Heparin binding protein detection kit

Abstract

The invention provides a heparin binding protein detection kit, the kit comprises a reagent I and a reagent II, the reagent I is a buffer solution containing a surfactant and a macromolecular polymer; and the reagent II is a buffer solution of polystyrene latex microspheres containing monoclonal antibody pairs coated with anti-human heparin binding protein. The invention solves the technical problem that in the prior art, the latex enhanced immunoturbidimetry is adopted to improve the linear upper limit, so that the low-value repeatability is poor, and the positive and negative judgment in clinical diagnosis is influenced.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection, and in particular, to a kit for detecting heparin-binding protein. Background Art

[0002] Heparin Binding Protein (HBP) is a granule protein secreted by neutrophils. It was discovered and successfully isolated by Professor Shafer in 1984 and named CAP37 (cationic antimicrobial protein of 37000). Later, it was named heparin-binding protein (Heparin-bindin protein, HBP) because of its heparin-binding ability.

[0003] Heparin-binding protein is a multifunctional protein that can change vascular permeability by binding to vascular epithelial cells, induce plasma leakage and inflammatory responses. Heparin-binding protein can also chemotax and activate monocytes and macrophages to respond to bacterial infections and participate in the regulation of immune responses. When an infection occurs, pathogenic microorganisms stimulate neutrophils to release HBP, resulting in an increase in the content of HBP in the blood. HBP is one of the earliest elevated markers in bacterial infections, and local or mild bacterial infections can also cause a rapid increase in HBP, while the level of HBP does not increase in non-bacterial infections. At the same time, HBP has a short half-life and can quickly reflect the improvement or deterioration of the patient's condition and the effect of antibiotic treatment, and evaluate whether it is necessary to change antibiotics or stop taking drugs.

[0004] Studies have shown that the level of HBP in the blood of patients with severe sepsis and septic shock will increase significantly. Therefore, monitoring the level of HBP in critically ill patients can assist in judging the severity of the condition, effectively predict the occurrence of sepsis and septic shock, and is a good clinical diagnostic indicator.

[0005] Currently, the detection kits for heparin-binding protein widely used clinically are fluorescence immunoassay dry method and latex enhanced immunoturbidimetry. The fluorescence immunoassay dry method is cumbersome to operate and has low detection efficiency; the latex enhanced immunoturbidimetry is suitable for automatic biochemical analyzers, is convenient to operate, and has rapid detection.

[0006] Heparin-binding protein is a granule protein secreted by neutrophils. The content of heparin-binding protein in the blood of normal people is very low, and its normal value is usually less than 10 ng / ml. When patients have bacterial infections, septic shock, circulatory failure and other conditions, the level of heparin-binding protein in the plasma increases sharply, and the plasma HBP concentration in patients with severe sepsis infections can be as high as more than 300 ng / mL. Therefore, in order to meet the purpose of clinical diagnosis, it is particularly necessary to develop a heparin-binding protein detection reagent with higher sensitivity and wider linear range.

[0007] The methods for expanding the linear range of latex - enhanced immunoturbidimetry include: increasing the upper linear limit. In the prior art, the methods for increasing the upper linear limit mainly include increasing the amount of antibody, using antibodies with low titer, or using polystyrene microspheres with smaller particle sizes cross - linked with antibodies. These methods all come at the cost of sacrificing the detection limit of the reagent, resulting in poor repeatability of low values and affecting the judgment of positive and negative in clinical diagnosis.

[0008] Therefore, the object of the present invention is to develop a heparin - binding protein detection kit that can not only meet the high - linearity requirements but also improve the repeatability of low values. Summary of the Invention

[0009] The technical problem solved by the present invention is that in the prior art, when using latex - enhanced immunoturbidimetry to increase the upper linear limit, it will lead to poor repeatability of low values and affect the judgment of positive and negative in clinical diagnosis.

[0010] To solve the above - mentioned problems, the present invention provides a heparin - binding protein detection kit, which includes: Reagent 1 and Reagent 2. Reagent 1 is a buffer solution containing a surfactant and a macromolecular polymer; Reagent 2 is a buffer solution containing polystyrene latex microspheres coated with a monoclonal antibody pair against human heparin - binding protein.

[0011] Compared with the prior art, the technical effects achieved by adopting this technical solution: The heparin - binding protein detection kit provided by the present invention has high sensitivity, good repeatability, and high accuracy. Under the requirements of meeting high linearity and high hook - effect, it greatly improves the low detection repeatability, can more accurately reflect the changes of heparin - binding protein in the human body, and better reflects the clinical application value of heparin - binding protein.

[0012] In an example of the present invention, the concentration ratio of the surfactant to the macromolecular polymer is 3:1.

[0013] Compared with the prior art, the technical effects achieved by adopting this technical solution: By adding a surfactant and a macromolecular polymer with a concentration ratio of 3:1 to Reagent 1, the reagent has good detection repeatability.

[0014] In an example of the present invention, the concentration of the surfactant is 0.3 g / L - 30 g / L, and the concentration of the macromolecular polymer is 0.1 g / L - 10 g / L.

[0015] In an example of the present invention, the surfactant is diphenylstyryl phenol polyoxyethylene ether - 18.8; and / or the macromolecular polymer is poly(ethylene glycol) - block - poly(propylene glycol) - block - poly(ethylene glycol).

[0016] Compared with the prior art, the technical effects achieved by adopting this technical solution are as follows: 4,4'-bis(2-styrylphenyl) polyoxyethylene ether-18.8 can block other non-specific reactions except for the specific binding of heparin-binding protein and monoclonal antibody against human heparin-binding protein; while specifically recognizing heparin-binding protein, the monoclonal antibody against human heparin-binding protein can better improve the linearity of the reagent; poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) can better improve the efficiency of specific binding between heparin-binding protein and monoclonal antibody against human heparin-binding protein, and improve the sensitivity and repeatability of the reagent.

[0017] In one example of the present invention, the particle size of the polystyrene latex microspheres is 300 nm to 450 nm.

[0018] Compared with the prior art, the technical effects achieved by adopting this technical solution are as follows: The particle size of the polystyrene latex microspheres is 300 nm to 450 nm. The polystyrene latex within this particle size range has a high carboxyl content and a high efficiency of binding antibodies, which can improve the sensitivity of the reagent.

[0019] In one example of the present invention, the monoclonal antibody pair against human heparin-binding protein includes two or more monoclonal antibodies against human heparin-binding protein.

[0020] Compared with the prior art, the technical effects achieved by adopting this technical solution are as follows: The monoclonal antibody pair against human heparin-binding protein has high specificity. While specifically recognizing heparin-binding protein, it can better improve the linearity of the reagent.

[0021] In one example of the present invention, the preparation method of the polystyrene latex microspheres coated with the monoclonal antibody pair against human heparin-binding protein includes: S100, Activation: Activate the polystyrene latex microspheres and prepare a latex activation solution; Activate the monoclonal antibody against human heparin-binding protein and prepare an antibody activation solution; S200, Coupling: Mix the latex activation solution and the antibody activation solution; S300, Blocking: After the coupling reaction is completed, centrifuge to remove the supernatant and add BSA for blocking; S400, Dissolution: After blocking is completed, centrifuge to remove the supernatant and add a dispersion solution for dissolution to obtain the polystyrene latex microspheres coated with the monoclonal antibody pair against human heparin-binding protein.

[0022] Compared with the prior art, the technical effects achieved by adopting this technical solution are as follows: By adopting this preparation method, the monoclonal antibody against human heparin-binding protein can be more firmly bound to the surface groups of the polystyrene latex microspheres and is not easily detached, thereby increasing the sensitivity and stability of the reagent.

[0023] In an example of the present invention, S100 includes: preparing a latex activation solution: diluting polystyrene latex microspheres with a buffer solution to 0.15%, adding EDC and NHS, centrifuging to remove the supernatant after reaction at room temperature to obtain a first precipitate; suspending the first precipitate in the buffer solution, ultrasonically dispersing, adding AAD and reacting at room temperature, and centrifuging again to remove the supernatant to obtain a second precipitate; suspending the second precipitate in the buffer solution and ultrasonically dispersing to obtain a latex activation solution; and / or preparing an antibody activation solution: adding two or more anti-human heparin-binding protein monoclonal antibodies to a buffer solution, mixing evenly, adding sodium periodate and reacting at room temperature to obtain an antibody activation solution.

[0024] Compared with the prior art, the technical effects achieved by adopting this technical solution are as follows: The carboxyl groups on the surface of polystyrene latex microspheres are activated into hydrazides by EDC and NHS to prepare a latex activation solution. The pair of anti-human heparin-binding protein monoclonal antibodies is composed of anti-human heparin-binding protein monoclonal antibodies mixed in a ratio of 1:1, reacting it with sodium periodate to prepare an antibody activation solution; then mixing and reacting the latex activation solution and the antibody activation solution. This coupling method can make the anti-human heparin-binding protein monoclonal antibody more firmly bound to the surface groups of polystyrene latex microspheres and not easily fall off, thereby increasing the sensitivity and stability of the reagent.

[0025] In an example of the present invention, reagent two further contains a protective agent, and the protective agent is one or more of sucrose, glycerol, bovine serum albumin, and glycine; and / or reagent two further contains a buffer solution, and the buffer solution is any one of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer solution, phosphate buffer solution, and tris(hydroxymethyl)aminomethane hydrochloride buffer solution; and / or reagent two further contains a preservative, and the preservative is Proclin-300 or sodium azide.

[0026] Compared with the prior art, the technical effects achieved by adopting this technical solution are as follows: The protective agent can increase the long-term stability of the reagent.

[0027] In an example of the present invention, the kit further includes a calibrator, and the calibrator is a recombinant human heparin-binding protein solution with 5 to 6 different concentrations, and the concentration of the recombinant human heparin-binding protein solution is 0 ng / mL to 600 ng / mL.

[0028] After adopting the technical solution of the present invention, the following technical effects can be achieved: (1) Mix reagent one containing surfactant and macromolecular polymer with the sample to be tested. The surfactant in reagent one can effectively inhibit the adsorption of non-specific substances such as conjugated bilirubin, unconjugated bilirubin, triglyceride, and hemoglobin in the sample to be tested onto polystyrene latex microspheres. Then mix reagent two containing polystyrene latex microspheres coated with monoclonal antibody pairs against human heparin-binding protein with the mixture of reagent one and the sample to be tested, so that the heparin-binding protein in the sample to be tested specifically binds to the monoclonal antibody pairs against human heparin-binding protein, forming an insoluble polystyrene latex microglobulin complex-antibody-polystyrene latex microglobulin complex emulsion, generating a certain turbidity, and the level of the turbidity is proportional to the concentration of heparin-binding protein in the sample. Among them, the macromolecular polymer contained in reagent one can greatly improve the binding of heparin-binding protein and monoclonal antibody against human heparin-binding protein, greatly increasing the turbidity of the formed complex emulsion, thus greatly improving the sensitivity and repeatability of the reagent; (2) The kit of the present invention adopts latex-enhanced immunoturbidimetry technology, which can be efficiently tested on an automatic biochemical analyzer, and has the characteristics of high cost-effectiveness and fast analysis speed. Specific embodiments

[0029] To make the above objects, features, and advantages of the present invention more obvious and understandable, the following detailed description of the specific embodiments of the present invention is provided.

[0030] The present invention provides a kit for detecting heparin-binding protein, and the kit includes: reagent one and reagent two. Reagent one is a buffer solution containing surfactant and macromolecular polymer; reagent two is a buffer solution containing polystyrene latex microspheres coated with monoclonal antibody pairs against human heparin-binding protein.

[0031] Furthermore: The reaction principle of the present invention will be described in combination with the actual use of the kit of the present invention. First, reagent one containing a surfactant and a macromolecular polymer is mixed with the test sample. The surfactant in reagent one can effectively inhibit the adsorption of non-specific substances such as conjugated bilirubin, unconjugated bilirubin, triglycerides, and hemoglobin in the test sample onto the polystyrene latex microspheres. Then, reagent two containing polystyrene latex microspheres coated with a monoclonal antibody pair against human heparin-binding protein is mixed with the mixture of reagent one and the test sample, so that the heparin-binding protein in the test sample specifically binds to the monoclonal antibody pair against human heparin-binding protein, forming an insoluble polystyrene latex microglobulin complex-antibody-polystyrene latex microglobulin complex emulsion, generating a certain turbidity, and the level of the turbidity is directly proportional to the concentration of heparin-binding protein in the sample. Among them, the macromolecular polymer contained in reagent one can greatly improve the binding of heparin-binding protein and the monoclonal antibody against human heparin-binding protein, greatly increasing the turbidity of the formed complex emulsion, thereby greatly improving the sensitivity and repeatability of the reagent, more accurately reflecting the changes of HBP in the human body, and better reflecting the clinical application value of HBP.

[0032] In a specific embodiment of the present invention, the concentration ratio of the surfactant to the macromolecular polymer is 3:1.

[0033] Furthermore: In some embodiments, the concentration of the surfactant is 3 g / L and the concentration of the macromolecular polymer is 1 g / L. Among them, the surfactant is diphenylstyryl phenol polyoxyethylene ether-18.8; the macromolecular polymer is poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol); according to the results in Table 1-5, when the concentration of the surfactant is 3 g / L and the concentration of the macromolecular polymer is 1 g / L, the repeatability is good.

[0034] In a specific embodiment of the present invention, the concentration of the surfactant is 0.3 g / L to 30 g / L, and the concentration of the macromolecular polymer is 0.1 g / L to 10 g / L.

[0035] In a specific embodiment of the present invention, the surfactant is diphenylstyryl phenol polyoxyethylene ether-18.8; and / or the macromolecular polymer is poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol).

[0036] Further: The surfactant is specifically selected as styrylphenol polyoxyethylene ether - 18.8; the macromolecular polymer is specifically selected as poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol); among them, styrylphenol polyoxyethylene ether - 18.8 can block other non-specific reactions except for the specific binding of heparin-binding protein and monoclonal antibody against human heparin-binding protein; the monoclonal antibody against human heparin-binding protein can improve the linearity of the reagent better while specifically recognizing heparin-binding protein; poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) can better improve the efficiency of specific binding of heparin-binding protein and monoclonal antibody against human heparin-binding protein, and improve the sensitivity and repeatability of the reagent.

[0037] In a specific embodiment of the present invention, the particle size of the polystyrene latex microspheres is 300 nm to 450 nm.

[0038] Further: The particle size of the polystyrene latex microspheres is 300 nm to 450 nm. The polystyrene latex microspheres within this particle size range have a high carboxyl content and a high antibody-binding efficiency, which can improve the sensitivity of the reagent.

[0039] In a specific embodiment of the present invention, the monoclonal antibody pair against human heparin-binding protein includes two or more monoclonal antibodies against human heparin-binding protein.

[0040] Further: The monoclonal antibody pair against human heparin-binding protein is formed by mixing monoclonal antibodies against human heparin-binding protein paired at two or more sites. In some embodiments, the monoclonal antibody pair against human heparin-binding protein is formed by mixing two monoclonal antibodies against human heparin-binding protein in a ratio of 1:1. The monoclonal antibody pair against human heparin-binding protein can improve the linearity of the reagent better while specifically recognizing heparin-binding protein.

[0041] In a specific embodiment of the present invention, the preparation method of the polystyrene latex microspheres coated with the monoclonal antibody pair against human heparin-binding protein includes: S100, Activation: Activate the polystyrene latex microspheres and prepare a latex activation solution; Activate the monoclonal antibody against human heparin-binding protein and prepare an antibody activation solution; S200, Coupling: Mix the latex activation solution and the antibody activation solution; S300, Blocking: After the coupling reaction is completed, centrifuge to remove the supernatant and add BSA for blocking; S400, Dissolution: After blocking, centrifuge to remove the supernatant and add a dispersion solution for dissolution to obtain the polystyrene latex microspheres coated with the monoclonal antibody pair against human heparin-binding protein.

[0042] Further: A method for preparing polystyrene latex microspheres coated with a pair of monoclonal antibodies against human heparin-binding protein. In some embodiments, the latex activation solution and the antibody activation solution are mixed and incubated overnight at room temperature. After centrifugation at 12,000 rpm for 20 min, the supernatant is removed. The precipitate is suspended in 20 mM HEPES (pH 7.4) buffer, ultrasonically dispersed, 1% BSA is added, and it is blocked at 25 °C for 1 hour, followed by centrifugation to remove the supernatant; the latex is dissolved with the dispersion solution and ultrasonically dispersed; wherein, the dispersion solution includes 20 mM HEPES (pH 7.4) buffer, 10% Tween 20, 10% sucrose, 0.2% BSA, and 0.1% Proclin 300. By adopting this preparation method, the monoclonal antibody against human heparin-binding protein can be more firmly bound to the surface groups of the polystyrene latex microspheres and is not easily detached, thereby increasing the sensitivity and stability of the reagent.

[0043] In a specific embodiment of the present invention, S100 includes: preparing a latex activation solution: diluting polystyrene latex microspheres to 0.15% with a buffer solution, adding EDC and NHS, reacting at room temperature and then centrifuging to remove the supernatant to obtain precipitate one; suspending precipitate one in the buffer solution, ultrasonically dispersing, adding AAD and reacting at room temperature, and centrifuging again to remove the supernatant to obtain precipitate two; suspending precipitate two in the buffer solution and ultrasonically dispersing to obtain the latex activation solution; and / or preparing an antibody activation solution: adding two or more monoclonal antibodies against human heparin-binding protein to the buffer solution, mixing evenly, adding sodium periodate and reacting at room temperature to obtain the antibody activation solution.

[0044] Further: The Chinese name of EDC is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and the Chinese name of NHS is N-hydroxysuccinimide; the activation principle of the latex activation solution is that the carboxyl groups on the surface of the polystyrene latex microspheres are activated into hydrazides through EDC and NHS; in some embodiments, the buffer solution is 20 mM HEPES (pH 7.4) buffer solution, the concentration of EDC is 0.5 mg / ml, the concentration of NHS is 0.3 mg / ml, and the concentration of AAD is 3 mg / ml; the polystyrene latex particles with a particle size of 350 nm are diluted to a concentration of 0.15% with 20 mM HEPES (pH 7.4) buffer solution for preparing the latex activation solution; the concentration of the monoclonal antibody against human heparin-binding protein is 0.1 mg / ml. Two monoclonal antibodies against human heparin-binding protein are mixed in a ratio of 1:1 and then reacted with sodium periodate to prepare the antibody activation solution. By this preparation method, the monoclonal antibody against human heparin-binding protein can be more firmly bound to the surface groups of the polystyrene latex microspheres and is not easily detached, thereby increasing the sensitivity and stability of the reagent.

[0045] In a specific embodiment of the present invention, reagent two further contains a protective agent, and the protective agent is one or more of sucrose, glycerol, bovine serum albumin, and glycine; and / or reagent two further contains a buffer solution, and the buffer solution is any one of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer solution, phosphate buffer solution, and tris(hydroxymethyl)aminomethane hydrochloride buffer solution; and / or reagent two further contains a preservative, and the preservative is Proclin-300 or sodium azide.

[0046] Furthermore: In some embodiments, the buffer solution is selected as 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (abbreviated as HEPES) buffer solution; the protective agent is selected as 10% sucrose; the preservative is selected as 0.1% Proclin-300.

[0047] In a specific embodiment of the present invention, the kit further includes a calibrator, and the calibrator is a recombinant human heparin-binding protein solution with 5 to 6 different concentrations, and the concentration of the recombinant human heparin-binding protein solution is 0 ng / mL - 600 ng / mL.

[0048] Example 1 A heparin-binding protein detection kit: It includes a calibrator, reagent one, and reagent two; The components and their corresponding contents contained in the calibrator and reagent one are: Calibrator: 15 mmol / L phosphate buffer solution (pH 7.4); 9 g / L sodium chloride; 20 g / L BSA; 1 g / L Proclin-300; recombinant heparin-binding protein; Reagent one: 100 mmol / L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer solution; 30 g / L sodium chloride; 1 g / L Proclin 300; 2 g / L bovine serum albumin; 3 g / L surfactant, 1 g / L macromolecular polymer; Reagent two: Preparation method of polystyrene latex microspheres labeled with monoclonal antibody pairs against human heparin-binding protein: S100. Dilute polystyrene latex particles with a particle size of 350 nm to a concentration of 0.15% with 20 mM HEPES (pH 7.4) buffer solution, add 0.5 mg / ml EDAC and 0.3 mg / ml NHS, react at room temperature for 0.5 hour, centrifuge at 12,000 revolutions per minute for 20 min, remove the supernatant, suspend the precipitate in 20 mM HEPES (pH 7.4), ultrasonically disperse, add 3 mg / ml AAD, react at room temperature for 3 hours, centrifuge again at 12,000 revolutions per minute for 20 min, remove the supernatant, suspend the precipitate in 20 mM HEPES pH 7.4, ultrasonically disperse to prepare a latex activation solution; Antibody activation solution was prepared by adding 0.1 mg / ml of anti-human heparin binding protein monoclonal antibody 1 and 0.1 mg / ml of anti-human heparin binding protein monoclonal antibody 2 to 20 mM HEPES (pH 7.4) buffer, mixing well, then adding 1 mg / ml of NaIO4 and reacting at room temperature for 0.5 hour. S200: Mix the latex activation solution and the antibody activation solution at room temperature overnight. Centrifuge at 12,000 rpm for 20 minutes and remove the supernatant. S300, the precipitate was suspended in 20 mM HEPES (pH 7.4), dispersed by ultrasonication, and 1% BSA was added. The cells were blocked at 25°C for 1 hour, and the supernatant was removed by centrifugation. S400, dissolve the latex in a dispersion solution containing 20 mM HEPES (pH 7.4), 10% Tween 20, 10% sucrose, 0.2% BSA, and 0.1% Proclin 300, and disperse it by ultrasonication.

[0049] Example 2 The only difference from Example 1 is that the content of the surfactant in Reagent 1 is 3 g / L, the content of the macromolecular polymer is 3 g / L, and the contents of the other components are the same as in Example 1.

[0050] Example 3 The only difference from Example 1 is that the content of the surfactant in Reagent 1 is 1 g / L, the content of the macromolecular polymer is 1 g / L, and the contents of the other components are the same as in Example 1.

[0051] Comparative Example 1 The only difference from Example 1 is that Reagent 1 does not contain surfactant and macromolecular polymer, and the contents of other components are the same as in Example 1.

[0052] Comparative Example 2 The only difference from Example 1 is that the content of the surfactant in Reagent 1 is 1 g / L, and it does not contain macromolecular polymers. The contents of other components are the same as in Example 1.

[0053] Comparative Example 3 The only difference from Example 1 is that the content of the surfactant in Reagent 1 is 3 g / L, and it does not contain macromolecular polymers. The contents of other components are the same as in Example 1.

[0054] Comparative Example 4 The only difference from Example 1 is that the content of the macromolecular polymer in Reagent 1 is 1 g / L, and no surfactant is contained. The contents of other components are the same as in Example 1.

[0055] Comparative Example 5 It is only different from Example 1 in that the content of the macromolecular polymer in Reagent 1 is 3 g / L, and there is no surfactant, and the contents of other components are the same as those in Example 1.

[0056] Detection parameters The determination method is the two-point end-point method, and the detection wavelengths are the main wavelength of 600 nm respectively. The dosages of Reagent 1 and Reagent 2 are 200 μl and 50 μl respectively. The dosage of the sample to be tested is 6 μl; the reaction direction is upward, and the determination temperature is 37 °C. First, mix the sample to be tested with Reagent 1 and incubate for 5 minutes, then add Reagent 2, immediately read the absorbance value at the first point (A1), after timing the reaction for 5 minutes, read the absorbance value at the second point (A2), calculate the difference in absorbance between the two points, and obtain the content of heparin-binding protein in the sample to be tested according to the calibration curve.

[0057] Calibrate with the reagents and calibrators in Examples 1-3 and Comparative Examples 1-5 respectively to obtain their respective calibration curves. Then, use the reagents in Examples 1-3 and Comparative Examples 1-5 to detect clinical plasma samples at and below the concentration levels near the medical decision level. Each sample is repeated 10 times to obtain their respective absorbance differences, and then calculate the respective concentration values by the respective calibration curves for the absorbance differences. The results are shown in Tables 1-5.

[0058] Table 1: Repeatability of Sample 1 Table 2: Repeatability of Sample 2 Table 3: Repeatability of Sample 3 Table 4: Repeatability of Sample 4 Table 5: Repeatability of Sample 5 It can be seen from the detection results in Tables 1 to 5 that Example 1 has the smallest repeatability CV values in the same batch of samples, which are 1.5%, 1.4%, 2.6%, 3.8%, and 5.8% respectively, indicating that Reagent 1 contains 3 g / L of surfactant and 1 g / L of macromolecular polymer, and the reagent has good repeatability, and the improvement of repeatability is relatively obvious.

[0059] Although the present invention is disclosed as above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be subject to the scope defined by the claims.

Claims

1. A heparin-binding protein detection kit, characterized in that, the kit comprises: Reagent 1 and Reagent 2, Reagent 1 is a buffer solution containing a surfactant and a macromolecular polymer; Reagent 2 is a buffer solution containing polystyrene latex microspheres coated with a monoclonal antibody pair against human heparin-binding protein.

2. The kit according to claim 1, characterized in that, the concentration ratio of the surfactant to the macromolecular polymer is 3:

1.

3. The kit according to claim 1, characterized in that, the concentration of the surfactant is 0.3 g / L to 30 g / L, the concentration of the macromolecular polymer is 0.1 g / L to 10 g / L.

4. The kit according to claim 1, characterized in that, the surfactant is diphenylstyryl phenol polyoxyethylene ether-18.8; and / or the macromolecular polymer is poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol).

5. The kit according to claim 1, characterized in that, the particle size of the polystyrene latex microspheres is 300 nm to 450 nm.

6. The kit according to claim 1, characterized in that, the monoclonal antibody pair against human heparin-binding protein comprises two or more monoclonal antibodies against human heparin-binding protein.

7. The kit according to claim 1, characterized in that, the preparation method of the polystyrene latex microspheres coated with the monoclonal antibody pair against human heparin-binding protein comprises: S100, activation: Activate the polystyrene latex microspheres to prepare a latex activation solution; Activate the monoclonal antibody against human heparin-binding protein to prepare an antibody activation solution; S200, coupling: Mix the latex activation solution and the antibody activation solution; S300, blocking: After the coupling reaction is completed, centrifuge to remove the supernatant, and add BSA for blocking; S400, dissolution: After blocking is completed, centrifuge to remove the supernatant, and add a dispersion solution for dissolution to obtain the polystyrene latex microspheres coated with the monoclonal antibody pair against human heparin-binding protein.

8. The kit according to claim 7, characterized in that, S100 comprises: Preparation of the latex activation solution: Dilute the polystyrene latex microspheres with a buffer solution to 0.15%, add EDC and NHS, react at room temperature and then centrifuge to remove the supernatant to obtain precipitate 1; Suspend precipitate 1 in a buffer solution, ultrasonically disperse, add AAD and react at room temperature, and then centrifuge to remove the supernatant to obtain precipitate 2; Suspend precipitate 2 in a buffer solution and ultrasonically disperse to obtain the latex activation solution; and / or Preparation of the antibody activation solution: Add two or more monoclonal antibodies against human heparin-binding protein to a buffer solution, mix evenly, add sodium periodate and react at room temperature to obtain the antibody activation solution.

9. The kit according to claim 1, characterized in that, Reagent 2 further contains a protective agent, and the protective agent is one or more of sucrose, glycerol, bovine serum albumin and glycine; and / or The reagent II further contains a buffer solution, and the buffer solution is any one of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer solution, phosphate buffer solution, and tris(hydroxymethyl)aminomethane hydrochloride buffer solution; and / or The reagent II further contains a preservative, and the preservative is Proclin-300 or sodium azide.

10. The kit according to claim 1, wherein The kit further includes a calibrator, and the calibrator is a recombinant human heparin-binding protein solution with 5 to 6 different concentrations, and the concentration of the recombinant human heparin-binding protein solution is 0 ng / mL - 600 ng / mL.

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