B-type natriuretic peptide detection kit and preparation method thereof

Abstract

The present invention discloses a B-type natriuretic peptide detection kit and a preparation method thereof. The B-type natriuretic peptide detection kit comprises: a magnetic bead component: containing streptavidin magnetic beads coated with a B-type natriuretic peptide monoclonal antibody; an enzyme component: containing a B-type natriuretic peptide monoclonal antibody labeled with alkaline phosphatase; wherein the magnetic bead component is in a paste or solid state after drying. Based on the chemiluminescence technology, the present invention performs drying treatment on the magnetic bead component to make the magnetic bead component in a paste or solid state, which can avoid the problem of magnetic bead sedimentation and wall adhesion caused by the inversion and overturning of the reagent during transportation; the present invention adopts the amplification effect of chemiluminescence technology and biotin-streptavidin, which can obtain lower sensitivity and wider linear range, react in a liquid homogeneous environment, and obtain better repeatability. The present invention adopts single-person reagent independent packaging, which is discarded after use to avoid pollution and waste.

Description

Technical Field

[0001] The present invention relates to the technical field of detection kits, and particularly to a B-type natriuretic peptide detection kit and a preparation method thereof. Background Art

[0002] Immunochromatography or chemiluminescence is a commonly used method for measuring B-type natriuretic peptide at present. However, due to technical limitations, the immunochromatography reagent has low sensitivity and poor repeatability.

[0003] Chemiluminescence method: Using antibody labeling technology to link antibodies with alkaline phosphatase and magnetic beads to prepare reagent components, and detecting the analyte by the principle of antigen-antibody immune reaction. Specifically, monoclonal antibodies are linked to superparamagnetic microparticles, and alkaline phosphatase (ALP) is linked to another monoclonal antibody. First, after the analyte is incubated with magnetic microparticles and enzyme complexes, a sandwich structure of magnetic beads-analyte-enzyme complexes is formed. After washing to remove impurities, it is then contacted with the substrate for reaction. The enzyme catalyzes the luminescent substrate to emit a light signal, and the intensity of the signal is proportional to the content of the analyte. Through the calibration curve of concentration-relative light units (RLU), the content of the analyte can be calculated.

[0004] Chemiluminescence methods mainly include multi-person chemiluminescent reagents and single-person chemiluminescent reagents. Multi-person chemiluminescent reagents have problems such as reagent contamination, waste, high cost, and long specimen transportation cycle. Existing single-person chemiluminescent reagents use liquid reagents, and due to the inversion and overturning of the reagents during transportation, magnetic bead sedimentation, adhesion to the wall, etc. will ultimately lead to abnormal test results. Summary of the Invention

[0005] The purpose of the present invention is to provide a B-type natriuretic peptide detection kit and a preparation method thereof, so as to solve the problem of magnetic bead sedimentation and adhesion to the wall caused by the inversion and overturning of existing single-person chemiluminescent reagents.

[0006] The present invention is achieved through the following technical solutions:

[0007] A B-type natriuretic peptide detection kit, comprising:

[0008] Magnetic bead component: containing streptavidin magnetic beads coated with B-type natriuretic peptide monoclonal antibodies;

[0009] Enzyme component: containing B-type natriuretic peptide monoclonal antibodies labeled with alkaline phosphatase;

[0010] Among them, the magnetic bead component is in a paste or solid state after being dried.

[0011] Existing single-person chemiluminescent reagents use liquid reagents, and due to the inversion and overturning of the reagents during transportation, magnetic bead sedimentation, adhesion to the wall, etc. will ultimately lead to abnormal test results.

[0012] Based on chemiluminescence technology, the present invention dries the magnetic bead component to make it in a paste or solid state, which can avoid the problems of magnetic bead sedimentation and wall sticking caused by reagent inversion and tipping during transportation.

[0013] Furthermore, the magnetic bead component comprises the following components:

[0014] Streptavidin magnetic beads, monoclonal antibody against B-type natriuretic peptide, biotin ester, bovine serum albumin, Triton X-100, trehalose, sodium dihydrogen phosphate dihydrate, disodium hydrogen phosphate dodecahydrate, sodium chloride, ProClin 300, vitamin H, sucrose, glycerol, sorbitol, disodium EDTA, and hydrolyzed albumin.

[0015] In order to adapt to the drying treatment, the present invention improves the existing magnetic bead component. In addition to conventional substances, the magnetic bead component also contains sucrose, glycerol, trehalose, sorbitol, disodium EDTA, and hydrolyzed albumin, which can enhance the stability of the reagent. If the above substances are not added and the drying treatment is directly carried out, the signal value of the reagent will decrease.

[0016] The dosage of each reagent in the magnetic bead component is as follows:

[0017] PB (a mixture of sodium dihydrogen phosphate dihydrate and disodium hydrogen phosphate dodecahydrate) 0.02M, bovine serum albumin 5g / L - 20g / L, Triton X-100 1mL / L - 5mL / L, trehalose 3g / L - 10g / L, sodium chloride 9g / L - 27g / L, ProClin300 1mL / L - 2mL / L, sucrose 1g / L - 5g / L, glycerol 1mL / L - 5mL / L, sorbitol 1g / L - 5g / L, disodium EDTA 0.1g / L - 5g / L, hydrolyzed albumin 10mL / L - 100mL / L.

[0018] Among them, the dosages of streptavidin magnetic beads, monoclonal antibody against B-type natriuretic peptide, biotin ester, and vitamin H are determined according to the actual reaction.

[0019] Furthermore, the excess sites on the streptavidin magnetic beads coated with monoclonal antibody against B-type natriuretic peptide are blocked with vitamin H.

[0020] Furthermore, the specific process of the drying treatment is as follows:

[0021] Heat up to 37°C and keep warm for 1 - 1.5h, then heat up to 45°C and keep warm for 3 - 4h, and finally cool down to 37°C, and the cooling time is 1 - 1.5h or overnight.

[0022] When the cooling time is 1 - 1.5h, the magnetic bead component is in a high-viscosity paste state and adheres to the bottom of the reaction cup; when the cooling time is overnight, the magnetic bead component is completely dried into a solid state and adheres to the bottom of the reaction cup.

[0023] The parameter settings for the drying process are related to the formulation of the magnetic bead components. The applicant found through experiments that by improving the formulation with more magnetic bead components, during the drying process (at 37°C and 45°C), the reagent titer was not affected, enabling the reagent to be stored for a long time at room temperature.

[0024] The prepared magnetic bead components are aliquoted into reaction cups in a certain volume and dried. The drying parameters are shown in Table 1:

[0025] Table 1

[0026] Step Temperature (°C) Time (h) Heating up for preheating 37 1-1.5h High-temperature heating 45 3-4h Cooling stage 37 1-1.5h

[0027] If the temperature is higher, such as 60°C, the reagent stability may be affected again. Conducting directly at 37°C makes the preparation process slightly longer. If set for overnight, the energy consumption increases. To improve production efficiency, the intermediate temperature is set at 45°C.

[0028] Furthermore, the enzyme component includes the following components:

[0029] B-type natriuretic peptide monoclonal antibody, alkaline phosphatase, SMCC, 2-IT, casein, sodium dihydrogen phosphate dihydrate, disodium hydrogen phosphate dodecahydrate, sodium chloride, ProClin 300, zinc chloride, magnesium chloride, trehalose, and hydrolyzed albumin.

[0030] In addition to conventional substances, the enzyme component of the present invention also contains trehalose, hydrolyzed albumin, magnesium chloride, and zinc chloride.

[0031] Among them, adding magnesium chloride and zinc chloride can enhance the enzyme activity, and trehalose and hydrolyzed albumin can enhance the reagent stability

[0032] The dosages of each reagent in the enzyme component are as follows:

[0033] PB (mixed solution of sodium dihydrogen phosphate dihydrate and disodium hydrogen phosphate dodecahydrate) 0.02M; casein 5 g / L - 20 g / L; sodium chloride 9 g / L - 27 g / L; ProClin 300 1 mL / L - 2 mL / L; zinc chloride 0.001 mol / L - 0.01 mol / L; magnesium chloride 0.001 mol / L - 0.01 mol / L; trehalose 3 g / L - 10 g / L; hydrolyzed albumin 10 mL / L - 100 mL / L.

[0034] Among them, the dosages of B-type natriuretic peptide monoclonal antibody, alkaline phosphatase, and SMCC are determined according to the actual reaction.

[0035] Furthermore, it also includes:

[0036] Washing solution and AMPPD substrate.

[0037] Furthermore, the magnetic bead component is a single - person - portion reagent.

[0038] The preparation method of the B - type natriuretic peptide detection kit includes the following steps:

[0039] S1. Preparation of magnetic bead component: Link the B - type natriuretic peptide monoclonal antibody with biotin ester, and use the biotin - streptavidin reaction system to link the biotin ester linked with the monoclonal antibody to the superparamagnetic microparticles labeled with streptavidin. Block the excess sites on the magnetic microparticles, and then sub - package the magnetic bead component into reaction cups and dry it.

[0040] S2. Preparation of enzyme component: Link alkaline phosphatase ALP with cross - linker SMCC; link the B - type natriuretic peptide monoclonal antibody with cross - linker 2 - IT, and use the reaction between SMCC and 2 - IT to link them into an ALP - SMCC - 2 - IT - antibody complex.

[0041] Furthermore, in step S1, after the blocking treatment, trehalose and sucrose are added to the magnetic bead component.

[0042] Furthermore, in step S2, zinc chloride and magnesium chloride are added to the enzyme component.

[0043] Furthermore, the specific process of the drying treatment is as follows:

[0044] Keep it at 37°C for 1 - 1.5 h, then raise the temperature to 45°C and keep it for 3 - 4 h, and finally cool it to 37°C. The cooling time is 1 - 1.5 h or overnight.

[0045] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0046] 1. The present invention can not only meet the requirements of high sensitivity and good repeatability, but also avoid reagent contamination and waste, and avoid abnormal tests caused by the sedimentation and wall - sticking of magnetic beads, and better meet the use requirements of primary medical institutions.

[0047] 2. The present invention adopts chemiluminescence technology and the amplification effect of biotin - streptavidin, can obtain lower sensitivity and a wider linear range, reacts in a liquid homogeneous environment, and obtains better repeatability.

[0048] 3. The present invention uses single - person - portion reagents for independent sub - packaging, which can be discarded immediately after use, avoiding contamination and waste; and the instruments supporting the single - person - portion reagents are small in size, simple in structure, and convenient to operate. They can be used in multiple scenarios such as emergency departments, clinical laboratories, and primary medical institutions. Samples can be taken and tested immediately, with short time consumption, meeting the use requirements of chest pain centers. Specific embodiments

[0049] To make the objectives, technical solutions and advantages of the present invention more clear and understandable, the present invention will be further described in detail below in conjunction with embodiments. The illustrative embodiments of the present invention and their descriptions are only used to explain the present invention and are not intended to limit the present invention.

[0050] Example 1:

[0051] The B-type natriuretic peptide detection kit includes:

[0052] Magnetic bead component: containing streptavidin magnetic beads coated with B-type natriuretic peptide monoclonal antibody;

[0053] The magnetic bead component includes the following components:

[0054] Streptavidin magnetic beads, B-type natriuretic peptide monoclonal antibody, biotin ester, bovine serum albumin, Triton X-100, trehalose, sodium dihydrogen phosphate dihydrate, disodium hydrogen phosphate dodecahydrate, sodium chloride, ProClin 300, vitamin H, and sucrose.

[0055] Enzyme component: containing B-type natriuretic peptide monoclonal antibody labeled with alkaline phosphatase;

[0056] The enzyme component includes the following components:

[0057] B-type natriuretic peptide monoclonal antibody, alkaline phosphatase, SMCC, 2-IT, casein, sodium dihydrogen phosphate dihydrate, disodium hydrogen phosphate dodecahydrate, sodium chloride, ProClin 300, zinc chloride, and magnesium chloride.

[0058] It also includes:

[0059] Washing solution and AMPPD substrate;

[0060] Among them, the magnetic bead component is in paste or solid form after drying treatment, and each component is in single-person dose.

[0061] The preparation method of the B-type natriuretic peptide detection kit described in this example includes the following steps:

[0062] S1. Preparation of magnetic bead component: Link the B-type natriuretic peptide monoclonal antibody with biotin ester, and use the biotin-streptavidin reaction system to link the biotin ester linked with the monoclonal antibody to the superparamagnetic particles labeled with streptavidin, and block the excess sites on the magnetic particles with vitamin H. Add trehalose and sucrose, as well as PB, bovine serum albumin, Triton X-100, sodium chloride, ProClin 300, glycerol, sorbitol, disodium EDTA, and hydrolyzed albumin to the blocked magnetic beads, and dispense the magnetic bead component into reaction cups for drying.

[0063] The dosage of each reagent in the magnetic bead component is as follows:

[0064] PB (mixed solution of sodium dihydrogen phosphate dihydrate and disodium hydrogen phosphate dodecahydrate) 0.02 M, bovine serum albumin 5 g / L - 20 g / L, Triton X-100 1 mL / L - 5 mL / L, trehalose 5 g / L, sodium chloride 9 g / L - 27 g / L, ProClin 300 1 mL / L - 2 mL / L, sucrose 5 g / L, glycerol 1 mL / L - 5 mL / L, sorbitol 1 g / L - 5 g / L, disodium EDTA 0.1 g / L - 5 g / L, hydrolyzed albumin 10 mL / L - 100 mL / L.

[0065] Among them, the dosage of each reagent can be adjusted within the above range. Specifically, in this example, the dosage of each reagent in the magnetic bead component is as follows:

[0066] PB 0.02 M, bovine serum albumin 10 g / L, Triton X-100 3 mL / L, trehalose 5 g / L, sodium chloride 27 g / L,

[0067] ProClin 300 2 mL / L, sucrose 5 g / L, glycerol 4 mL / L, sorbitol 3 g / L, disodium EDTA 3 g / L, hydrolyzed albumin 50 mL / L.

[0068] The drying parameters are shown in Table 2:

[0069] Table 2

[0070] Step Temperature (°C) Time (h) Heating up for preheating 37 1 High-temperature heating 45 3 Cooling stage 37 1

[0071] When the cooling time is 1 h, the magnetic bead component is a high-viscosity paste and adheres to the bottom of the reaction cup; when the cooling time is overnight, the magnetic bead component is completely dried into a solid state and adheres to the bottom of the reaction cup.

[0072] S2. Preparation of the enzyme component: Link alkaline phosphatase (ALP) with the cross-linker SMCC, and link another B-type natriuretic peptide monoclonal antibody with the cross-linker 2-IT. Utilize the reaction between SMCC and 2-IT to link the two into an ALP-SMCC-2-IT-antibody complex, add zinc chloride and magnesium chloride to enhance the enzyme activity, and simultaneously add PB, casein, sodium chloride, ProClin 300, trehalose, and hydrolyzed albumin.

[0073] The dosage of each reagent in the enzyme component is as follows:

[0074] PB (mixture of sodium dihydrogen phosphate dihydrate and disodium hydrogen phosphate dodecahydrate) 0.02 M; casein 5 g / L - 20 g / L; sodium chloride 9 g / L - 27 g / L; ProClin 300 1 mL / L - 2 mL / L; zinc chloride 0.001 mol / L - 0.01 mol / L; magnesium chloride 0.001 mol / L - 0.01 mol / L; trehalose 3 g / L - 10 g / L; hydrolyzed albumin 10 mL / L - 100 mL / L.

[0075] Among them, the dosage of each reagent can be adjusted within the above range. Specifically, in this example, the dosage of each reagent in the enzyme component is as follows:

[0076] PB (mixture of sodium dihydrogen phosphate dihydrate and disodium hydrogen phosphate dodecahydrate) 0.02 M; casein 20 g / L; sodium chloride 27 g / L; ProClin 300 2 mL / L; zinc chloride 0.01 mol / L; magnesium chloride 0.01 mol / L; trehalose 5 g / L; hydrolyzed albumin 50 mL / L.

[0077] The titer of the magnetic bead component prepared in this example was tested, and the results are shown in Table 3:

[0078] Table 3

[0079]

[0080] Based on the chemiluminescence technology, the magnetic bead component of the present invention is dried to make the magnetic bead component in a paste or solid state, which can avoid the problems of magnetic bead sedimentation and wall sticking caused by reagent inversion and overturning during transportation. Moreover, by improving the formulation of the magnetic bead component, the reagent titer is not affected during the drying process (37 °C and 45 °C).

[0081] The present invention can not only meet the requirements of high sensitivity and good repeatability, but also avoid reagent contamination and waste, and avoid abnormal tests caused by magnetic bead sedimentation and wall sticking, thus better meeting the use requirements of primary medical institutions.

[0082] The specific application process of the B-type natriuretic peptide detection kit described in this example is as follows:

[0083] Each component such as enzyme, cleaning solution, and AMPPD substrate is dispensed into the predetermined holes of the integrated card strip, and the reaction cup containing the magnetic bead component is assembled onto the card strip. First, the analyte, magnetic particles, and enzyme complex are incubated to form a magnetic bead (linked monoclonal antibody)-analyte-enzyme (linked to another monoclonal antibody) immune complex. After washing to remove impurities, it is then contacted with the AMPPD substrate for reaction. The enzyme catalyzes the luminescent substrate to emit a light signal, and the intensity of the signal is proportional to the content of the analyte. The content of the analyte is calculated through the calibration curve of concentration-relative luminescence value (RLU).

[0084] A specific application process is as follows: Use the calibration product S0 to measure 20 times, obtain the signal values of the 20 measurement results, calculate their average value M and standard deviation SD, and obtain the signal value corresponding to (M + 2SD). Repeat the measurement 3 times with the calibration product S1 to obtain the average signal value of the 3 measurement results. Perform two-point regression fitting based on the concentration-signal value results between the calibration product S0 and the calibration product S1 to obtain a linear equation: Y = 242.08X + 830. Substitute the signal value corresponding to (M + 2SD) into the equation and calculate to obtain the corresponding concentration of 0.5 pg / mL.

[0085] The sensitivity test results and repeatability test results are shown in Table 4 and Table 5 respectively:

[0086] Table 4

[0087]

[0088]

[0089] Note: In Table 4, the calibration product S0 is the calibration product diluent, and the calibration product S1 is obtained by diluting the antigen with the calibration product diluent.

[0090] Table 5

[0091]

[0092] Note: Samples 1 and 2 in Table 5 are the analytes, and the data in Table 5 are not the results of testing the analytes with the kit of Example 1.

[0093] In summary, the present invention can not only meet the requirements of high sensitivity and good repeatability, but also avoid reagent contamination and waste, and avoid abnormal testing caused by the sedimentation and wall sticking of magnetic beads, and better meet the use requirements of primary medical institutions.

[0094] Comparative Example 1:

[0095] This comparative example is based on Example 1. The difference from Example 1 is that the magnetic bead formula is not improved, and other operations are the same. The specific formula of the magnetic beads in this comparative example is:

[0096] PB 0.02M, bovine serum albumin 10 g / L, Triton X-100 3 mL / L, sodium chloride 27 g / L, ProClin 300 2 mL / L.

[0097] Compared with Example 1, it does not contain trehalose, sucrose, glycerol, sorbitol, disodium EDTA, and hydrolyzed albumin.

[0098] The comparison of the signal values before and after drying in this comparative example is shown in Table 6:

[0099] Table 6

[0100]

[0101] The comparison of signal values before and after drying in Example 1 is shown in Table 7 as follows:

[0102] Table 7

[0103]

[0104] It can be seen from the data in Table 6 and Table 7 of Example 1 that:

[0105] If the drying treatment is carried out without improving the magnetic bead formula, the signal values before and after drying are quite different, that is, the stability is poor. After adding trehalose, sucrose, glycerol, sorbitol, disodium EDTA and hydrolyzed albumin to the magnetic bead formula, the stability can be improved, and the difference in signal values before and after drying is small.

[0106] Comparative Example 2:

[0107] This comparative example is based on Example 1. The difference from Example 1 is that the drying parameters are different, and other operations are the same. The specific drying parameters of this comparative example are shown in Table 8 as follows:

[0108] Table 8

[0109] Step Temperature (°C) Time (h) Heating up for preheating 37 1 High-temperature heating 37 8 Cooling stage 37 1

[0110] The potency results of this comparative example are shown in Table 9 as follows:

[0111] Table 9

[0112]

[0113] It can be seen from the data in Table 9 that:

[0114] Directly adopting 37°C for the drying parameters has basically no impact on the potency, but the preparation process is slightly longer, and it takes a total of 10 h to achieve solidification. To improve the production efficiency, the temperature is set to 45°C in the middle, and the duration can be shortened to about 5 h.

[0115] The specific embodiments described above have further elaborated on the purpose, technical solutions and beneficial effects of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not used to limit the protection scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.

Claims

1. A B-type natriuretic peptide detection kit, characterized in that: include: Magnetic bead components: Contains streptavidin magnetic beads coated with B-type natriuretic peptide monoclonal antibody; Enzyme component: contains B-type natriuretic peptide monoclonal antibody labeled with alkaline phosphatase; Wherein, the magnetic bead component is in a paste or solid state after drying; The magnetic bead composition includes the following components: Streptavidin magnetic beads, B-type natriuretic peptide monoclonal antibody, biotin ester, bovine serum albumin, Triton X-100, trehalose, sodium dihydrogen phosphate dihydrate, sodium hydrogen phosphate dodecahydrate, sodium chloride, ProClin 300, vitamin H, sucrose, glycerol, sorbitol, disodium EDTA, and hydrolyzed albumin; The preparation method of the B-type natriuretic peptide detection kit comprises the following steps: S1. Preparation of magnetic bead components: linking B-type natriuretic peptide monoclonal antibody to biotin ester, and using biotin-streptavidin reaction system, linking the biotin ester linked to the monoclonal antibody to superparamagnetic microparticles labeled with streptavidin, blocking the excess sites on the magnetic microparticles with vitamin H, adding trehalose and sucrose, as well as PB, bovine serum albumin, Triton X-100, sodium chloride, ProClin 300, glycerol, sorbitol, disodium EDTA and hydrolyzed albumin to the blocked magnetic beads, and packing the magnetic bead components into reaction cups for drying; S2. Preparation of enzyme components: linking alkaline phosphatase ALP with the cross-linking agent SMCC; linking B-type natriuretic peptide monoclonal antibody with the cross-linking agent 2-IT, and utilizing the reaction between SMCC and 2-IT to link the two into an ALP-SMCC-2-IT-antibody complex.

2. The B-type natriuretic peptide detection kit according to claim 1, characterized in that The specific process of drying treatment is: Keep warm at 37℃ for 1-1.5h, then raise the temperature to 45℃ and keep warm for 3-4h, and finally cool to 37℃ for 1-1.5h or overnight.

3. The B-type natriuretic peptide detection kit according to claim 1, characterized in that: The enzyme components include the following: B-type natriuretic peptide monoclonal antibody, alkaline phosphatase, SMCC, 2-IT, casein, sodium dihydrogen phosphate dihydrate, sodium dihydrogen phosphate dodecahydrate, sodium chloride, ProClin 300, zinc chloride, magnesium chloride, trehalose, and hydrolyzed albumin.

4. The B-type natriuretic peptide detection kit according to claim 1, characterized in that Also includes: Wash solution and AMPPD substrate.

5. The B-type natriuretic peptide detection kit according to any one of claims 1 to 4, characterized in that The magnetic bead components are provided as single-aliquot reagents.

6. The B-type natriuretic peptide detection kit according to claim 1, characterized in that: In step S1, trehalose and sucrose are added to the magnetic bead component after the blocking treatment; in step S2, zinc chloride and magnesium chloride are added to the enzyme component.

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