu-Ser) from position 155 to 164 of the full-length HBP protein, and the binding epitope of monoclonal antibody HBP-19# is the amino acid sequence GAYDLRRRERQSR (Gly-Ala-Tyr-Asp-Leu-Arg-Arg-Arg-Glu-Arg-Gln-Ser-Arg) from position 82 to 94 of the full-length HBP protein.

[0055] Table 2 Binding epitope information of monoclonal antibodies

[0056]

[0057] Example 6: Configuration of HBP detection reagents

[0058] (1) Preparation of antibody buffer: Add 50mM pH 7.3 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), 0.5% lauryl alcohol polyoxyethylene ether, 50mM tris(hydroxymethylaminomethane) (Tris), 2% sucrose, 4% arginine, 2.5% casein, and 0.1% sodium azide to prepare antibody buffer, and adjust the pH to 7.30±0.05 (25°C). Filter with a 0.8μm filter membrane to remove large particles.

[0059] (2) Preparation of magnetic bead storage buffer: Add 50mM pH 7.3 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), 0.5% lauryl alcohol polyoxyethylene ether, 50mM potassium dihydrogen phosphate, 20mM sodium chloride, 2% sucrose, 4% arginine, 1% bovine serum albumin, and 0.1% sodium azide to prepare magnetic bead storage buffer. The pH is corrected to 7.40±0.05 (25°C). Use a 0.8μm filter membrane to filter out large particles.

[0060] (3) Biotin labeling of antibodies: Dissolve biotin to 1 mg / ml using dimethyl sulfoxide solution, add 2 mg / mL of HBP capture antibody, i.e., HBP-10#, mix at a mass ratio of 1:10, and then dilute with an equal volume of carbonate buffer, pH 9.6, and incubate at 37°C for 2 hours to complete labeling. Then, ultrafiltration was performed using a 50 kDa ultrafiltration tube to replace the buffer with antibody buffer. The final concentration of biotin-labeled HBP capture antibody was 10 μg / mL.

[0061] (4) Coupling of magnetic bead reagent: After magnetic separation, 20 mg of concentrated streptavidin-labeled magnetic beads were washed three times with antibody buffer, and the above-mentioned biotin-labeled HBP capture antibody was added and mixed at a mass ratio of 50:1. The mixture was reacted in an antibody buffer environment at 30°C for 15 min to complete the coupling of the antibody and magnetic beads. After magnetic separation, the supernatant was discarded, and the beads were resuspended in magnetic bead storage buffer and diluted to a volume of 200 mL. This is the magnetic bead reagent that has completed the antibody coupling.

[0062] (5) Coupling of acridinium ester labeling reagent: Dissolve acridinium ester in dimethyl sulfoxide solution to 5 mg / mL, add 2 mg / mL of HBP detection antibody, i.e., HBP-19#, mix at a mass ratio of 1:10, and then dilute with an equal volume of carbonate buffer at pH 9.6, incubate at 37°C for 2 hours to complete labeling. Then, ultrafilter using a 50 kDa ultrafiltration tube, replace the buffer with antibody buffer, and dilute to 2 L to obtain the acridinium ester labeling reagent that has completed antibody coupling.

[0063] (6) Preparation of calibrators and quality control products: The recombinant HBP antigen was prepared into a series of calibrators with calibrator diluent, with concentrations of 0 ng / mL, 5 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, and 300 ng / mL. The composition of the calibrator diluent was 50 mM pH 7.3 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), 0.5% lauryl alcohol polyoxyethylene ether, 50 mM potassium chloride, 2% sucrose, 4% arginine, and 0.1% sodium azide. The high-concentration HBP sample (100 ng / mL) was diluted to 8 ng / mL and 50 ng / mL with the calibrator diluent, which were used as the low-value quality control product and the high-value quality control product, respectively.

[0064] Example 7: Accuracy of the kit

[0065] The accuracy of a reagent refers to the degree of agreement between the average value of a quantity obtained by repeated measurements an infinite number of times and a reference value.

[0066] Using the detection kit in Example 6, one portion of high concentration (149.47±7.96 ng / mL) and one portion of low concentration (15.05±0.72 ng / mL) of heparin binding protein corporate reference were tested, and the test was repeated three times. The test results are shown in Table 3. According to the relative deviation of the test results, the relative deviation of the three test results of the two reference products was within 5%, and it was considered that the kit had good accuracy.

[0067] Table 3 Test results of the HBP kit accuracy in Example 6

[0068]

[0069] Example 8: Linear range of the kit

[0070] The expected linear range of the HBP kit in Example 6 is 5.9-500 ng / mL. A high-value sample (501.24 ng / mL) and a low-value sample (5.71 ng / mL) are selected near the upper and lower limits of the range, and the high and low-value samples are accurately diluted in different proportions with the zero-value HBP calibrator to prepare the following 6 samples with different concentration levels (5.71 ng / mL, 22.94 ng / mL, 91.84 ng / mL, 177.97 ng / mL, 350.22 ng / mL, 501.24 ng / ml), which are tested using the HBP kit in Example 6.

[0071] The test results are shown in Table 4. After fitting curve and linear analysis, the linear regression equation of the test kit is: y=1.0075x +0.3224, R 2 =0.9998>0.995( Figure 2 ), it is preliminarily judged that it meets the expected linear range requirements; the relative deviation between the actual measured value and the theoretical value of each concentration sample is no more than 10%, and the expected linear range is acceptable.

[0072] Table 4 Detection results of the linear range of the HBP kit in Example 6

[0073]

[0074] Example 9: Precision of the kit

[0075] Precision refers to the degree of consistency between the labeled value or measured value obtained by repeated measurement of the same or similar object under specified conditions. The precision of the HBP kit in Example 6 was evaluated by repeatability, intermediate precision (intra-laboratory precision), and reproducibility (inter-laboratory precision). Three batches of samples with known concentrations of high-concentration quality control product 1 (target value of 20.73 ng / mL), low-concentration quality control product 2 (target value of 103.04 ng / mL), and mixed quality control product (target value of 61.89 ng / mL) were used to evaluate this performance.

[0076] In the same laboratory, using the same detection instrument, three batches of samples were tested separately, with two analysis batches tested every day. Two samples of each concentration in each batch were processed in parallel for testing to evaluate the in-laboratory precision of the kit.

[0077] In the same laboratory, three batches of samples were tested separately using three different instruments (NRM411, NORMAN-CL 5, NORMAN-CL 211) launched by Nanjing Norman Biotechnology Co., Ltd. and a single batch of detection kit. Each batch of samples was tested continuously for 5 days and each batch of samples was tested 5 times a day to evaluate the inter-laboratory precision of the kit.

[0078] The results of intra-laboratory precision and inter-laboratory precision tests were analyzed using a two-way ANOVA model. The ANOVA results are shown in Table 5, the standard deviation and CV analysis results are shown in Table 6, and the confidence interval results are shown in Table 7. All of the above results are in line with expectations, with no abnormal values.

[0079] Table 5 Precision variance analysis results of the HBP kit in Example 6

[0080]

[0081] In Table 5:

[0082] SS site: sum of squares - between laboratories (between instruments); MS site: mean square - between laboratories (between instruments); SS day: sum of squares - between days; MS day: mean square - between days; SS error: sum of squares - within batch; MS error: mean square - within batch; SS total: sum of squares - total.

[0083] Table 6 Precision analysis results of the HBP kit in Example 6

[0084]

[0085] In Table 6: s: standard deviation; CV: coefficient of variation.

[0086] Table 7 Confidence intervals for the precision of the HBP kit in Example 6 (95%, α=0.05).

[0087]

[0088] The present invention is described in detail above in conjunction with specific implementation methods and exemplary examples, but these descriptions cannot be understood as limiting the present invention. Those skilled in the art understand that, without departing from the spirit and scope of the present invention, various equivalent substitutions, modifications or improvements can be made to the technical solution of the present invention and its implementation methods, all of which fall within the scope of protection of the present invention.

Claims

1. A heparin binding protein antibody or an antigen binding fragment thereof, characterized in that: Include: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2; or, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

4.

2. The heparin-binding protein antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof can recognize and / or specifically bind to the epitope of the heparin-binding protein as shown in SEQ ID NO: 5 or SEQ ID NO:

6.

3. The heparin-binding protein antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody is a monoclonal antibody; the antigen-binding fragment of the antibody is selected from F(ab')2, F(ab)2, Fab', Fab, Fv or scFv.

4. A nucleic acid encoding the heparin binding protein antibody or antigen-binding fragment thereof according to claim 1.

5. An expression vector comprising the nucleic acid of claim 4.

6. A transgenic cell line or recombinant bacterium comprising the nucleic acid of claim 4 or the expression vector of claim 5.

7. An immunoconjugate, characterized in that: The invention comprises an antibody portion and a coupling portion coupled to the antibody portion, wherein the antibody portion comprises the heparin-binding protein antibody or an antigen-binding fragment thereof according to claim 1, and the coupling portion is selected from a fluorescent substance, a chemiluminescent substance, a colored substance, an enzyme or a combination thereof.

8. A heparin binding protein detection kit, characterized in that: It comprises a first antibody and / or a second antibody; the first antibody comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 1 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 2; the second antibody comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 3 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:

4.

9. The heparin binding protein detection kit according to claim 8, characterized in that: The kit is used for detecting heparin binding protein by immunochemiluminescence.

10. The heparin binding protein detection kit according to claim 8, characterized in that: It comprises a first antibody and a second antibody used in pair; the first antibody is used as a capture antibody and the second antibody is used as a detection antibody, or the first antibody is used as a capture antibody and the second antibody is used as a detection antibody.

11. The heparin binding protein detection kit according to claim 10, characterized in that: The kit comprises the magnetic particles coated with the capture antibody, the detection antibody marked with a luminescent marker, a substrate and an exciting liquid suitable for the luminescent marker, and a series of HBP antigen quality control products and calibration products.

12. Use of the heparin binding protein antibody or antigen binding fragment thereof according to any one of claims 1 to 3 in the preparation of a sepsis diagnostic reagent or kit.

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