A freeze-dried VB12 rapid detection kit and a VB12 detection method

Abstract

The application belongs to the technical field of medical immunodiagnostic reagents, and discloses a freeze-dried VB12 rapid detection kit and a VB12 detection method. In the preparation process of the freeze-dried microsphere detection reagent, trehalose, glucose, gelatin, triton, polyethylene glycol, Proclin 950, 5-bromo-5-nitro-1,3-dioxane, mannitol and glycine with specific mass ratios are used as the freeze-drying solution system, so as to reduce the stress damage of VB12 binding protein and VB12 complex antibody in the freeze-drying process, improve the stability of the reagent in the preparation and storage process, and realize the rapid reconstitution of the freeze-dried reagent. The detection system formed by compounding the reagent storage solution, the freeze-dried microsphere reconstitution solution and the sample dissociation solution has excellent anti-interference ability. The obtained detection kit has excellent detection sensitivity, reliability and stability, and has very excellent practical application prospect.

Description

Technical Field

[0001] This invention belongs to the technical field of medical immunodiagnostic reagents, and particularly relates to a lyophilized VB12 rapid detection kit and a method for detecting VB12. Background Technology

[0002] Vitamin B12 (cobalamin, VB12) is a key nutrient for hematopoiesis and nervous system health. Deficiency can lead to megaloblastic anemia and irreversible nerve damage. VB12 detection has significant clinical value in the management of anemia, neuropathy, and chronic diseases in the elderly. Currently, most mainstream chemiluminescence immunoassay kits for VB12 detection use potassium cyanide (KCN) as the VB12 dissociation agent, posing significant safety risks. The detection process is cumbersome, requiring sample pre-incubation, a competitive delayed one-step reaction, and signal detection, making it complex, time-consuming, and reliant on high-precision equipment. Furthermore, the results are easily interfered with by substances such as homocysteine ​​and folic acid, resulting in high false positive rates and poor low-value recovery rates. Its weak anti-interference ability fails to adequately meet the application needs of emergency departments and primary healthcare settings, thus exhibiting significant limitations. Summary of the Invention

[0003] The primary objective of this invention is to address the problems existing in the application of chemiluminescence methods to VB12 detection, such as cumbersome sample pretreatment steps, the need for toxic reagents, long detection time, and weak anti-interference ability of the detection system, and to provide a lyophilized VB12 rapid detection kit.

[0004] A second objective of this invention is to provide a method for detecting VB12 for non-diagnostic purposes.

[0005] Specifically, the lyophilized VB12 rapid detection kit provided by this invention includes: a lyophilized microsphere detection reagent, comprising magnetic microparticle lyophilized microspheres, acridil ester lyophilized microspheres, and sample-processing lyophilized microspheres; the magnetic microparticle lyophilized microspheres are obtained by treating VB12-binding protein coated with magnetic beads in a reagent storage solution with liquid nitrogen droplet freezing; the acridil ester lyophilized microspheres are obtained by treating acridil ester-labeled VB12 complex antibody in a reagent storage solution with liquid nitrogen droplet freezing; the sample-processing lyophilized microspheres are obtained by treating a reducing agent in a reagent storage solution with liquid nitrogen droplet freezing, wherein the reducing agent is selected from one of dithiothreitol, tris(2-carboxyethyl)phosphine hydrochloride, reduced glutathione, cysteine, β-mercaptoethanol, and mercaptoglycerol; wherein the reagent storage solution comprises a mass ratio of (2~10):(2~10):(0.05~1):(0.05~1):(0.1~0.5): Trehalose, glucose, gelatin, Triton, polyethylene glycol, Proclin 950, 5-bromo-5-nitro-1,3-dioxane, mannitol, and glycine in a mass ratio of (0.05~1):(0.05~1):(1~5):(0.05~1); Lyophilized microsphere reconstitution solution comprising surfactant S9, Triton, Proclin 950, and 5-bromo-5-nitro-1,3-dioxane in a mass ratio of (0.05~1):(0.05~1):(0.05~1):(0.9~1.1); Sample dissociation solution comprising sodium hydroxide, ethylenediaminetetraacetic acid, potassium cobalt cyanide, and Proclin in a mass ratio of (1.8~2.2):(1.7~2):(6.5~75):(0.4~0.6):(0.4~0.6). 950 and 5-bromo-5-nitro-1,3-dioxane.

[0006] Further, the reagent stock solution comprises 2% (w / v) to 10% (w / v) trehalose, 2% (w / v) to 10% (w / v) glucose, 0.05% (w / v) to 1% (w / v) gelatin, 0.05% (w / v) to 1% (w / v) Triton, 0.1% (w / v) to 0.5% (w / v) polyethylene glycol, 0.05% (w / v) to 1% (w / v) Proclin 950, 0.05% (w / v) to 1% (w / v) 5-bromo-5-nitro-1,3-dioxane, 1% (w / v) to 5% (w / v) mannitol and 0.05% (w / v) to 1% (w / v) glycine.

[0007] Further, the reagent stock solution comprises 0.8% (w / v) to 0.9% (w / v) sodium chloride, 4% (w / v) to 6% (w / v) BSA, 4% (w / v) to 6% (w / v) trehalose, 4% (w / v) to 6% (w / v) glucose, 0.1% (w / v) to 0.3% (w / v) gelatin, 0.05% (w / v) to 0.06% (w / v) Triton X-100, 0.1% (w / v) to 0.11% (w / v) PEG6000, and 0.4% (w / v) to 0.6% (w / v) Proclin 950, 0.4% (w / v) to 0.6% (w / v) of 5-bromo-5-nitro-1,3-dioxane, 2.8% (w / v) to 3.2% (w / v) of mannitol and 0.4% (w / v) to 0.5% (w / v) of glycine.

[0008] Further, the preparation of the magnetic microparticle lyophilized microspheres includes: (1) activating the magnetic beads with 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to obtain activated magnetic beads; (2) coupling the activated magnetic beads with VB12 binding protein and blocking them in a magnetic bead blocking solution to obtain the VB12 binding protein coated on the magnetic beads; (3) mixing the VB12 binding protein coated on the magnetic beads with a reagent storage solution to obtain a lyophilized solution of magnetic beads microspheres; and subjecting the lyophilized solution of magnetic beads microspheres to liquid nitrogen droplet freezing treatment at -200℃ to -190℃ to obtain the magnetic microparticle lyophilized microspheres.

[0009] Furthermore, in the preparation of the freeze-dried magnetic microspheres, in step (1), the particle size of the magnetic beads is 1.5µm~3.0µm.

[0010] Furthermore, in the preparation of the lyophilized magnetic microspheres, in step (1), the concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 0.05 mg / mL to 50 mg / mL, the concentration of N-hydroxysuccinimide is 0.05 mg / mL to 50 mg / mL, and the concentration of the magnetic beads is 5 mg / mL to 100 mg / mL.

[0011] Furthermore, in the preparation of the freeze-dried magnetic microspheres, in step (1), the activation treatment temperature is 18℃~30℃ and the time is 10min~60min.

[0012] Furthermore, in the preparation of the lyophilized magnetic microspheres, in step (2), the concentration of the activated magnetic beads is 5 mg / mL to 100 mg / mL, and the concentration of the VB12 binding protein is 10 μg / mL to 1000 μg / mL.

[0013] Furthermore, in the preparation of the freeze-dried magnetic microspheres, in step (2), the coupling treatment temperature is 18℃~30℃ and the time is 2h~3h.

[0014] Furthermore, in the preparation of the lyophilized magnetic microspheres, in step (2), the magnetic bead sealing liquid includes 1 g / L to 10 g / L of BSA and 0.01% (v / v) to 1% (v / v) of Tween 20.

[0015] Furthermore, in the preparation of the freeze-dried magnetic microspheres, in step (2), the concentration of the activated magnetic beads after the coupling treatment is 5 mg / mL to 20 mg / mL.

[0016] Furthermore, in the preparation of the freeze-dried magnetic microspheres, in step (2), the sealing treatment temperature is 18℃~37℃ and the time is 10min~60min.

[0017] Furthermore, in the preparation of the lyophilized magnetic microspheres, in step (3), the concentration of the lyophilized magnetic microsphere solution is 0.1 mg / mL to 2 mg / mL.

[0018] Furthermore, in the preparation of the magnetic microparticle freeze-dried microspheres, in step (3), the amount of the added magnetic microsphere freeze-drying solution in the liquid nitrogen droplet freezing treatment is 10 μL / microsphere to 30 μL / microsphere.

[0019] Furthermore, the particle size of the freeze-dried magnetic microspheres is 1 mm to 5 mm.

[0020] Further, the preparation of the acridine ester lyophilized microspheres includes: (1) coupling acridine ester with a VB12 complex antibody solution, blocking it in the presence of lysine, and then dialysis to obtain the acridine ester-labeled VB12 complex antibody; (2) mixing the acridine ester-labeled VB12 complex antibody with a reagent storage solution to obtain an acridine ester microsphere lyophilized solution; and subjecting the acridine ester microsphere lyophilized solution to liquid nitrogen droplet freezing treatment at -200℃ to -190℃ to obtain the acridine ester lyophilized microspheres.

[0021] Furthermore, in the preparation of the acridinium ester lyophilized microspheres, in step (1), the concentration of the VB12 complex antibody solution is 0.5 mg / mL to 2 mg / mL.

[0022] Furthermore, in the preparation of the acridine ester lyophilized microspheres, in step (1), the molar ratio of the added VB12 complex antibody to acridine ester is 1:(5~20).

[0023] Furthermore, in the preparation of the acridinium ester freeze-dried microspheres, in step (1), the coupling treatment temperature is 20℃~30℃ and the time is 2h~3h.

[0024] Furthermore, in the preparation of the acridine ester freeze-dried microspheres, in step (1), the molar ratio of lysine to acridine ester is (100~150):1.

[0025] Furthermore, in the preparation of the acridinium ester freeze-dried microspheres, in step (1), the sealing treatment temperature is 20℃~30℃ and the time is 10min~60min.

[0026] Furthermore, in the preparation of the acridinium ester lyophilized microspheres, in step (1), the molecular weight cutoff of the dialysis membrane during the dialysis treatment is 40KD~60KD.

[0027] Furthermore, in the preparation of the acridinium ester lyophilized microspheres, in step (2), during the liquid nitrogen droplet freezing treatment, the amount of acridinium ester microsphere lyophilization solution added is 10 μL / sphere to 30 μL / sphere.

[0028] Furthermore, the acridine ester lyophilized microspheres have a particle size of 1 mm to 5 mm.

[0029] Further, the preparation of the sample-processed lyophilized microspheres includes: (1) mixing the reducing agent and the reagent storage solution to obtain a sample processing solution; (2) subjecting the sample processing solution to liquid nitrogen droplet freezing treatment at -200℃ to -190℃ to obtain the sample-processed lyophilized microspheres.

[0030] Furthermore, in the preparation of the sample-processed lyophilized microspheres, in step (1), the concentration of the reducing agent used is 0.1% (w / v) to 0.5% (w / v).

[0031] Furthermore, in the preparation of the sample-processed lyophilized microspheres, in step (2), the amount of sample processing solution added in the liquid nitrogen droplet freezing bead treatment is 10 μL / bead to 30 μL / bead.

[0032] Furthermore, the particle size of the freeze-dried microspheres processed in the sample is 1 mm to 5 mm.

[0033] Further, the lyophilized microsphere reconstitution solution comprises 0.04% (w / v) to 0.06% (w / v) of surfactant S9, 0.04% (w / v) to 0.06% (w / v) of Triton X-100, 0.04% (w / v) to 0.06% (w / v) of Proclin 950 and 0.04% (w / v) to 0.06% (w / v) of 5-bromo-5-nitro-1,3-dioxane.

[0034] Furthermore, the sample dissociation solution comprises 45mM~55mM sodium hydroxide, 4mM~6mM ethylenediaminetetraacetic acid, 6g / L~8g / L potassium cobalt cyanide, 0.04%(w / v)~0.06%(w / v) Proclin 950 and 0.04%(w / v)~0.06%(w / v) 5-bromo-5-nitro-1,3-dioxane.

[0035] The non-diagnostic VB12 detection method provided by this invention uses the above-mentioned lyophilized VB12 rapid detection kit to detect the sample to be tested, specifically including the following steps: S1, incubating the sample dissociation solution with the sample to be tested in incubation treatment I to obtain a sample processing reaction solution; S2, mixing the sample processing reaction solution with lyophilized reconstitution solution, magnetic microparticle lyophilized microspheres, acrid ester lyophilized microspheres and sample processing lyophilized microspheres until completely reconstituted, then incubating in incubation treatment II, measuring the luminescence value, and obtaining the VB12 detection result.

[0036] Further, in step S1, the mixing volume ratio of the sample dissociation solution to the sample to be tested is (1~5):1.

[0037] Furthermore, in step S1, the temperature of the incubation treatment I is 30℃~40℃, and the time is 1min~5min.

[0038] Further, in step S2, the addition ratio of the sample processing reaction solution, the lyophilized reconstituted solution, the magnetic microparticle lyophilized microspheres, the acridinium ester lyophilized microspheres, and the sample processing lyophilized microspheres is (50~100)μL: (100~200)μL: 1 microsphere: 1 microsphere: 1 microsphere.

[0039] Furthermore, in step S2, the temperature of the incubation treatment II is 30℃~40℃, and the time is 5min~20min.

[0040] Beneficial effects:

[0041] In the lyophilized VB12 rapid detection kit provided by this invention, during the lyophilization process of VB12 binding protein coated with magnetic beads and VB12 complex antibody labeled with acrid ester, trehalose, glucose, gelatin, triton, polyethylene glycol, and proclin are introduced in specific mass ratios. A reagent stock solution containing 950, 5-bromo-5-nitro-1,3-dioxane, mannitol, and glycine was used as the freeze-drying solution system. Through the synergistic effect of these substances and a special process of liquid nitrogen droplet freezing, excellent protection of VB12 binding protein and VB12 complex antibody was achieved. The resulting lyophilized microsphere detection reagent exhibited low signal attenuation and excellent stability after freeze-drying and accelerated destructive testing at 45°C. Secondly, the reagent stock solution, in synergy with a lyophilized microsphere reconstitution solution containing surfactants S9, Triton, Proclin 950, and 5-bromo-5-nitro-1,3-dioxane in specific mass ratios, not only rapidly reconstituted the lyophilized microsphere detection reagent but also quickly restored its activity by optimizing the structure of VB12 binding protein and VB12 complex antibody. Furthermore, the sample dissociation solution contained sodium hydroxide, ethylenediaminetetraacetic acid, potassium cobalt cyanide, Proclin 950, and... 5-Bromo-5-nitro-1,3-dioxane can work synergistically with the reducing agent introduced into the sample-processed lyophilized microspheres to achieve rapid and complete release of VB12 from the test sample. This method offers advantages such as high safety, simple sample processing steps, excellent VB12 dissociation, and high processing efficiency. Furthermore, the detection solution system, composed of the reagent storage solution, sample-processed lyophilized microspheres, lyophilized microsphere reconstitution solution, and sample dissociation solution, effectively enhances the specific recognition and binding of VB12-binding proteins to VB12, and improves the recognition of VB12-protein complexes by VB12 complex antibodies. This reduces the non-specific binding of homocysteine, folic acid, and other substances to VB12-binding proteins, giving the detection system better anti-interference capabilities and combining excellent detection sensitivity, reliability, and stability. This lyophilized VB12 rapid detection kit can well meet the application needs of practical testing scenarios such as emergency medicine and primary healthcare, and has excellent application prospects. Attached Figure Description

[0042] Figure 1 This is a physical image of the lyophilized microsphere detection reagent provided in the embodiments of the present invention;

[0043] Figure 2 The standard curve of the lyophilized VB12 rapid detection kit provided in the embodiments of the present invention;

[0044] Figure 3The figure shows the experimental results of the correlation test of Roche-assigned serum samples with the lyophilized VB12 rapid detection kit provided in the embodiments of the present invention. Detailed Implementation

[0045] In a first aspect, the lyophilized VB12 rapid detection kit provided by the present invention specifically includes: lyophilized microsphere detection reagent, lyophilized microsphere reconstitution solution, and sample dissociation solution. Specifically, the lyophilized microsphere detection reagent includes magnetic microparticle lyophilized microspheres, acridinium ester lyophilized microspheres, and sample-processing lyophilized microspheres.

[0046] In this invention, a reagent storage solution with a specific composition is introduced as a lyophilization solvent system during the preparation of the lyophilized microsphere detection reagent. This effectively reduces stress damage to VB12 binding protein and VB12 complex antibody during lyophilization, improving the stability of the reagent during preparation and storage. Furthermore, the introduction of this reagent storage solution into the detection solution system also enables rapid reconstitution of the reagent and enhances the system's anti-interference ability. More specifically, the reagent storage solution includes trehalose, glucose, gelatin, Triton, polyethylene glycol, Proclin 950, 5-bromo-5-nitro-1,3-dioxane, mannitol, and glycine. The specific mass ratio of 950, 5-bromo-5-nitro-1,3-dioxane, mannitol, and glycine is (2~10):(2~10):(0.05~1):(0.05~1):(0.1~0.5):(0.05~1):(0.05~1):(1~5):(0.05~1), such as 2:2:0.05:0.05:0.1:0.05:0.05:1:0.05, 2:5:1:1:0.1:0.2:0.1:0.1:2:0.1, 5:5:1:1:0.5:1:1:5:1, 10:10:1:1:0.5:1:1:5:1, or any value between them.

[0047] In some specific embodiments, the reagent stock solution preferably includes: 2% (w / v) to 10% (w / v) of trehalose, such as 2% (w / v), 2.5% (w / v), 2.8% (w / v), 3% (w / v), 4% (w / v), 5% (w / v), 7% (w / v), 9% (w / v), 10% (w / v) or any value between them; 2% (w / v) to 10% (w / v) of... Glucose, such as 2% (w / v), 2.5% (w / v), 2.8% (w / v), 3% (w / v), 4% (w / v), 5% (w / v), 7% (w / v), 9% (w / v), 10% (w / v) or any value between them; gelatin from 0.05% (w / v) to 1% (w / v), such as 0.05% (w / v), 0.08% (w / v), 0.1% (w / v), 0.25% (w / v), 0.5% (w / v), 0.75% (w / v), 1% (w / v) or any value between them; 0.05% (w / v) to 1% (w / v) of triatomine, such as 0.05% (w / v), 0.07% (w / v), 0.09% (w / v), 0.1% (w / v), 0.2% (w / v), 0.35% (w / v), 0.5% (w / v), 0.6% (w / v) 0.8% (w / v), 1% (w / v), or any value between them; 0.1% (w / v) to 0.5% (w / v) of polyethylene glycol, such as 0.1% (w / v), 0.2% (w / v), 0.25% (w / v), 0.3% (w / v), 0.48% (w / v), 0.5% (w / v), or any value between them; 0.05% (w / v) to 1% (w / v) of Proclin 950, such as 0.05% (w / v), 0.1% (w / v), 0.2% (w / v), 0.3% (w / v), 0.6% (w / v), 0.8% (w / v), 1% (w / v) or any value between them; 0.05% (w / v) to 1% (w / v) of 5-bromo-5-nitro-1,3-dioxane, such as 0.05% (w / v), 0.08% (w / v), 0.1% (w / v), 0.2% (w / v), 0.4% (w / v), 0.6% (w / v), 0.8% (w / v), 1% (w / v), etc. Mannitol at concentrations of 1% (w / v) to 5% (w / v), such as 1% (w / v), 1.5% (w / v), 1.8% (w / v), 2% (w / v), 2.6% (w / v), 3% (w / v), 4% (w / v), 5% (w / v), or any value between them; and glycine at concentrations of 0.05% (w / v) to 1% (w / v), such as 0.05% (w / v), 0.08% (w / v), 0.1% (w / v), 0.15% (w / v), 0.25% (w / v), 0.4% (w / v), 0.6% (w / v), 0.8% (w / v), 1% (w / v), or any value in between.

[0048] In some preferred embodiments, the reagent stock solution more preferably includes: 0.8% (w / v) to 0.9% (w / v) of sodium chloride, such as 0.8% (w / v), 0.83% (w / v), 0.85% (w / v), 0.87% (w / v), 0.9% (w / v), or any value between therewith; and 4% (w / v) to 6% (w / v) of BSA, such as 4% (w / v), 4.5% (w / v), 4.8% (w / v), 5% (w / v), 5.2% (w / v), 5.5% (w / v). 5.8% (w / v), 6% (w / v), or any value between them; 4% (w / v) to 6% (w / v) trehalose, such as 4% (w / v), 4.3% (w / v), 4.5% (w / v), 4.8% (w / v), 5% (w / v), 5.3% (w / v), 5.5% (w / v), 6% (w / v), or any value between them; 4% (w / v) to 6% (w / v) glucose, such as 4% (w / v), 4.3% (w / v), 4.5% (w / v), 4.8% (w / v), 5% (w / v), 5.3% (w / v), 5.5% (w / v), 6% (w / v), or any value between them. 5% (w / v), 5.3% (w / v), 5.6% (w / v), 6% (w / v) or any value between them; 0.1% (w / v) to 0.3% (w / v) gelatin, such as 0.1% (w / v), 0.13% (w / v), 0.15% (w / v), 0.18% (w / v), 0.2% (w / v), 0.25% (w / v), 0.3% (w / v) or any value between them; 0.05% (w / v) to 0.06% (w / v) Triton X-100, such as 0 0.05% (w / v), 0.053% (w / v), 0.058% (w / v), 0.06% (w / v) or any value between them; PEG6000 at 0.1% (w / v) to 0.11% (w / v), such as 0.1% (w / v), 0.101% (w / v), 0.103% (w / v), 0.105% (w / v), 0.108% (w / v), 0.11% (w / v) or any value between them; Proclin at 0.4% (w / v) to 0.6% (w / v). 950, such as 0.4% (w / v), 0.43% (w / v), 0.45% (w / v), 0.48% (w / v), 0.5% (w / v), 0.53% (w / v), 0.55% (w / v), 0.6% (w / v) or any value between them; 0.4% (w / v) to 0.6% (w / v) of 5-bromo-5-nitro-1,3-dioxane, such as 0.4% (w / v), 0.45% (w / v), 0.48% (w / v), 0.5% (w / v), 0.53% (w / v), 0.55% (w / v), 0.58% (w / v), 0.6% (w / v) or any value between them; 2.8% (w / v) to 3.2% (w / v) mannitol, such as 2.8% (w / v), 2.85% (w / v), 2.9% (w / v), 3% (w / v), 3.05% (w / v), 3.1% (w / v), 3.2% (w / v), or any value between therewith; and 0.4% (w / v) to 0.5% (w / v) glycine, such as 0.4% (w / v), 0.43% (w / v), 0.48% (w / v), 0.5% (w / v), or any value between therewith.

[0049] In this invention, the solvent system of the reagent storage solution is a pH buffer system that can maintain the pH value of the solution within a relatively stable range. Specific examples include, but are not limited to, one or more of PBS buffer, Tris-HCl buffer, HEPES buffer, and MES buffer.

[0050] In some specific embodiments, the solvent system of the reagent storage solution is preferably HEPES buffer; more specifically, the concentration of the HEPES buffer is preferably 10mM~30mM, and the pH value is preferably 7.0~8.0.

[0051] In this invention, the magnetic microparticle lyophilized microspheres include a VB12-binding protein for capturing VB12 in the test sample, and the VB12-binding protein is coupled to magnetic beads, existing in the magnetic microparticle lyophilized microspheres in the form of VB12-binding protein coated with magnetic beads. The VB12-binding protein is a commonly used reagent in existing VB12 detection methods. Those skilled in the art can make adaptive selections from existing known reagents according to actual usage requirements. Furthermore, the detection solution environment provided by the lyophilized VB12 rapid detection kit claimed in this invention has excellent applicability to different VB12-binding proteins, and this invention does not impose any particular limitation on the VB12-binding protein.

[0052] In this invention, the magnetic microparticle lyophilized microspheres are specifically obtained by subjecting the VB12-binding protein coated with magnetic beads to liquid nitrogen droplet freezing treatment in a reagent storage solution. More specifically, the preparation of the magnetic microparticle lyophilized microspheres includes the following steps:

[0053] (1) Activate the magnetic beads by taking 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to obtain activated magnetic beads;

[0054] (2) The activated magnetic beads were coupled with VB12 binding protein and then blocked in magnetic bead blocking solution to obtain the VB12 binding protein coated with magnetic beads.

[0055] (3) Take the VB12 binding protein coated by the magnetic beads and mix it with the reagent storage solution to obtain a lyophilized solution of magnetic beads microspheres; take the lyophilized solution of magnetic beads microspheres and perform liquid nitrogen droplet freezing treatment at -200℃~-190℃ to obtain the lyophilized magnetic microparticles microspheres.

[0056] In some specific embodiments, in step (1), the particle size of the magnetic beads is preferably 1.5µm to 3.0µm, specifically 1.5µm, 1.8µm, 2µm, 2.3µm, 2.5µm, 2.8µm, 3.0µm or any value between them.

[0057] In some specific embodiments, in step (1), the concentration of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide is preferably 0.05 mg / mL to 50 mg / mL, such as 0.05 mg / mL, 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 10 mg / mL, 5 mg / mL, 20 mg / mL, 50 mg / mL, or any value between them; the concentration of the N-hydroxysuccinimide is preferably 0.05 mg / mL to 50 mg / mL, such as 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, or any value between them. / mL, 0.4mg / mL, 1mg / mL, 3mg / mL, 5mg / mL, 10mg / mL, 20mg / mL, 30mg / mL, 40mg / mL, 50mg / mL or any value between them; the preferred concentration of the magnetic beads is 5mg / mL to 100mg / mL, such as 5mg / mL, 7mg / mL, 9mg / mL, 10mg / mL, 12mg / mL, 15mg / mL, 20mg / mL, 30mg / mL, 50mg / mL, 75mg / mL, 100mg / mL or any value between them.

[0058] In some specific embodiments, in step (1), the activation treatment conditions specifically include a temperature preferably of 18℃~30℃, such as 18℃, 20℃, 23℃, 25℃, 28℃, 30℃ or any value between them; and a time preferably of 10min~60min, such as 10min, 15min, 18min, 20min, 25min, 30min, 40min, 50min, 60min or any value between them.

[0059] In some specific embodiments, in step (1), the activation treatment is preferably carried out in the solution environment provided by the magnetic bead cleaning solution. More specifically, the preparation of the magnetic bead cleaning solution includes: adding Proclin 300 to MES buffer at a final concentration of 0.05 mL / L to 0.5 mL / L and mixing evenly to obtain the magnetic bead cleaning solution; wherein the concentration of the MES buffer is 0.1 M to 0.5 M and the pH is 5.5 to 6.5.

[0060] In some specific embodiments, in step (2), the concentration of the activated magnetic beads is preferably 5 mg / mL to 100 mg / mL, such as 5 mg / mL, 5.5 mg / mL, 8 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 80 mg / mL, 100 mg / mL or any value between them; the concentration of the VB12 binding protein is preferably 10 μg / mL to 1000 μg / mL, specifically it can be 10 μg / mL, 20 μg / mL, 50 μg / mL, 80 μg / mL, 100 μg / mL, 120 μg / mL, 150 μg / mL, 300 μg / mL, 500 μg / mL, 800 μg / mL, 1000 μg / mL or any value between them.

[0061] In some specific implementations, in step (2), the conditions for the coupling process include a temperature preferably of 18°C ​​to 30°C, such as 18°C, 20°C, 23°C, 25°C, 28°C, 30°C or any value between them; and a time preferably of 2h to 3h, such as 2h, 2.3h, 2.5h, 2.8h, 3h or any value between them.

[0062] In some specific embodiments, in step (2), the coupling treatment is preferably performed in the solution environment provided by the magnetic bead cleaning solution. More specifically, the preparation of the magnetic bead cleaning solution includes: adding Proclin 300 to MES buffer at a final concentration of 0.05 mL / L to 0.5 mL / L and mixing evenly to obtain the magnetic bead cleaning solution; wherein the concentration of the MES buffer is 0.1 M to 0.5 M and the pH is 5.5 to 6.5.

[0063] In some specific embodiments, in step (2), the magnetic bead sealing liquid preferably includes 1 g / L to 10 g / L of BSA, such as 1 g / L, 1.5 g / L, 2 g / L, 4 g / L, 5 g / L, 6 g / L, 8 g / L, 9 g / L, 10 g / L or any value between them; and 0.01% (v / v) to 1% (v / v) of Tween 20, such as 0.01% (v / v), 0.05% (v / v), 0.1% (v / v), 0.5% (v / v), 0.8% (v / v), 1% (v / v) or any value between them.

[0064] In some specific embodiments, in step (2), the concentration of the activated magnetic beads after the coupling treatment is 5 mg / mL to 20 mg / mL, such as 5 mg / mL, 6 mg / mL, 8 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL or any value between them.

[0065] In some specific embodiments, in step (2), the sealing conditions include a temperature preferably of 18°C ​​to 37°C, such as 18°C, 20°C, 23°C, 25°C, 28°C, 30°C, 37°C or any value between them; and a time preferably of 10 min to 60 min, such as 10 min, 12 min, 14.5 min, 18 min, 20 min, 25 min, 30 min, 40 min, 50 min, 60 min or any value between them.

[0066] In some specific embodiments, in step (2), the buffer solvent system of the magnetic bead blocking solution is preferably Tris-HCl buffer; more specifically, the concentration of the Tris-HCl buffer is preferably 0.001M~0.05M, and the pH value is preferably 7.0~9.0.

[0067] In some specific embodiments, in step (3), the concentration of the lyophilized magnetic bead microsphere solution is preferably 0.1 mg / mL to 2 mg / mL, such as 0.1 mg / mL, 0.15 mg / mL, 0.2 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.8 mg / mL, 1 mg / mL, 1.3 mg / mL, 1.5 mg / mL, 1.9 mg / mL, 2 mg / mL or any value between them.

[0068] In some specific embodiments, in step (3), during the liquid nitrogen droplet freezing treatment, the amount of the lyophilized magnetic bead solution added is 10 μL / bead to 30 μL / bead, such as 10 μL / bead, 12.5 μL / bead, 15 μL / bead, 18 μL / bead, 20 μL / bead, 23.5 μL / bead, 25 μL / bead, 28 μL / bead, 30 μL / bead, or any value between them. In this case, the particle size of the resulting lyophilized magnetic microparticles is approximately 1 mm to 5 mm.

[0069] In this invention, the acridine ester lyophilized microspheres include a VB12 complex antibody for specifically recognizing and binding to the VB12-protein complex, and the VB12 complex antibody is labeled with acridine ester. The acridine ester-labeled VB12 complex antibody exists in the acridine ester lyophilized microspheres. The VB12 complex antibody is a commonly used reagent in existing VB12 detection methods. Those skilled in the art can make adaptive selections from known reagents according to actual usage requirements. Furthermore, the detection solution environment provided by the lyophilized VB12 rapid detection kit claimed in this invention has excellent adaptability to different VB12 complex antibodies, and this invention does not impose any particular limitation on the VB12 complex antibody.

[0070] In this invention, the acridine ester lyophilized microspheres are specifically obtained by performing liquid nitrogen droplet freezing treatment on acridine ester-labeled VB12 complex antibody in a reagent storage solution. More specifically, the preparation of the acridine ester lyophilized microspheres includes the following steps:

[0071] (1) Acridinium ester and VB12 complex antibody solution were coupled and blocked in the presence of lysine, and then dialyzed to obtain the acridinium ester-labeled VB12 complex antibody.

[0072] (2) The acridinium ester-labeled VB12 complex antibody was mixed with the reagent storage solution to obtain an acridinium ester microsphere lyophilized solution; the acridinium ester microsphere lyophilized solution was subjected to liquid nitrogen droplet freezing treatment at -200℃ to -190℃ to obtain the acridinium ester lyophilized microspheres.

[0073] In some specific embodiments, in step (1), the concentration of the VB12 complex antibody solution is preferably 0.5 mg / mL to 2 mg / mL, such as 0.5 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, 1.2 mg / mL, 1.5 mg / mL, 1.8 mg / mL, 2 mg / mL, or any value between them. More specifically, the solvent system of the VB12 complex antibody solution is preferably PBS buffer, and the concentration of the PBS buffer is 0.01 M to 0.05 M, and the pH value is 6.0 to 8.0.

[0074] In some specific embodiments, in step (1), the molar ratio of the added VB12 complex antibody and acridine ester is preferably 1:(5~20), such as 1:5, 1:8, 1:10, 1:12.5, 1:15, 1:20 or any value between them.

[0075] In some specific implementations, in step (1), the conditions for the coupling process specifically include a temperature preferably of 20℃~30℃, such as 20℃, 21.5℃, 23℃, 2.5℃, 28℃, 30℃ or any value between them; and a time preferably of 2h~3h, such as 2h, 2.3h, 2.4h, 2.5h, 2.7h, 2.9h, 3h or any value between them.

[0076] In some specific embodiments, in step (1), the molar ratio of lysine to acridine ester is preferably (100~150):1, such as 100:1, 105:1, 110:1, 112.5:1, 120:1, 125:1, 130:1, 150:1 or any value between them.

[0077] In some specific embodiments, in step (1), the sealing conditions specifically include a temperature preferably between 20°C and 37°C, such as 20°C, 21.5°C, 22°C, 23°C, 25°C, 26.5°C, 28°C, 30°C, 37°C or any value between them; and a time preferably between 10 min and 60 min, such as 10 min, 12.5 min, 13 min, 15 min, 20 min, 25 min, 30 min, 40 min, 50 min, 60 min or any value between them.

[0078] In some specific embodiments, in step (1), the molecular weight cutoff of the dialysis membrane during the dialysis treatment is preferably 40KD~60KD, such as 40KD, 42.5KD, 43KD, 45KD, 48KD, 50KD, 52KD, 53.5KD, 55KD, 58KD, 60KD, or any value between them. More specifically, the dialysis solution used in the dialysis treatment is preferably PBS buffer, and the concentration of the PBS buffer is preferably 0.01M~0.05M, and the pH value is preferably 7.0~8.0. More specifically, the dialysis treatment time is preferably 2h~4h, and the number of repetitions is preferably 3~5 times.

[0079] In some specific embodiments, in step (2), the concentration of the acridinium ester microsphere lyophilization solution is preferably 0.1 μg / mL to 2.5 μg / mL, such as 0.1 μg / mL, 0.12 μg / mL, 0.125 μg / mL, 0.13 μg / mL, 0.2 μg / mL, 0.5 μg / mL, 0.8 μg / mL, 1 μg / mL, 1.5 μg / mL, 2 μg / mL, 2.5 μg / mL or any value between them.

[0080] In some specific embodiments, in step (2), during the liquid nitrogen droplet freezing bead treatment, the amount of acridine ester microsphere lyophilization solution added is preferably 10 μL / bead to 30 μL / bead, such as 10 μL / bead, 12.5 μL / bead, 15 μL / bead, 20 μL / bead, 25 μL / bead, 30 μL / bead, or any value between them. In this case, the particle size of the resulting acridine ester lyophilized microspheres is approximately 1 mm to 5 mm.

[0081] In this invention, the sample-processed lyophilized microspheres include a reducing agent to help release VB12, specifically selected from one of dithiothreitol, tris(2-carboxyethyl)phosphine hydrochloride, reduced glutathione, cysteine, β-mercaptoethanol, and mercaptoglycerol.

[0082] In this invention, the sample-processed lyophilized microspheres are specifically obtained by treating a reducing agent in a reagent storage solution with liquid nitrogen droplets for freezing. More specifically, the preparation of the sample-processed lyophilized microspheres includes the following steps:

[0083] (1) Mix the reducing agent and the reagent storage solution to obtain the sample processing solution;

[0084] (2) The sample treatment solution was subjected to liquid nitrogen droplet freezing treatment at -200℃ to -190℃ to obtain the sample treatment freeze-dried microspheres.

[0085] In some specific embodiments, in step (1), the concentration of the reducing agent is preferably 0.1% (w / v) to 0.5% (w / v), such as 0.1% (w / v), 0.125% (w / v), 0.15% (w / v), 0.2% (w / v), 0.35% (w / v), 0.4% (w / v), 0.5% (w / v) or any value between them.

[0086] In some specific embodiments, in step (2), during the liquid nitrogen droplet freezing bead treatment, the amount of sample treatment solution added is preferably 10 μL / bead to 30 μL / bead, such as 10 μL / bead, 12.5 μL / bead, 13 μL / bead, 15 μL / bead, 18 μL / bead, 20 μL / bead, 23 μL / bead, 25 μL / bead, 30 μL / bead, or any value between them. In this case, the particle size of the resulting sample-treated lyophilized microspheres is approximately 1 mm to 5 mm.

[0087] In this invention, the liquid nitrogen droplet freezing treatment in the preparation of the magnetic microparticle freeze-dried microspheres, the preparation of the acrid ester freeze-dried microspheres, and the sample processing freeze-dried microspheres is a conventional freeze-drying technique in the prior art. Those skilled in the art can make adaptive selections according to actual needs, and this invention does not impose any particular limitations on it.

[0088] In this invention, the lyophilized microsphere reconstitution solution is used to achieve rapid reconstitution of the aforementioned lyophilized microsphere detection reagent, and also has the functions of restoring the activity of VB12 binding protein and VB12 complex antibody, and improving the anti-interference ability of the detection system. More specifically, the lyophilized microsphere reconstitution solution specifically includes: surfactant S9 with a preferred concentration of 0.04% (w / v) to 0.06% (w / v), such as 0.04% (w / v), 0.042% (w / v), 0.043% (w / v), 0.046% (w / v), 0.048% (w / v), 0.05% (w / v), 0.053% (w / v), 0.059% (w / v), 0.0 6% (w / v) or any value therebetween; preferably 0.04% (w / v) to 0.06% (w / v) of Triton X-100, such as 0.04% (w / v), 0.045% (w / v), 0.05% (w / v), 0.055% (w / v), 0.06% (w / v) or any value therebetween; preferably 0.04% (w / v) to 0.06% (w / v) of Proclin. 950, such as 0.04% (w / v), 0.045% (w / v), 0.05% (w / v), 0.055% (w / v), 0.06% (w / v) or any value therein; and, preferably, 0.04% (w / v) to 0.06% (w / v) of 5-bromo-5-nitro-1,3-dioxane, such as 0.04% (w / v), 0.045% (w / v), 0.05% (w / v), 0.055% (w / v), 0.06% (w / v) or any value therein.

[0089] In this invention, the solvent system of the lyophilized microsphere reconstitution solution is specifically a pH buffer, and specific examples of the pH buffer include, but are not limited to, one or more of PBS buffer, Tris-HCl buffer, HEPES buffer and MES buffer.

[0090] In some specific embodiments, the solvent system for the reconstitution solution of the lyophilized microspheres is preferably PB buffer. More specifically, the concentration of the PB buffer is preferably 480 mM to 50 mM, and the pH value is preferably 4.8 to 5.2.

[0091] In this invention, the sample dissociation solution is used to fully release VB12 from the sample to be tested, and also has the functions of restoring the activity of VB12 binding protein and VB12 complex antibody, as well as improving the anti-interference ability of the detection system. More specifically, the sample dissociation solution preferably includes: sodium hydroxide at a concentration of 45 mM to 55 mM, such as 45 mM, 47.5 mM, 49 mM, 50 mM, 52.5 mM, 55 mM, or any value between them; ethylenediaminetetraacetic acid at a concentration of 4 mM to 6 mM, such as 4 mM, 4.3 mM, 4.5 mM, 4.8 mM, 5 mM, 5.2 mM, 5.5 mM, 5.8 mM, 6 mM, or any value between them; potassium cobalt cyanide at a concentration of 6 g / L to 8 g / L, such as 6 g / L, 6.3 g / L, 6.5 g / L, 6.8 g / L, 7 g / L, 7.2 g / L, 7.5 g / L, 8 g / L, or any value between them; and Proclin at a concentration of 0.04% (w / v) to 0.06% (w / v). 950, such as 0.04% (w / v), 0.045% (w / v), 0.05% (w / v), 0.055% (w / v), 0.6% (w / v) or any value therein; and, preferably, 0.04% (w / v) to 0.06% (w / v) of 5-bromo-5-nitro-1,3-dioxane, such as 0.04% (w / v), 0.045% (w / v), 0.05% (w / v), 0.055% (w / v), 0.6% (w / v) or any value therein.

[0092] In this invention, the solvent system of the sample dissociation solution can specifically be a pH buffer and / or pure water. In some specific embodiments, the solvent system of the sample dissociation solution is preferably water.

[0093] Secondly, the non-diagnostic VB12 detection method provided by the present invention uses the above-mentioned lyophilized VB12 rapid detection kit to detect the sample to be tested, specifically including the following steps:

[0094] S1. Take the sample dissociation solution and the sample to be tested and incubate them together to obtain the sample treatment reaction solution;

[0095] S2. Take the sample treatment reaction solution, lyophilized reconstituted solution, magnetic microparticle lyophilized microspheres, acrid ester lyophilized microspheres and sample treatment lyophilized microspheres, mix them completely and reconstitute them, then perform incubation treatment II, measure the luminescence value, and obtain the VB12 detection result.

[0096] In this invention, in step S1, the preferred mixing volume ratio of the sample dissociation solution to the sample to be tested is (1~5):1, such as 1:1, 1.2:1, 1.5:1, 2:1, 2.5:1, 3:1, 4:1, 5:1 or any value between them.

[0097] In this invention, in step S1, the conditions for the incubation treatment I include a temperature preferably of 30°C to 40°C, such as 30°C, 31.5°C, 33°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C or any value between them; and a time preferably of 1 min to 5 min, such as 1 min, 1.3 min, 1.5 min, 1.8 min, 2 min, 2.5 min, 3 min, 3.5 min, 4 min, 5 min or any value between them.

[0098] In this invention, in step S2, the addition ratio of the sample processing reaction solution, the lyophilized reconstitution solution, the magnetic microparticle lyophilized microspheres, the acridinium ester lyophilized microspheres, and the sample processing lyophilized microspheres is (50~100) μL : (100~200) μL : 1 microsphere : 1 microsphere : 1 microsphere, that is, each lyophilized microsphere detection reagent (including 1 magnetic microparticle lyophilized microsphere, 1 acridinium ester lyophilized microsphere, and 1 sample processing lyophilized microsphere) is mixed with 50 μL~100 μL of sample processing reaction solution and 100 μL~200 μL of lyophilized reconstitution solution.

[0099] In this invention, the incubation treatment II conditions include a temperature preferably of 30°C to 40°C, such as 30°C, 32.5°C, 33°C, 35°C, 37°C, 38°C, 40°C or any value between them; and a time preferably of 5 min to 20 min, such as 5 min, 6 min, 7.5 min, 9 min, 10 min, 12.5 min, 15 min, 20 min or any value between them.

[0100] In this invention, the method for measuring the luminescence value is a conventional technique used in existing chemiluminescence detection. Those skilled in the art can make an adaptive selection according to actual needs, and this invention does not impose any particular limitations on it.

[0101] In this invention, the detection method uses the above-mentioned lyophilized VB12 rapid detection kit to detect the sample. It only requires mixing the dissociation solution with the sample and incubating for a few minutes to process the sample, which greatly simplifies the sample processing steps. The analytical blank limit is as low as 30.53 pmol / L, the coefficient of variation for repeated detection of the same sample is not higher than 5%, and the concordance rate between the detection results and Roche-assigned serum samples is not less than 0.9907. It has excellent detection sensitivity, reliability and stability, and can well meet the actual needs of rapid detection of clinical samples.

[0102] The embodiments of the present invention are described in detail below. These embodiments are intended to explain the present invention and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0103] Example

[0104] This embodiment illustrates a lyophilized VB12 rapid detection kit and its preparation method. The kit specifically includes a reagent storage solution, magnetic microparticle lyophilized microspheres, acrid ester lyophilized microspheres, sample processing lyophilized microspheres, a lyophilized microsphere reconstitution solution, and a sample dissociation solution. The preparation process includes the following steps:

[0105] 1. Preparation of reagent storage solution

[0106] Take NaCl, BSA, trehalose, glucose, gelatin, Triton X-100, PEG6000, Proclin 950 preservative (PC950), 10% 5-bromo-5-nitro-1,3-dioxane solution (BND-10), mannitol and glycine according to the concentrations shown in Table 1, add them to HEPES buffer (20mM, pH=7.4) and mix well to obtain the reagent stock solution.

[0107] Table 1.

[0108]

[0109] 2. Preparation of lyophilized microsphere detection reagents

[0110] (1) Preparation of lyophilized magnetic microspheres: a. Magnetic beads (JSR, catalog number MS300 / Carboxyl, particle size 3.0µm) were washed three times with magnetic bead cleaning solution and then resuspended to prepare a magnetic bead resuspension with a concentration of 10mg / mL; 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were added to the magnetic bead resuspension at final concentrations of 0.1mg / mL and 0.2mg / mL, respectively, and activated at 25℃ for 30min to obtain activated magnetic beads; the activated magnetic beads were washed three times with magnetic bead cleaning solution and then resuspended to obtain an activated magnetic bead resuspension with a concentration of 10mg / mL;

[0111] The preparation of the magnetic bead cleaning solution includes: adding Proclin 300 to MES buffer (0.2M, pH=6.0) at a final concentration of 0.1mL / L and mixing thoroughly to obtain the magnetic bead cleaning solution.

[0112] b. VB12 binding protein (Nanjing Oukai Biotechnology Co., Ltd., catalog number: intrinsic factor (IF) C1770) was added to the activated magnetic bead resuspension at a final concentration of 100 μg / mL and coupled at 25℃ for 3 h. Then, the coupled activated magnetic beads and magnetic bead blocking solution were mixed at an addition rate of 10 mg / mL and blocked at 25℃ for 25 min to obtain magnetic bead-coated VB12 binding protein. The preparation of the magnetic bead blocking solution included adding BSA and Tween 20 to Tris-HCl buffer (0.01 M, pH=7.4) at a final concentration of 5 g / L and 0.1% (v / v) and mixing them evenly to obtain the magnetic bead blocking solution.

[0113] c. The VB12 binding protein coated with magnetic beads was washed three times with reagent storage solution and then resuspended to obtain a lyophilized solution of magnetic beads with a concentration of 0.25 mg / mL. 20 μL of the lyophilized solution of magnetic beads was subjected to liquid nitrogen droplet freezing treatment at -196℃ to obtain lyophilized magnetic microparticles (counted as 1 part, with a particle size of about 3.2 mm).

[0114] (2) Preparation of acridine ester lyophilized microspheres: a. VB12 complex antibody (Nanjing Oukai Biotechnology Co., Ltd., catalog number H835e1) was added to CB buffer (0.02M, pH=9.6) at a final concentration of 1 mg / mL and mixed evenly to prepare an antibody solution; b. Acridine ester was added to the antibody solution at a molar ratio of 1:20 between VB12 complex antibody and acridine ester, and the mixture was coupled at 25℃ for 3 h. Then, lysine was added to the solution at a molar ratio of 140:1 between lysine and acridine ester, and the mixture was blocked at 25℃ for 30 min. Then, the blocked solution was dialyzed 5 times in a 50KD dialysis bag using PBS buffer (0.02M, pH=6.5) (each dialyzing time was 3 h) to obtain acridine ester-labeled VB12 complex antibody.

[0115] c. The acridine ester-labeled VB12 complex antibody was diluted with reagent stock solution to obtain a lyophilized solution of acridine ester microspheres with a concentration of 0.125 μg / mL; 20 μL of the lyophilized solution of acridine ester microspheres was subjected to liquid nitrogen droplet freezing treatment at -196℃ to obtain lyophilized acridine ester microspheres (counted as 1 part, with a particle size of approximately 3.2 mm).

[0116] (3) Preparation of sample-treated lyophilized microspheres: Tris(2-carboxyethyl)phosphine hydrochloride was added to the reagent storage solution at a final concentration of 0.125% (w / v) and mixed evenly to obtain the sample treatment solution; 20 μL of the sample treatment solution was subjected to liquid nitrogen droplet freezing treatment at -196℃ to obtain sample-treated lyophilized microspheres (counted as 1 part, with a particle size of about 3.2 mm).

[0117] (4) Reference Figure 1 One part of magnetic microparticle lyophilized microspheres, one part of acridinium ester lyophilized microspheres, and one part of sample processing lyophilized microspheres were packaged in the same vacuum reagent tube to obtain one part of lyophilized microsphere detection reagent.

[0118] 3. Preparation of reconstituted solutions of lyophilized microspheres

[0119] According to the concentrations shown in Table 2, surfactants S9, Triton X-100, PC950 and BND-10 were added to PB buffer (500mM, pH=5.0) and mixed evenly to obtain the lyophilized microsphere reconstituted solution.

[0120] Table 2.

[0121]

[0122] 4. Preparation of sample dissociation solution

[0123] According to the concentrations shown in Table 3, NaOH, ethylenediaminetetraacetic acid (EDTA), K3Co(CN)6, PC950 and BND-10 were added to ddH2O and mixed evenly to obtain the sample dissociation solution.

[0124] Table 3.

[0125]

[0126] Comparative Example 1

[0127] The lyophilized VB12 rapid test kit provided in this comparative example is basically the same as that in the example, except that the reagent storage solution is set to HEPES buffer (20mM, pH=8.0); other conditions are kept the same to obtain the lyophilized VB12 rapid test kit.

[0128] Comparative Example 2

[0129] The lyophilized VB12 rapid detection kit provided in this comparative example is basically the same as that in the example, except that the lyophilized microsphere reconstitution solution is set to PB buffer (500mM, pH=5.0); other conditions are kept the same to obtain the lyophilized VB12 rapid detection kit.

[0130] Comparative Example 3

[0131] The lyophilized VB12 rapid detection kit provided in this comparative example is basically the same as that in the example, except that the lyophilized VB12 rapid detection kit does not include sample-processed lyophilized microspheres. Other conditions remain the same to obtain the lyophilized VB12 rapid detection kit.

[0132] Comparative Example 4

[0133] The lyophilized VB12 rapid test kit provided in this comparative example is basically the same as the example, except that the preparation of the reagent storage solution is different. Specifically, mannitol, trehalose, casein, Triton X-100, gelatin and PC950 are added to Tris-HCl buffer (50mM, pH=7.4) according to the concentrations shown in Table 4 to obtain the reagent storage solution; other conditions are kept the same to obtain the lyophilized VB12 rapid test kit.

[0134] Table 4.

[0135]

[0136] Test case

[0137] This test example illustrates the detection performance of the lyophilized VB12 rapid test kit provided in the above embodiments and comparative examples. The specific tests include:

[0138] 1. Effects of reagent stock solutions and lyophilized microsphere reconstitution solutions on the activity of VB12-binding protein and VB12 complex antibodies.

[0139] (1) Take sucrose, trehalose, PC950 and BND-10 according to the concentrations shown in Table 5 and add them to PBS buffer (50mM, pH=7.4) and mix well to obtain the calibrator dilution solution.

[0140] Table 5.

[0141]

[0142] (2) Take the calibrator diluent and dilute the VB12 standard (concentration of 2 mg / mL, national standard, catalog number 100248, the same below) to 158.2 poml / L and 542.8 poml / L respectively to obtain the low-value quality control product and the high-value quality control product.

[0143] (3) Take the low-value quality control sample (or high-value quality control sample) and the sample dissociation solution at a volume ratio of 1:3 and mix them evenly. Incubate at 37°C for 2 min to obtain the sample treatment reaction solution. Then, take the sample treatment reaction solution and the lyophilized reconstitution solution at a volume ratio of 60 μL: 125 μL and add them to a vacuum reagent tube (containing 1 part of magnetic microparticle lyophilized microspheres, 1 part of acridinium ester lyophilized microspheres and 1 part of sample treatment lyophilized microspheres). Mix evenly until completely reconstituted. Incubate at 37°C for 10 min and then use a chemiluminescence analyzer to detect the luminescence value (RLU). Use the lyophilized solution of magnetic microspheres that has not been treated with liquid nitrogen droplets, the lyophilized solution of acridinium ester, and the sample treatment solution (diluted with lyophilized reconstitution solution) as the original control (the operation and conditions are kept consistent). The results are shown in Table 6.

[0144] Table 6.

[0145]

[0146] As shown in Table 6, compared with Comparative Example 1, introducing the reagent storage solution provided in the present invention during the preparation of magnetic microparticle lyophilized microspheres and acridine ester lyophilized microspheres endows the finally prepared magnetic microparticle lyophilized microspheres and acridine ester lyophilized microspheres with both excellent detection activity and rapid reconstitution ability. The detection results of low-value quality control samples and high-value quality control samples are highly consistent with the lyophilized solutions of magnetic microspheres and acridine ester lyophilized microspheres that have not undergone liquid nitrogen droplet freezing treatment.

[0147] As shown in Table 6, compared with Comparative Example 2, the reconstitution of the two lyophilized preparations using the lyophilized microsphere reconstitution solution provided in this embodiment of the invention not only achieves rapid reconstitution but also restores their activity by optimizing the structure of VB12 binding protein and VB12 complex antibody. The detection results of the low-value and high-value quality control products are highly consistent with the lyophilized magnetic bead microsphere solution and the lyophilized acridinium ester microsphere solution that have not undergone liquid nitrogen droplet freezing treatment.

[0148] As shown in Table 6, compared with Comparative Example 3, the presence of the sample-processed lyophilized microspheres introduced in this embodiment of the invention can synergistically work with the lyophilized microsphere reconstitution solution to achieve rapid reconstitution while also restoring the activity of VB12 binding protein and VB12 complex antibody by optimizing their structure. The detection results of low-value and high-value quality control products are highly consistent with the lyophilized solutions of magnetic bead microspheres and acridinium ester microspheres that have not undergone liquid nitrogen droplet freezing treatment.

[0149] As shown in Table 6, compared to Comparative Example 4, the reagent storage solution provided in this embodiment of the invention contains NaCl, BSA, trehalose, glucose, gelatin, Triton X-100, PEG6000, PC950, BND-10, mannitol, and glycine in specific proportions, forming an organic whole. The synergistic cooperation among these substances is necessary to endow the obtained magnetic microparticle lyophilized microspheres and acridinium ester lyophilized microspheres with excellent detection activity and rapid reconstitution ability. Furthermore, the casein introduced in Comparative Example 4 leads to non-specific binding, resulting in a significant increase in luminescence value.

[0150] 2. Detection standard curve of the lyophilized VB12 rapid test kit

[0151] (1) The calibrator diluent was prepared according to the method provided in “1. Effect of reagent storage solution and lyophilized microsphere reconstitution solution on the activity of VB12 binding protein and VB12 complex antibody”.

[0152] (2) Take the calibrator diluent and dilute the VB12 standard to 30.36 pmol / L, 60.10 poml / L, 109.71 poml / L, 309.72 poml / L, 541.10 poml / L, 1173.67 poml / L and 1491.53 poml / L respectively to obtain a series of calibrator solutions.

[0153] (3) Mix 30 μL of calibrator solution with 90 μL of sample dissociation solution and incubate at 37 °C for 2 min to obtain sample processing reaction solution; add 60 μL of sample processing reaction solution and 125 μL of lyophilized reconstitution solution to a vacuum reagent tube (containing 1 part magnetic microparticle lyophilized microspheres, 1 part acridinium ester lyophilized microspheres and 1 part sample processing lyophilized microspheres), incubate at 37 °C for 10 min, and then detect the luminescence value (RLU) using a chemiluminescence analyzer. The results are shown in Table 7 and Figure 2 As shown.

[0154] Table 7.

[0155]

[0156] From Table 7 and Figure 2 The results show that the lyophilized VB12 rapid detection kit provided in this embodiment of the invention, when used to detect a series of calibrator solutions, yields a standard curve of Y = (-6415.4808 - 2451074.0213) / (1 + (X / 1924.7168)^1.1110) + 2451074.0213, R 2 The value is close to 1, indicating a high degree of fit to the standard curve.

[0157] 3. Detection sensitivity of the lyophilized VB12 rapid test kit

[0158] (1) The calibrator diluent was prepared according to the method provided in “1. Effect of reagent storage solution and lyophilized microsphere reconstitution solution on the activity of VB12 binding protein and VB12 complex antibody”.

[0159] (2) Mix 30 μL of calibrator diluent with 90 μL of sample dissociation solution and incubate at 37 °C for 2 min to obtain sample processing reaction solution; add 60 μL of sample processing reaction solution and 125 μL of lyophilized reconstitution solution to a vacuum reagent tube (containing 1 part magnetic microparticle lyophilized microspheres, 1 part acridinium ester lyophilized microspheres and 1 part sample processing lyophilized microspheres), incubate at 37 °C for 10 min, and then detect the luminescence value (RLU) using a chemiluminescence analyzer. Repeat the test 20 times in parallel, calculate the average value (X) and standard deviation (SD) of the luminescence value, and substitute the calculated value of X+2SD into the... Figure 2 The analytical sensitivity was calculated from the standard curve shown, and the results are shown in Table 8.

[0160] Table 8.

[0161]

[0162] As shown in Table 8, the analytical sensitivity (blank limit) of the lyophilized VB12 rapid detection kit provided in this embodiment of the invention is 30.53 pmol / L, which meets the detection requirements (<36.9 pmol / L).

[0163] 4. Repeatability test of the lyophilized VB12 rapid test kit

[0164] (1) Following the method provided in “1. Effects of reagent storage solution and lyophilized microsphere reconstitution solution on the activity of VB12 binding protein and VB12 complex antibody”, low-value quality control (VB12 concentration of 158.2 poml / L) and high-value quality control (VB12 concentration of 542.8 poml / L) were prepared.

[0165] (2) Take 30 μL of low-value quality control sample (or high-value quality control sample) and mix it evenly with 90 μL of sample dissociation solution. Incubate at 37°C for 2 min to obtain the sample treatment reaction solution. Take 60 μL of the sample treatment reaction solution and 125 μL of lyophilized reconstitution solution and add them to a vacuum reagent tube (containing 1 part magnetic microparticle lyophilized microspheres, 1 part acridinium ester lyophilized microspheres and 1 part sample treatment lyophilized microspheres). Incubate at 37°C for 10 min. Detect the luminescence value (RLU) using a chemiluminescence analyzer. Substitute the measured luminescence value into the... Figure 2 The VB12 test concentration was obtained from the standard curve shown. The test was repeated 10 times in parallel, and the intra-batch coefficient of variation (CV) was calculated. The results are shown in Table 9.

[0166] Table 9.

[0167]

[0168] As shown in Table 9, the lyophilized VB12 rapid detection kit provided in this embodiment of the invention showed that the intra-assay coefficient of variation was only 4% when the low-value quality control sample was repeatedly tested 10 times, while the intra-assay coefficient of variation was only 2% when the high-value quality control sample was repeatedly tested 10 times, demonstrating excellent repeatability.

[0169] 5. Accelerated thermal stability of the lyophilized VB12 rapid test kit

[0170] The lyophilized VB12 rapid test kit was placed in an electric thermostatic incubator at 45℃±2℃ for one month. Following the method described in "1. Effects of reagent storage solution and lyophilized microsphere reconstitution solution on the activity of VB12 binding protein and VB12 complex antibody", low, medium, and high value antigen samples (prepared with VB12 national standard, VB12 concentrations of 158.2 poml / L, 446.8 poml / L, and 759.0 poml / L) and low, medium, and high value samples (assigned with Roche reagents, VB12 detection concentrations of 251.3 poml / L, 568.9 poml / L, and 1005.2 poml / L) were tested. The kit before storage was used as a control. The results are shown in Table 10.

[0171] Table 10.

[0172]

[0173] As shown in Table 10, the lyophilized VB12 rapid detection kit provided in this embodiment of the invention, after being stored at 45℃±2℃ for 30 days, showed a decrease in the luminescence value of the same sample by less than 10%, indicating excellent accelerated thermal stability.

[0174] 6. Clinical sample detection performance of the lyophilized VB12 rapid test kit

[0175] Using a lyophilized VB12 rapid detection kit, and following the method described in "1. Effects of reagent stock solution and lyophilized microsphere reconstitution solution on the activity of VB12 binding protein and VB12 complex antibody," 33 serum samples tested using the Roche Vitamin B12 Detection Kit (electrochemiluminescence assay) (catalog number: 07212771 190) were analyzed. The Roche reagent results were used as a control to compare the detection concordance rate. Results are shown in Table 11 and... Figure 3 As shown.

[0176] From Table 11 and Figure 3 The results show that, using the lyophilized VB12 rapid detection kit provided in this embodiment of the invention, the signal gradient of 33 Roche-assigned serum samples with concentrations ranging from 18 pmol / L to 140 pmol / L was obvious, and the sample concordance rate (R) was high. 2 With a value of 0.9907, it has excellent reliability and stability, and can well meet the actual needs of rapid clinical sample testing.

[0177] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

[0178] Table 11.

[0179]

Claims

1. A lyophilized VB12 rapid detection kit, characterized in that, The test kit includes: The lyophilized microsphere detection reagent comprises magnetic microparticle lyophilized microspheres, acridil ester lyophilized microspheres, and sample processing lyophilized microspheres. The magnetic microparticle lyophilized microspheres are obtained by treating VB12-binding protein coated with magnetic beads in a reagent storage solution with liquid nitrogen droplet freezing. The acridil ester lyophilized microspheres are obtained by treating acridil ester-labeled VB12 complex antibody in a reagent storage solution with liquid nitrogen droplet freezing. The sample processing lyophilized microspheres are obtained by treating a reducing agent in a reagent storage solution with liquid nitrogen droplet freezing. The reducing agent is selected from dithiothreitol, tris(2-carboxyethyl)phosphine hydrochloride, reduced glutathione, cysteine, and β-mercaptoethanol. The reagent stock solution comprises one of the following: sodium chloride (0.8% w / v) to 0.9% w / v), BSA (4% w / v) to 6% w / v), trehalose (4% w / v) to 6% w / v), glucose (4% w / v) to 6% w / v), gelatin (0.1% w / v) to 0.3% w / v), Triton X-100 (0.05% w / v) to 0.06% w / v), PEG6000 (0.1% w / v) to 0.11% w / v), and Proclin (0.4% w / v) to 0.6% w / v. 950, 0.4% (w / v) to 0.6% (w / v) of 5-bromo-5-nitro-1,3-dioxane, 2.8% (w / v) to 3.2% (w / v) of mannitol and 0.4% (w / v) to 0.5% (w / v) of glycine; The lyophilized microsphere reconstituted solution comprises 0.04% (w / v) to 0.06% (w / v) surfactant S9, 0.04% (w / v) to 0.06% (w / v) Triton X-100, 0.04% (w / v) to 0.06% (w / v) Proclin 950 and 0.04% (w / v) to 0.06% (w / v) 5-bromo-5-nitro-1,3-dioxane; The sample dissociation solution consisted of 45 mM to 55 mM sodium hydroxide, 4 mM to 6 mM ethylenediaminetetraacetic acid, 6 g / L to 8 g / L potassium cobalt cyanide, 0.04% (w / v) to 0.06% (w / v) Proclin 950, and 0.04% (w / v) to 0.06% (w / v) 5-bromo-5-nitro-1,3-dioxane.

2. The lyophilized VB12 rapid detection kit according to claim 1, characterized in that, The preparation of the magnetic microparticle freeze-dried microspheres includes: (1) activating the magnetic beads with 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to obtain activated magnetic beads; (2) coupling the activated magnetic beads with VB12 binding protein and blocking them in a magnetic bead blocking solution to obtain the VB12 binding protein coated on the magnetic beads; (3) mixing the VB12 binding protein coated on the magnetic beads with a reagent storage solution to obtain a freeze-dried solution of magnetic beads; and subjecting the freeze-dried solution of magnetic beads to liquid nitrogen droplet freezing treatment at -200℃ to -190℃ to obtain the magnetic microparticle freeze-dried microspheres.

3. The lyophilized VB12 rapid detection kit according to claim 2, characterized in that, In step (1), the particle size of the magnetic beads is 1.5µm~3.0µm.

4. The lyophilized VB12 rapid detection kit according to claim 2, characterized in that, In step (1), the concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide used is 0.05 mg / mL to 50 mg / mL, the concentration of N-hydroxysuccinimide used is 0.05 mg / mL to 50 mg / mL, and the concentration of magnetic beads used is 5 mg / mL to 100 mg / mL.

5. The lyophilized VB12 rapid detection kit according to claim 2, characterized in that, In step (1), the activation treatment temperature is 18℃~30℃ and the time is 10min~60min.

6. The lyophilized VB12 rapid detection kit according to claim 2, characterized in that, In step (2), the concentration of the activated magnetic beads is 5 mg / mL to 100 mg / mL, and the concentration of the VB12 binding protein is 10 μg / mL to 1000 μg / mL.

7. The lyophilized VB12 rapid detection kit according to claim 2, characterized in that, In step (2), the coupling treatment is performed at a temperature of 18°C ​​to 30°C for 2 hours to 3 hours.

8. The lyophilized VB12 rapid detection kit according to claim 2, characterized in that, In step (2), the magnetic bead blocking solution includes 1 g / L to 10 g / L of BSA and 0.01% (v / v) to 1% (v / v) of Tween 20.

9. The lyophilized VB12 rapid detection kit according to claim 2, characterized in that, In step (2), the concentration of activated magnetic beads after the coupling treatment is 5 mg / mL to 20 mg / mL.

10. The lyophilized VB12 rapid detection kit according to claim 2, characterized in that, In step (2), the temperature of the sealing treatment is 18℃~37℃ and the time is 10min~60min.

11. The lyophilized VB12 rapid detection kit according to claim 2, characterized in that, In step (3), the concentration of the lyophilized magnetic bead microsphere solution is 0.1 mg / mL to 2 mg / mL.

12. The lyophilized VB12 rapid detection kit according to claim 2, characterized in that, In step (3), the amount of the lyophilized magnetic bead solution added in the liquid nitrogen droplet freezing treatment is 10 μL / bead to 30 μL / bead.

13. The lyophilized VB12 rapid detection kit according to claim 2, characterized in that, The particle size of the freeze-dried magnetic microspheres is 1 mm to 5 mm.

14. The lyophilized VB12 rapid detection kit according to claim 1, characterized in that, The preparation of the acridine ester lyophilized microspheres includes: (1) coupling acridine ester with a VB12 complex antibody solution, blocking the solution in the presence of lysine, and then dialysis to obtain the acridine ester-labeled VB12 complex antibody; (2) mixing the acridine ester-labeled VB12 complex antibody with a reagent storage solution to obtain an acridine ester microsphere lyophilized solution; and subjecting the acridine ester microsphere lyophilized solution to liquid nitrogen droplet freezing treatment at -200℃ to -190℃ to obtain the acridine ester lyophilized microspheres.

15. The lyophilized VB12 rapid detection kit according to claim 14, characterized in that, In step (1), the concentration of the VB12 complex antibody solution is 0.5 mg / mL to 2 mg / mL.

16. The lyophilized VB12 rapid detection kit according to claim 14, characterized in that, In step (1), the molar ratio of the VB12 complex antibody to acridine ester is 1:(5~20).

17. The lyophilized VB12 rapid detection kit according to claim 14, characterized in that, In step (1), the coupling treatment is performed at a temperature of 20°C to 30°C for 2 hours to 3 hours.

18. The lyophilized VB12 rapid detection kit according to claim 14, characterized in that, In step (1), the molar ratio of lysine to acridine ester is (100~150):

1.

19. The lyophilized VB12 rapid detection kit according to claim 14, characterized in that, In step (1), the temperature of the sealing treatment is 20℃~37℃ and the time is 10min~60min.

20. The lyophilized VB12 rapid detection kit according to claim 14, characterized in that, In step (1), the molecular weight cutoff of the dialysis membrane in the dialysis treatment is 40KD~60KD.

21. The lyophilized VB12 rapid detection kit according to claim 14, characterized in that, In step (2), the concentration of the acridinium ester microsphere lyophilization solution is 0.1 μg / mL to 2.5 μg / mL.

22. The lyophilized VB12 rapid detection kit according to claim 14, characterized in that, In step (2), the amount of acridine ester microsphere freeze-drying solution added in the liquid nitrogen droplet freezing treatment is 10 μL / microsphere to 30 μL / microsphere.

23. The lyophilized VB12 rapid detection kit according to claim 14, characterized in that, The acridine ester freeze-dried microspheres have a particle size of 1 mm to 5 mm.

24. The lyophilized VB12 rapid detection kit according to claim 1, characterized in that, The preparation of the sample-processed lyophilized microspheres includes: (1) mixing the reducing agent and the reagent storage solution to obtain a sample processing solution; (2) subjecting the sample processing solution to liquid nitrogen droplet freezing treatment at -200℃ to -190℃ to obtain the sample-processed lyophilized microspheres.

25. The lyophilized VB12 rapid detection kit according to claim 24, characterized in that, In step (1), the concentration of the reducing agent used is 0.1% (w / v) to 0.5% (w / v).

26. The lyophilized VB12 rapid detection kit according to claim 24, characterized in that, In step (2), the amount of sample treatment solution added in the liquid nitrogen droplet freezing bead treatment is 10 μL / bead to 30 μL / bead.

27. The lyophilized VB12 rapid detection kit according to claim 24, characterized in that, The sample-processed freeze-dried microspheres have a particle size of 1 mm to 5 mm.

28. A method for detecting VB12 for non-diagnostic purposes, characterized in that, The detection method uses the lyophilized VB12 rapid detection kit described in claim 1 to detect the sample to be tested, and specifically includes the following steps: S1. Take the sample dissociation solution and the sample to be tested and incubate them together to obtain the sample treatment reaction solution; S2. Take the sample treatment reaction solution, lyophilized reconstituted solution, magnetic microparticle lyophilized microspheres, acrid ester lyophilized microspheres and sample treatment lyophilized microspheres, mix them completely and reconstitute them, then perform incubation treatment II, measure the luminescence value, and obtain the VB12 detection result.

29. The method for detecting VB12 for non-diagnostic purposes according to claim 28, characterized in that, In step S1, the mixing volume ratio of the sample dissociation solution to the sample to be tested is (1~5):

1.

30. The method for detecting VB12 for non-diagnostic purposes according to claim 28, characterized in that, In step S1, the temperature of the incubation treatment I is 30℃~40℃, and the time is 1min~5min.

31. The method for detecting VB12 for non-diagnostic purposes according to claim 28, characterized in that, In step S2, the addition ratio of the sample processing reaction solution, the lyophilized reconstituted solution, the magnetic microparticle lyophilized microspheres, the acridinium ester lyophilized microspheres, and the sample processing lyophilized microspheres is (50~100)μL:(100~200)μL:1 microsphere:1 microsphere:1 microsphere.

32. The method for detecting VB12 for non-diagnostic purposes according to claim 28, characterized in that, In step S2, the temperature of the incubation treatment II is 30℃~40℃, and the time is 5min~20min.

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