Heparin binding protein detection kit and preparation method thereof

Abstract

The invention provides a heparin binding protein detection kit and a preparation method thereof, belonging to the field of biological detection technology. The kit comprises a reagent R1 and a reagent R2 which are independent of each other, characterized in that the components and corresponding contents thereof are as follows: reagent R1: tris hydroxymethylaminomethane buffer 30-60mmol / L; polyethylene glycol 6000 1-3g / L; surfactant A 15-25g / L; sodium azide 0.05-0.15g / L; reagent R2: tris hydroxymethylaminomethane buffer 30-60mmol / L; latex particles coated with mouse anti-human heparin binding protein antibody 1-3g / L; surfactant B 15-25g / L; stabilizer 5-10g / L; sodium azide 0.05-0.15g / L; the kit has higher precision and stability through the selection of surfactants and stabilizers in reagents 1 and 2, and the anti-interference ability is also significantly improved.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and particularly relates to a heparin binding protein detection kit based on latex immunoturbidimetry and a preparation method thereof. Background Art

[0002] Heparin-binding protein (HBP), also known as azurocidin or CAP37, is a granule protein derived from neutrophils. The chemotactic activity, bactericidal ability and heparin binding ability of HBP are related to its strong positive charge. As a chemotactic substance, HBP can activate monocytes and macrophages, induce vascular leakage and tissue edema, and induce Ca2+ in vitro. 2+ HBP can increase the permeability of vascular endothelial cells because HBP has a strong positive charge, which contacts with endothelial cells and causes the intracellular Ca2+ of endothelial cells to increase. 2+ Rapid activation forms actin tension fibers, which leads to leakage of the paracellular pathway. Excessive concentration of HBP can lead to severe vascular leakage, causing hypotension or even shock. In addition, HBP is also associated with a variety of infections such as soft tissue infections, which indicates that HBP is closely related to severe bacterial infections and is likely to become a clinical diagnostic indicator and drug treatment target.

[0003] At present, the commonly used methods for detecting the content of heparin binding protein in clinical practice are enzyme-linked immunosorbent assay, immunoturbidimetry and immunofluorescence assay. The detection sensitivity of enzyme-linked immunosorbent assay is high, but it has the defects of cumbersome operation process, long test cycle and high labor cost; immunoturbidimetry has low detection sensitivity, narrow linear range and long test cycle, so it cannot effectively meet clinical needs; therefore, the fluorescent immunoassay is more commonly used to detect heparin binding protein, but this method also has the defects of large amount of antibody used, high reagent cost, low sensitivity of most products in the low value area and poor repeatability near the clinical reference value, which makes it difficult to accurately and quantitatively detect heparin binding protein.

[0004] Chinese patent CN115980331A discloses a kit for detecting heparin-binding protein. It contains a sample treatment agent and a detection kit; the sample treatment agent includes: sodium alginate with a mass concentration of 0.01-0.5%, polyoxyethylene lauryl ether of 0.1-10g / L, and Tris buffer of 30-100mmol / L; the pH of the sample treatment agent is 7.8-8.2. The kit for detecting heparin-binding protein of the invention can detect heparin-binding protein in serum, plasma and whole blood at the same time, and does not require centrifugation of blood samples, which greatly shortens the detection time; and can more efficiently extract heparin-binding protein and reduce background interference, and has the advantages of high sensitivity, good repeatability, good specificity, wide linear range, low cost, etc.

[0005] Chinese patent CN115656518A discloses a detection kit for detecting heparin-binding protein in human plasma and urine, comprising reagent R1, reagent R2 and a calibrator, wherein the reagent R1 comprises a blocker, salts and a preservative to resist interference from homologous proteins in human plasma and urine; the reagent R2 comprises a first polystyrene latex microsphere, a second polystyrene latex microsphere, a protective agent and a preservative. The kit can eliminate interference from homologous samples and can be used for urine sample detection, and has the characteristics of fast detection speed, good linear range and good stability.

[0006] However, the precision, stability and anti-interference ability of the existing test kits cannot better meet the requirements. Therefore, it is necessary to develop a heparin-binding protein detection kit based on latex immunoturbidimetry with high precision, stability and good linearity and a preparation method thereof. Summary of the invention

[0007] Based on the deficiencies in the prior art, the present invention aims to provide a heparin binding protein detection kit based on latex immunoturbidimetry and a preparation method thereof. The kit has high precision and stability and good linear relationship by selecting surfactants in reagents 1 and 2.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] In one aspect, the present invention provides a kit for detecting heparin binding protein (latex immunoturbidimetry), comprising a reagent R1 and a reagent R2 which are independent of each other, and the components and corresponding contents thereof are as follows:

[0010] Reagent R1: Tris buffer 30-60mmol / L; polyethylene glycol 6000 1-3g / L; surfactant A 15-25g / L; sodium azide 0.05-0.15g / L;

[0011] Reagent R2: Tris buffer 30-60 mmol / L; latex particles coated with mouse anti-human heparin binding protein antibody 1-3 g / L; surfactant B 15-25 g / L; stabilizer 5-10 g / L; sodium azide 0.05-0.15 g / L.

[0012] Preferably, the kit comprises reagent R1 and reagent R2 which are independent of each other, and the components and corresponding contents thereof are as follows:

[0013] Reagent R1: Tris buffer 50mmol / L; polyethylene glycol 6000 2g / L; surfactant A 20g / L; sodium azide 0.1g / L;

[0014] Reagent R2: Tris buffer 50 mmol / L; latex particles coated with mouse anti-human heparin binding protein antibody 2 g / L; surfactant B 20 g / L; stabilizer 6 g / L; sodium azide 0.1 g / L.

[0015] Wherein, the surfactant A is a mixture of Tween 20 and sodium lauroyl glutamate; the concentration ratio of the two is 6-8:1; preferably 7:1.

[0016] The surfactant A in reagent 1 is a mixture of Tween 20 and sodium lauroyl glutamate. Tween 20 has the function of renaturing antigens, which can improve the specific recognition ability, will not destroy the protein structure, and can reduce the damage to the original interaction between proteins; sodium lauroyl glutamate contains long hydrophilic and hydrophobic chains, which will cause steric hindrance and thus affect the interaction between proteins. However, during the implementation of the present invention, it was unexpectedly found that adding a mixture of Tween 20 and sodium lauroyl glutamate as a surfactant to reagent 1 and controlling the concentration ratio of the two to 6-8:1 can significantly improve the precision and sensitivity of the test kit.

[0017] The surfactant B is Triton X-100;

[0018] The stabilizer is a mixture of mannitol, trehalose and 1-aminocyclohexanecarboxylic acid, and the concentration ratio of the three is 1-3:5:1-2; preferably 2:5:1.

[0019] The invention uses a mixture of mannitol, trehalose and 1-aminocyclohexanecarboxylic acid as a stabilizer and is used in combination with sodium azide, which can not only protect the structure of antibody protein and prevent protein degradation, but also inhibit the growth of microorganisms and has a good antibacterial effect; and the invention controls the concentration ratio of mannitol, trehalose and 1-aminocyclohexanecarboxylic acid to be 1-3:5:1-2, and the stability of the reagent can be better improved through the interaction between the three, thereby improving the accuracy and anti-interference performance of the kit.

[0020] As some preferred embodiments, the kit includes reagent R1 and reagent R2 which are independent of each other, and the components and corresponding contents thereof are as follows:

[0021] Reagent R1: Tris buffer 30-60mmol / L; polyethylene glycol 6000 1-3g / L; Tween 2014-22g / L; sodium lauroyl glutamate 1-3g / L; sodium azide 0.05-0.15g / L;

[0022] Reagent R2: Tris buffer 30-60mmol / L; latex particles coated with mouse anti-human heparin binding protein antibody 1-3g / L; Triton X-100 15-25g / L; stabilizer 5-10g / L; sodium azide 0.05-0.15g / L;

[0023] The stabilizer is a mixture of mannitol, trehalose and 1-aminocyclohexanecarboxylic acid in a concentration ratio of 1-3:5:1-2.

[0024] As a preferred embodiment, the kit comprises reagent R1 and reagent R2 which are independent of each other, and the components and corresponding contents thereof are as follows:

[0025] Reagent R1: Tris buffer 50mmol / L; polyethylene glycol 6000 2g / L; Tween 20 17.5g / L; sodium lauroyl glutamate 2.5g / L; sodium azide 0.1g / L;

[0026] Reagent R2: Tris buffer 50mmol / L; latex particles coated with mouse anti-human heparin binding protein antibody 2g / L; Triton X-100 20g / L; mannitol 1.5g / L, trehalose 3.75g / L; 1-aminocyclohexanecarboxylic acid 0.75g / L; sodium azide 0.1g / L.

[0027] In the kit, the volume ratio of the sample, reagent R1 and reagent R2 is 1:18-22:4-6, preferably 1:20:5.

[0028] Furthermore, the heparin binding protein detection kit also includes a calibrator and a quality control product, and the calibrator and the quality control product both include phosphate buffer, sodium azide and HBP antigen.

[0029] On the other hand, the present invention also provides a method for preparing the above-mentioned kit, comprising the following steps:

[0030] (1) Preparation of Reagent 1: Prepare a buffer solution and adjust the pH of the buffer solution to 6.0-8.0. Add polyethylene glycol 6000, surfactant A and sodium azide to the buffer solution and dissolve them to obtain Reagent 1.

[0031] (2) Preparation of Reagent 2: Prepare a buffer solution and adjust the pH of the buffer solution to 6.0-8.0. Add latex particles coated with mouse anti-human heparin binding protein antibody, surfactant B, stabilizer and sodium azide to the buffer solution and dissolve them to obtain Reagent 2.

[0032] In another aspect, the present invention also provides a method for using the above kit:

[0033] This kit is suitable for:

[0034] Hitachi series: 7080 / 7100 / 7180 / 7600 / 3100 / 3110 / 3500 / LST006 / LST008AS / LST008a;

[0035] Beckman Series:

[0036] AU400 / AU480 / AU640 / AU680 / AU2700 / AU5400 / AU5421 / AU5800, DXC 700AU, DXC800;

[0037] Toshiba series: TBA-40FR / 120FR / 2000FR;

[0038] Roche series: MODULAR, Cobas c311, Cobas c501, Cobas c502, Cobas c701, Cobasc702;

[0039] Mindray series: BS320 / 380 / 400 / 420 / 2800(M) / 2000(M);

[0040] Meilian series: MS-480, MS-480B, MS-880, MS-880B, MS-300, MS-200, MS-1280, MS-2080, MS-1880, MS-1680, MS-680, MS-600, MS-520, MS-450, MS-L8080, MS-L7280, MS-L8060, MS-L8000, MS-380, MS-400, MS-420, MS-380P, MS-400P, MS-420P;

[0041] Runkang series: RC-480, RC-460, RC-400, RC-680, RC-600, RC-520, RC-450, RC-880, RC-860, RC-800, RC-2080, RC-1880, RC1680;

[0042] Siemens series: Dimension RXL, Dimension EXL, Dimension X-PAND, ADVIA, XPT, ADVIA 1800, ADVIA 2400, Atellica;

[0043] Sysmex series: BX-3010, BX-4000;

[0044] Abbott series: c8000 / c4000 / c16000 / ci4100 / ci8200 / ci16200 / Alinity;

[0045] Canon series: TBA-FX8, TBA-1500FR.

[0046] Test conditions:

[0047]

[0048]

[0049] Steps:

[0050]

[0051] The principle of this method is: the heparin-binding protein HBP in the sample combines with the corresponding antibody bound to the latex particles in the reagent, an agglutination reaction occurs, an antigen-antibody complex is formed, and a certain turbidity is produced within the linear range. The level of this turbidity is proportional to the concentration of HBP in the sample when sufficient antibodies are present. The concentration of HBP in the blood can be calculated by measuring the absorbance value at a specific wavelength and referring to the calibration curve.

[0052] Compared with the prior art, the technical effects of the present invention are:

[0053] (1) The present invention adds surfactants to both reagent 1 and reagent 2, thereby improving the uniformity of the reagents; the mixture of Tween 20 and sodium lauroyl glutamate used in reagent 1 has the function of renaturing antigens, which can improve the specific recognition ability, will not destroy the protein structure, and can reduce the damage to the original interaction between proteins; sodium lauroyl glutamate contains long hydrophilic and hydrophobic chains, which will cause steric hindrance and thus affect the interaction between proteins. However, during the implementation of the present invention, it was unexpectedly found that adding a mixture of Tween 20 and sodium lauroyl glutamate as a surfactant to reagent 1 and controlling the concentration ratio of the two to 6-8:1 can significantly improve the precision and sensitivity of the test kit.

[0054] (2) The present invention adds a stabilizer to reagent 2. The present invention uses a mixture of mannitol, trehalose and 1-aminocyclohexanecarboxylic acid as a stabilizer, and sodium azide is added to reagent 2 to not only protect the structure of the antibody protein and prevent protein degradation, but also inhibit the growth of microorganisms, and has a good antibacterial effect; and the present invention controls the concentration ratio of mannitol, trehalose and 1-aminocyclohexanecarboxylic acid to be 1-3:5:1-2. Through the interaction between the three, the stability of the reagent can be better improved, thereby improving the accuracy and anti-interference performance of the kit. DETAILED DESCRIPTION

[0055] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0056] The latex particles coated with mouse anti-human heparin binding protein antibody used in the following examples were purchased from Shaoxing Chuangye Biotechnology Co., Ltd. with a specification of ≥50g / L.

[0057] Example 1 A kit for detecting heparin binding protein (latex immunoturbidimetry)

[0058] The invention comprises reagent R1 and reagent R2 which are independent of each other, and the components and corresponding contents thereof are as follows:

[0059] Reagent R1: Tris buffer 30mmol / L; polyethylene glycol 6000 1g / L; Tween 20 13.2g / L; sodium lauroyl glutamate 2.2g / L; sodium azide 0.05g / L;

[0060] Reagent R2: Tris buffer 30mmol / L; latex particles coated with mouse anti-human heparin binding protein antibody 1g / L; Triton X-100 15g / L; mannitol 0.8g / L; trehalose 4g / L; 1-aminocyclohexanecarboxylic acid 0.8g / L; sodium azide 0.05g / L.

[0061] The volume ratio of sample, reagent R1 and reagent R2 is 1:18:4, that is, 12 μL of sample, 216 μL of reagent R1 and 48 μL of reagent R2.

[0062] The preparation method comprises the following steps:

[0063] (1) Preparation of reagent 1: prepare tris(hydroxymethylaminomethane) buffer solution, adjust the pH of the buffer solution to 7.0, add polyethylene glycol 6000, Tween 20, sodium lauroyl glutamate and sodium azide to the buffer solution, respectively, and dissolve them to obtain reagent 1;

[0064] (2) Preparation of Reagent 2: Prepare tris(hydroxymethylaminomethane) buffer and adjust the pH of the buffer to 7.0. Add latex particles coated with mouse anti-human heparin binding protein antibody, Triton X-100, mannitol, trehalose, 1-aminocyclohexanecarboxylic acid and sodium azide to the buffer and dissolve them to obtain Reagent 2.

[0065] Example 2 A kit for detecting heparin binding protein (latex immunoturbidimetry)

[0066] The invention comprises reagent R1 and reagent R2 which are independent of each other, and the components and corresponding contents thereof are as follows:

[0067] Reagent R1: Tris buffer 60mmol / L; polyethylene glycol 6000 3g / L; Tween 20 21.6g / L; sodium lauroyl glutamate 2.7g / L; sodium azide 0.15g / L;

[0068] Reagent R2: Tris buffer 60mmol / L; latex particles coated with mouse anti-human heparin binding protein antibody 3g / L; Triton X-100 25g / L; mannitol 3g / L; trehalose 5g / L; 1-aminocyclohexanecarboxylic acid 2g / L; sodium azide 0.15g / L.

[0069] The volume ratio of sample, reagent R1 and reagent R2 is 1:22:6, that is, 12 μL of sample, 264 μL of reagent R1 and 72 μL of reagent R2.

[0070] The preparation method comprises the following steps:

[0071] (1) Preparation of reagent 1: prepare tris(hydroxymethylaminomethane) buffer solution, adjust the pH of the buffer solution to 7.0, add polyethylene glycol 6000, Tween 20, sodium lauroyl glutamate and sodium azide to the buffer solution, respectively, and dissolve them to obtain reagent 1;

[0072] (2) Preparation of Reagent 2: Prepare tris(hydroxymethylaminomethane) buffer and adjust the pH of the buffer to 7.0. Add latex particles coated with mouse anti-human heparin binding protein antibody, Triton X-100, mannitol, trehalose, 1-aminocyclohexanecarboxylic acid and sodium azide to the buffer and dissolve them to obtain Reagent 2.

[0073] Example 3 A kit for detecting heparin binding protein (latex immunoturbidimetry)

[0074] The invention comprises reagent R1 and reagent R2 which are independent of each other, and the components and corresponding contents thereof are as follows:

[0075] Reagent R1: Tris buffer 50mmol / L; polyethylene glycol 6000 2g / L; Tween 20 17.5g / L; sodium lauroyl glutamate 2.5g / L; sodium azide 0.1g / L;

[0076] Reagent R2: Tris buffer 50mmol / L; latex particles coated with mouse anti-human heparin binding protein antibody 2g / L; Triton X-100 20g / L; mannitol 1.5g / L, trehalose 3.75g / L; 1-aminocyclohexanecarboxylic acid 0.75g / L; sodium azide 0.1g / L.

[0077] The volume ratio of sample, reagent R1 and reagent R2 is 1:20:5, that is, 12 μL of sample, 240 μL of reagent R1 and 60 μL of reagent R2.

[0078] The preparation method comprises the following steps:

[0079] (1) Preparation of reagent 1: prepare tris(hydroxymethylaminomethane) buffer solution, adjust the pH of the buffer solution to 7.0, add polyethylene glycol 6000, Tween 20, sodium lauroyl glutamate and sodium azide to the buffer solution, respectively, and dissolve them to obtain reagent 1;

[0080] (2) Preparation of Reagent 2: Prepare tris(hydroxymethylaminomethane) buffer and adjust the pH of the buffer to 7.0. Add latex particles coated with mouse anti-human heparin binding protein antibody, Triton X-100, mannitol, trehalose, 1-aminocyclohexanecarboxylic acid and sodium azide to the buffer and dissolve them to obtain Reagent 2.

[0081] Comparative Example 1

[0082] The difference from Example 3 is that the surfactant in reagent R1 is only Tween 20, that is, Tween 20 20 g / L, and the contents of other components are the same as in Example 3.

[0083] Comparative Example 2

[0084] The difference from Example 3 is that the mass ratio of Tween 20 to sodium lauroyl glutamate in reagent R1 is 1:7, that is, 17.5 g / L of sodium lauroyl glutamate and 2.5 g / L of Tween 20, and the contents of other components are the same as those in Example 3.

[0085] Comparative Example 3

[0086] The difference from Example 3 is that the stabilizers in reagent R2 are mannitol and trehalose, namely, 1 g / L mannitol and 5 g / L trehalose, and the contents of other components are the same as those in Example 3.

[0087] Comparative Example 4

[0088] The difference from Example 3 is that the mass ratio of mannitol, trehalose and 1-aminocyclohexanecarboxylic acid is 2:1:3, i.e., 2 g / L mannitol, 1 g / L trehalose and 3 g / L 1-aminocyclohexanecarboxylic acid, and the contents of other components are the same as those in Example 3.

[0089] Comparative Example 5

[0090] The difference from Example 3 is that the mass ratio of mannitol, trehalose and 1-aminocyclohexanecarboxylic acid is 4:1:1, that is, 4 g / L mannitol, 1 g / L trehalose and 1 g / L 1-aminocyclohexanecarboxylic acid, and the contents of other components are the same as in Example 3.

[0091] Comparative Example 6

[0092] The difference from Example 3 is that the volume ratio of the sample, reagent R1 and reagent R2 is 3:40:10, that is, 15 μL of sample, 200 μL of reagent R1 and 50 μL of reagent R2, and the contents of other components are the same as in Example 3.

[0093] Effect test:

[0094] 1. Linear

[0095] Experimental requirements: Within the linear range of 6-340 ng / mL, the correlation coefficient r should be ≥ 0.990.

[0096] Experimental methods

[0097] The high-value sample (340ng / mL) close to the upper limit of the linear range was mixed with the zero-concentration sample to form 8 dilution concentrations, and the theoretical concentrations were 340ng / mL, 250ng / mL, 200ng / mL, 150ng / mL, 100ng / mL, 50ng / mL, 20ng / mL, and 6ng / mL. The sample of each concentration was tested 3 times to obtain the concentration value, and the measurement results of each sample were recorded, and the average value (yi) of the 3 measurement values ​​of each sample was calculated. The dilution concentration (x i ) is the independent variable, and the mean value (y i ) is the dependent variable to obtain the linear regression equation. The linear regression correlation coefficient (r) is calculated according to formula (1), which should meet the experimental requirements. The test data is shown in Table 1 below.

[0098]

[0099] Where:

[0100] r - correlation coefficient;

[0101] xi - dilution ratio;

[0102] y i -The mean value of the test results of each sample;

[0103] -mean of the dilution ratios;

[0104] -The overall mean of the sample measurement results.

[0105] Table 1

[0106] Correlation coefficient r Example 1 0.9985 Example 2 0.9988 Example 3 0.9995 Comparative Example 1 0.9856 Comparative Example 2 0.9972 Comparative Example 3 0.9978 Comparative Example 4 0.9980 Comparative Example 5 0.9981 Comparative Example 6 0.9863

[0107] According to the test results in Table 1 above, it can be seen that the experimental results of the linear correlation coefficient (r) of the kits prepared in Examples 1-3 of the present invention are not less than 0.990, and the linear relationship is good, especially the experimental result of the linear correlation coefficient (r) of the kit prepared in Example 3 is 0.9995, and the linear relationship is good. Therefore, the linear range of the kits prepared in Examples 1-3 can be defined as 6-340ng / mL; while the test kits prepared in Comparative Examples 1-6 are used for detection, the experimental results of the linear correlation coefficient (r) are lower than 0.9900, and although the linear relationship is good, it does not meet the requirements.

[0108] 2. Accuracy (stability)

[0109] The heparin-binding protein detection kits described in Examples 1-3 and Comparative Examples 1-6 were prepared respectively, and stored at 2°C-8°C in a light-proof environment without corrosive gases. The heparin-binding protein antigen sample reagents were tested at 1 month, 3 months, 6 months, 12 months, and 24 months of storage, and the accuracy was tested on the 1st of each month. The test data are shown in Table 2.

[0110] Experimental methods

[0111] A heparin binding protein antigen standard solution A with a concentration of 300 ng / mL (allowable deviation ±10%) was added to a sample B with a concentration of 180 ng / mL. The volume ratio between the added heparin binding protein antigen standard solution and the sample B was 1:9. The recovery rate R was calculated according to formula (2). The recovery rate should be within the range of 85%-115%. The specific test results are shown in Table 2 below.

[0112]

[0113] Where:

[0114] R—recovery rate;

[0115] V-volume of standard A solution;

[0116] V0-volume of sample B liquid;

[0117] C- the average of three measurements after adding the sample B solution to the standard A solution;

[0118] C0 - average of 3 measurements of sample B liquid;

[0119] C S -Concentration of standard solution A.

[0120] Table 2

[0121] 1 month 3 months 6 months 12 months 24 months Example 1 96.12％ 97.54％ 97.85％ 98.27％ 99.35％ Example 2 96.46％ 97.85％ 97.47％ 98.14％ 98.72％ Example 3 98.14％ 96.28％ 97.16％ 98.76％ 100.24％ Comparative Example 1 98.63％ 97.56％ 95.42％ 108.36％ 120.53％ Comparative Example 2 97.27％ 98.12％ 97.34％ 104.76％ 118.42％ Comparative Example 3 88.48％ 92.15％ 94.28％ 88.46％ 125.34％ Comparative Example 4 92.45％ 95.26％ 97.42％ 85.63％ 84.12％ Comparative Example 5 96.41％ 97.24％ 98.38％ 105.72％ 120.45％ Comparative Example 6 95.64％ 97.58％ 80.12％ 89.47％ 78.26％

[0122] According to the test results in Table 2 above, it can be seen that the test kits prepared in Examples 1-3 of the present invention, when the heparin binding protein antigen standard of known concentration is added to the low-value sample, have a recovery rate in the range of 85%-115%, which meets the requirements, and the test kits can still meet the conditions after being placed for 24 months, indicating that the stability of the test kits is relatively high; after the test kits prepared in Comparative Examples 1-6 were placed for 24 months, the detection accuracy of the test kits decreased to varying degrees, and the recovery rates obviously did not meet the requirements, indicating that changing the type or mass ratio of the reagents in the test kits will significantly affect the stability of the test kits, and the recovery rates were lower than 85% or higher than 115% after being placed for 24 months, which did not meet the requirements.

[0123] 3. Precision test

[0124] The heparin binding protein detection kits prepared in Examples 1-3 and Comparative Examples 1-5 were used to repeatedly test the same sample to be tested in the batch with a content of 250 ng / mL, and the SD and CV of the obtained results were calculated. The detection data are shown in Table 3.

[0125] Table 3

[0126]

[0127]

[0128] According to the test results in Table 3 above, it can be seen that the detection kits prepared in Examples 1-3 of the present invention, for repeated determinations of the same sample to be tested with an intra-batch content of 250 ng / mL, have a CV≤5%, which meets the requirements, indicating that the precision of the kit is relatively high; the precision of the repeated determinations of the same sample to be tested by the kits prepared in Comparative Examples 1-5 all decreased to varying degrees, indicating that changing the type or mass ratio of the reagents in the kit will significantly affect the precision of the kit.

[0129] It should be emphasized that the embodiments described in the present invention are illustrative rather than restrictive. Therefore, the present invention includes but is not limited to the embodiments described in the specific implementation manner. Any other implementation manner derived by those skilled in the art without departing from the technical solution of the present invention also falls within the scope of protection of the present invention.

Claims

1. A kit for detecting heparin-binding protein, comprising a reagent R1 and a reagent R2 which are independent of each other, characterized in that: The ingredients and corresponding contents are: Reagent R1: Tris buffer 30-60mmol / L; polyethylene glycol 6000 1-3g / L; surfactant A 15-25g / L; sodium azide 0.05-0.15g / L; Reagent R2: Tris buffer 30-60mmol / L; latex particles coated with mouse anti-human heparin binding protein antibody 1-3g / L; surfactant B 15-25g / L; stabilizer 5-10g / L; sodium azide 0.05-0.15g / L; The volume ratio of sample, reagent R1 and reagent R2 is 1:20:5; The surfactant A is a mixture of Tween 20 and sodium lauroyl glutamate; the concentration ratio of Tween 20 to sodium lauroyl glutamate is 6-8:1; The surfactant B is Triton X-100; The stabilizer is a mixture of mannitol, trehalose and 1-aminocyclohexanecarboxylic acid, and the concentration ratio of the mannitol, trehalose and 1-aminocyclohexanecarboxylic acid is 1-3:5:1-2.

2. The heparin binding protein detection kit according to claim 1, characterized in that: The invention comprises reagent R1 and reagent R2 which are independent of each other, and the components and corresponding contents thereof are as follows: Reagent R1: Tris buffer 50mmol / L; polyethylene glycol 6000 2g / L; surfactant A 20g / L; sodium azide 0.1g / L; Reagent R2: Tris buffer 50 mmol / L; latex particles coated with mouse anti-human heparin binding protein antibody 2 g / L; surfactant B 20 g / L; stabilizer 6 g / L; sodium azide 0.1 g / L.

3. The heparin binding protein detection kit according to claim 1, characterized in that: The concentration ratio of Tween 20 to sodium lauroyl glutamate is 7:

1.

4. The heparin binding protein detection kit according to claim 1, characterized in that: The concentration ratio of mannitol, trehalose and 1-aminocyclohexanecarboxylic acid is 2:5:

1.

5. The heparin binding protein detection kit according to any one of claims 1 to 4, characterized in that: The invention comprises reagent R1 and reagent R2 which are independent of each other, and the components and corresponding contents thereof are as follows: Reagent R1: Tris buffer 50mmol / L; polyethylene glycol 6000 2g / L; Tween 20 17.5g / L; sodium lauroyl glutamate 2.5g / L; sodium azide 0.1g / L; Reagent R2: Tris buffer 50mmol / L; latex particles coated with mouse anti-human heparin binding protein antibody 2g / L; Triton X-100 20g / L; mannitol 1.5g / L, trehalose 3.75g / L; 1-aminocyclohexanecarboxylic acid 0.75g / L; sodium azide 0.1g / L.

6. The method for preparing a heparin binding protein detection kit according to any one of claims 1 to 4, characterized in that: The steps include: (1) Preparation of Reagent 1: prepare a buffer solution and adjust the pH of the buffer solution to 6.0-8.

0. Add polyethylene glycol 6000, surfactant A and sodium azide to the buffer solution and dissolve them to obtain Reagent 1. (2) Preparation of Reagent 2: Prepare a buffer solution and adjust the pH of the buffer solution to 6.0-8.

0. Add latex particles coated with mouse anti-human heparin binding protein antibody, surfactant B, stabilizer and sodium azide into the buffer solution and dissolve them to obtain Reagent 2.

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