CN110967493A - Vitamin B12 determination kit and preparation method thereof - Google Patents
Vitamin B12 determination kit and preparation method thereof Download PDFInfo
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- CN110967493A CN110967493A CN201811153247.1A CN201811153247A CN110967493A CN 110967493 A CN110967493 A CN 110967493A CN 201811153247 A CN201811153247 A CN 201811153247A CN 110967493 A CN110967493 A CN 110967493A
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Abstract
The invention belongs to the technical field of biomolecule chemiluminescence detection, and particularly relates to a vitamin B12 determination kit and a preparation method thereof. The preparation method of the vitamin B12 determination kit provided by the invention comprises the following steps: reacting the magnetic beads with streptavidin to prepare streptavidin-coated magnetic beads; coupling the detection antibody with alkaline phosphatase to prepare a detection antibody marked by the alkaline phosphatase; then, carrying out phosphorylation modification on the detection antibody marked by the alkaline phosphatase by adopting an organic phosphate compound to obtain an enzyme-marked antibody compound; coupling the activated biotin to vitamin B12 to prepare biotin-labeled vitamin B12. The stability of the vitamin B12 determination kit prepared by the preparation method is obviously improved.
Description
Technical Field
The invention belongs to the technical field of biomolecule chemiluminescence detection, and particularly relates to a vitamin B12 determination kit and a preparation method thereof.
Background
Vitamin B12(Vitamin B12, VB12), also known as cyanocobalamin, cobalamin, exogenous factors, animal protein factors, is the only Vitamin containing mineral substances and is a micronutrient indispensable for maintaining the normal metabolism and function of human body. Vitamin B12, molecular weight 1355, is a polycyclic compound containing cobalt in3 valencies, with 4 reduced pyrrole rings linked together to become a1 corrin macrocycle (similar to a porphyrin) which is the core of the vitamin B12 molecule, and compounds containing such rings are commonly referred to as corrinoids. The vitamin B12 is a light red needle crystal, is easily soluble in water and ethanol, is most stable under a weak acid condition with the pH value of 4.5-5.0, is decomposed in a strong acid (pH <2) or an alkaline solution, can be damaged to a certain extent when being heated, has small high-temperature disinfection loss in a short time, and is easily damaged when being subjected to strong light or ultraviolet rays. Vitamin B12 is an important cofactor for transferring one carbon unit in human body, so it has synergistic effect in many biochemical reactions, is an important coenzyme in DNA synthesis process, and can reduce homocysteine level in blood plasma. The lack of VB12 can affect the utilization of folic acid by human body, and can also cause peripheral neuritis and anemia in children. Human VB12 deficiency is also a cause of megaloblastic anemia, and is also associated with senile dementia, depression, coronary heart disease and the like.
At present, methods for detecting the VB12 level in human bodies in vitro mainly comprise an enzyme-linked immunosorbent assay, a chemiluminescence assay and a liquid chromatography tandem mass spectrometry, and the chemiluminescence assay is high in sensitivity and specificity and is rapid in detection, so that the chemiluminescence assay is deeply emphasized by researchers at home and abroad in recent years and gradually becomes one of international mainstream in-vitro detection means.
According to the detection principle, the chemiluminescence method can be divided into a sandwich method and a competition method, the sandwich method takes magnetic particles as a carrier to couple antibodies, and the antigen to be detected is rapidly captured by utilizing the characteristic that magnetic beads are attracted by a magnetic field and mechanically move under the action of magnetic force. After a sample is added, an antigen to be detected of the sample is combined with an antibody on a magnetic bead, an Alkaline Phosphatase (AP) labeled detection antibody is added at the moment to form a magnetic bead coated antibody-antigen to be detected-AP labeled antibody compound, after unbound antibody is washed away, a luminous substrate solution of AP is added, the luminous substrate is catalyzed by the AP on the compound to continuously and stably emit photons, the emitted photons are recorded by a system, and the photon energy intensity is converted into the concentration of the antigen to be detected on a standard curve through software. The competition method is to use magnetic beads as carriers to coat antigens, and after a sample is added, the antigens to be detected of the sample and the antigens coupled with the magnetic beads are combined with the AP marked detection antibodies in a competitive mode to form a magnetic bead coated antigen-AP marked antibody compound.
The chemiluminescence method can be divided into direct luminescence and enzymatic chemiluminescence according to the principle of luminescence. Enzymatic chemiluminescence is mainly divided into a horseradish peroxidase luminescent system and an alkaline phosphatase luminescent system, a chemiluminescent substrate of the horseradish peroxidase luminescent system is easy to oxidize and self-luminesce, the background is relatively high, the signal-to-noise ratio is low, the influence factors are complex, and the result is relatively unstable. The chemiluminescence substrate of the alkaline phosphatase luminescence system has long time for reaching the plateau phase, low background luminescence, high signal-to-noise ratio, no catalysis for reaction, simple system, less interference factors and low detection cost, so the method can overcome the defects. However, since alkaline phosphatase has high stability under alkaline conditions and the labeled detection antibody is stable under acidic environments, the detection antibody labeled with alkaline phosphatase is easily affected by the environments, which results in low stability of the enzyme label, not only reduces the validity period of the product, but also requires frequent calibration during use, increasing the cost and unknown risks.
Disclosure of Invention
The invention mainly aims to provide a preparation method of a vitamin B12 determination kit, and aims to solve the technical problems that alkaline phosphatase in a vitamin B12 determination kit prepared by the prior art is easily influenced by the environment, the stability is low and the like.
In order to solve the above technical problems, in one aspect, the present invention provides a method for preparing a vitamin B12 assay kit, comprising:
reacting the magnetic beads with streptavidin to prepare streptavidin-coated magnetic beads;
coupling the detection antibody with alkaline phosphatase to prepare a detection antibody marked by the alkaline phosphatase; then, carrying out phosphorylation modification on the detection antibody marked by the alkaline phosphatase by adopting an organic phosphate compound to obtain an enzyme-marked antibody compound;
biotin was coupled to vitamin B12 to prepare biotin-labeled vitamin B12.
In another aspect, the present invention provides a vitamin B12 assay kit, comprising: magnetic separation reagent, antibody reagent and enzyme labeling reagent;
the magnetic separation reagent comprises magnetic beads coated with streptavidin;
the enzyme-labeled reagent comprises an enzyme-labeled antibody compound, and the enzyme-labeled antibody compound is a product obtained by carrying out phosphorylation modification on an alkaline phosphatase-labeled detection antibody by an organic phosphate compound;
the antigen reagent comprises biotin-labeled vitamin B12 and a biotin-labeled working solution.
Compared with the prior art, the invention adopts the organic phosphate compound to carry out phosphorylation modification on the detection antibody marked by the alkaline phosphatase, the organic phosphate compound is connected with the active site of the alkaline phosphatase to form an enzyme-marked antibody complex, the free energy of the alkaline phosphatase is reduced, and thus the conformation of the marked enzyme in the enzyme-marked antibody complex is more stable, the four-dimensional structure of the enzyme-marked antibody complex can be maintained even under the condition of smaller pH value of the solution environment or larger pH fluctuation range, and the stability of the determination kit is greatly improved. Through experimental detection, the stability of the vitamin B12 determination kit prepared by the preparation method is excellent.
Drawings
FIG. 1 is a correlation fitting curve of the VB12 assay kit and the Beckmann VB12 assay kit of the present invention in the test examples.
Detailed Description
The method aims to solve the technical problems that alkaline phosphatase in a vitamin B12 determination kit prepared by the prior art is easily influenced by the environment, the stability is low and the like. The embodiment of the invention provides a preparation method of a vitamin B12 determination kit, and the VB12 determination kit obtained by the method has excellent stability.
The preparation method of the vitamin B12 determination kit comprises the following steps:
s01, reacting the magnetic beads with streptavidin to prepare streptavidin-coated magnetic beads;
s02, coupling the detection antibody with alkaline phosphatase to prepare a detection antibody marked by the alkaline phosphatase; then, carrying out phosphorylation modification on the detection antibody marked by the alkaline phosphatase by adopting an organic phosphate compound to obtain an enzyme-marked antibody compound;
s03, coupling biotin to vitamin B12, and preparing the biotin-labeled vitamin B12.
The invention adopts the organic phosphate compound to carry out phosphorylation modification on the detection antibody marked by the alkaline phosphatase, the organic phosphate compound is connected with the active site of the alkaline phosphatase to form an enzyme-marked antibody compound, and the free energy near the active site of the marker enzyme is reduced, so that the conformation of the marker enzyme in the enzyme-marked antibody compound is more stable, the four-dimensional structure of the enzyme-marked antibody compound can be maintained even under the condition of low pH solution environment or large change of pH fluctuation range, and the stability of the determination kit is greatly improved.
Specifically, in step S01, the magnetic beads are super-strong paramagnetic solid carriers, can be rapidly aggregated in a magnetic field, and can be uniformly dispersed in a manner of facilitating magnetic separation after leaving the magnetic field, and may be selected from, but not limited to, carboxyl magnetic beads, amino magnetic beads, sulfonic magnetic beads, and hydroxyl magnetic beads.
The magnetic beads are provided with abundant surface active groups, streptavidin is coupled with the magnetic beads through the surface active groups of the magnetic beads, the streptavidin can be combined with biotin in a highly specific manner, the affinity between the streptavidin and the biotin is extremely strong, the streptavidin can be used as a coupling molecule to connect the magnetic beads with vitamin B12 marked by the biotin and further combine with biochemical substances to form a combination, and the combination is separated from a sample to be detected under the action of an external magnetic field.
Preferably, the mass ratio of the magnetic beads to the streptavidin is 10 (0.2 to 0.5).
In the embodiment of the present invention, step S01 may be performed by referring to the conventional technical means in the art. In step S01, before the magnetic beads are reacted with streptavidin, the surface active groups of the magnetic beads must be activated by an activation process. The magnetic bead activation treatment can be carried out by a method conventionally used in the art, for example, by performing activation treatment using an activating agent such as 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (abbreviated as EDC) or N-hydroxysuccinimide (abbreviated as NHS).
Preferably, in the embodiment of the present invention, before the magnetic beads are reacted with streptavidin, EDC is used to activate the magnetic beads; wherein the mass ratio of the magnetic beads to the EDC is preferably 10 (0.2-0.5); the magnetic beads are selected from carboxyl magnetic beads.
Currently, most magnetic beads used in chemiluminescence detection are composed of small metal particles (Fe)3O4) The core is wrapped with a layer of hydrophobic polymer material (such as polystyrene and polyvinyl chloride), the outermost layer is loaded with modification functional groups (such as carboxyl, hydroxyl, amino, aldehyde group and the like), the surface is hydrophobic, the core is provided with a plurality of cavities, and the area of the bonding interface of the magnetic beads and the aqueous solution is smaller than the surface area of the magnetic beads. The biotin marker is a water-soluble component, the higher the hydrophobicity of the surface of the magnetic bead is, the smaller the reaction area of the contact between the biotin marker and the surface of the magnetic bead is, and the weaker the binding efficiency between the biotin marker and the magnetic bead is.
In a preferred embodiment, the surface of the magnetic beads is modified by hydrophilization in order to increase the reaction area between the biotin labeling substance and the surface of the magnetic beads in the reaction system. Specifically, when the streptavidin-coated magnetic beads are prepared, a hydrophilic reagent is added into a reaction system for mixing reaction, so that hydrophilic groups are modified on the surfaces of the streptavidin-coated magnetic beads.
Through carrying out the hydrophilization modification to the magnetic bead surface, can make magnetic bead and biotin label have good reaction interface, improve the magnetic bead of streptavidin peridium and biotin label's binding rate, improve the detection signal to noise ratio, and then improve the sensitivity of kit, and difficult hardening under the condition of long-term storage, easily mixing.
Preferably, the hydrophilic agent is at least one of polyethyleneimine, polyethyleneimine derivatives, ethylenediamine ethanesulfonate, and ethylenediamine ethanesulfonate derivatives. The polyethyleneimine and the derivatives thereof, the ethylenediamine ethanesulfonate and the derivatives thereof have high solubility in solution and stable chemical properties, and have amino groups capable of being coupled with the AP enzyme.
In the embodiment of the invention, a hydrophilic reagent and streptavidin are simultaneously added into a magnetic bead suspension for reaction, so that streptavidin-labeled magnetic beads and magnetic bead hydrophilization modification are simultaneously carried out, a part of binding sites on the surfaces of the magnetic beads obtained after the reaction is terminated are connected with the streptavidin, a part of the binding sites are connected with hydrophilic groups, and a part of the binding sites are to be blocked.
In the embodiment of the invention, the mass ratio of the streptavidin to the hydrophilic reagent is (0.2-0.5): (0.02-0.1). When the mass ratio of the streptavidin to the hydrophilic reagent is less than 0.2:0.1, the coupling efficiency of the streptavidin and the magnetic beads is low, and the detection effect of the determination kit is influenced; when the mass ratio of the streptavidin to the hydrophilic reagent is greater than 0.5:0.02, the coupling efficiency of the streptavidin and the magnetic beads cannot be increased progressively, so that resource waste is caused.
In the embodiment of the present invention, the reaction conditions for reacting the magnetic beads with streptavidin are as follows: reacting for 1-3 h at room temperature. When the reaction time is less than 1h, the coupling efficiency of the magnetic beads and the streptavidin is low, and the detection effect of the detection kit is influenced; when the reaction time is longer than 3h, the reaction time is too long, and the coupling efficiency of the magnetic beads and the streptavidin cannot be increased progressively, so that resource waste is caused.
It is noted that room temperature for the examples of the present invention means 25. + -. 5 ℃. The coupling effect of the magnetic beads and the streptavidin at room temperature in the embodiment of the invention is better than that at 4 ℃ and 37 ℃.
In the step S02, the detection antibody is coupled to alkaline phosphatase to obtain an enzyme-labeled antibody, and during the detection process, the antigen in the sample to be detected and the biotin-labeled vitamin B12 competitively bind to the enzyme-labeled antibody to form a "magnetic bead-coated antigen-AP-labeled antibody" complex. After the unbound antibody is removed by washing, adding a luminous substrate solution of AP, catalyzing the luminous substrate by the AP on the compound, continuously and stably emitting photons, recording the emitted photons by a system, and converting the photon energy intensity on a standard curve into the concentration of the antigen to be detected by software. Wherein, the detection antibody can be selected from but not limited to intrinsic factor protein, vitamin B12 monoclonal antibody or vitamin B12 polyclonal antibody.
In the present embodiment, the process of coupling the detection antibody to alkaline phosphatase and preparing the detection antibody labeled with alkaline phosphatase in step S02 can be performed by a method conventionally used in the art. The method specifically comprises the following steps: adding an alkaline phosphatase solution (200-500 mu L, 5.0mg/mL) into the detection antibody, uniformly mixing, carrying out a light-shielding reaction at room temperature for 2h, adding a Tris buffer solution (0.1M, pH7.4) containing 1% BSA, and carrying out a blocking reaction at room temperature for 30min to block unbound sites on the detection antibody.
In step S02, before the detection antibody is coupled to the alkaline phosphatase, the detection antibody must be activated by an activation treatment in order to improve the enzyme labeling efficiency. The method of detecting the treatment for antibody activation can be performed by referring to the conventional technical means in the art, for example, the activation treatment is performed by using an activating agent such as EDC, NHS, etc.
Preferably, the mass ratio of the alkaline phosphatase to the detection antibody is 5:1 to 10: 1. When the mass ratio of the alkaline phosphatase to the detection antibody is less than 5:1, the coupling efficiency of the alkaline phosphatase and the detection antibody is low, and the detection effect of the determination kit is influenced; when the mass ratio of alkaline phosphatase to detection antibody is greater than 10:1, waste of resources is caused.
As a preferred embodiment, in order to improve the stability of the enzyme-labeled antibody, the present example uses an organophosphate compound as a phosphorylation modification raw material reversibly linked to the alkaline phosphatase active site by a chemical force, and improves the conformational stability without affecting the catalytic activity of the alkaline phosphatase-labeled detection antibody, thereby improving the stability of the assay kit.
Preferably, the organic phosphate compound is one or more of diethyl phosphate, dimethyl phosphate, triethyl phosphate, and dimethyl phosphate and derivatives thereof. Diethyl phosphate, dimethyl phosphate, triethyl phosphate and dimethyl phosphate and derivatives thereof form dissociable conjugates with the AP enzyme. In the bound state, the thermostability of the AP enzyme is significantly increased.
In the embodiment of the invention, the mass ratio of the alkaline phosphatase to the organic phosphate compound is 100 (3-5). When the mass ratio of the alkaline phosphatase to the organic phosphate compound is less than 100:5, the phosphorylation modification degree of the detection antibody marked by the alkaline phosphatase is insufficient, and the thermal stability effect of the detection kit is general; when the mass ratio of the alkaline phosphatase to the organophosphate compound is greater than 100:3, resources are wasted.
In the present example, the time for phosphorylation modification is 30min or more. The phosphorylation modification time is less than 30min, which affects the phosphorylation modification degree of the detection antibody marked by alkaline phosphatase, and the stability is low.
In step S03, the affinity between biotin and streptavidin is extremely strong, and when detection is performed, biotin-labeled vitamin B12 binds to streptavidin-coated magnetic beads to form magnetic bead-coated antigens.
In the present embodiment, biotin means biotin subjected to activation treatment. The method of activation treatment according to the embodiments of the present invention may be performed by referring to the conventional technical means in the art, for example, activation treatment is performed by using an activating agent such as EDC, NHS, or the like.
As a preferable embodiment, in order to overcome the problem that VB12 is sensitive to light, β -cyclodextrin is added into biotin-labeled vitamin B12, β -cyclodextrin has a unique cyclic structure, the photolysis effect of VB12 can be inhibited to a certain extent, the stability of VB12 is improved, and the stability of the detection kit is further improved.
The biotin-labeled vitamin B12 in the embodiment of the invention is an aqueous solution, and β -cyclodextrin has good solubility in water.
Preferably, in the biotin-labeled vitamin B12 solution, the mass percentage of β -cyclodextrin is 1% -5%, when the mass percentage of β -cyclodextrin is lower than 1%, the photolysis effect of VB12 is not obviously reduced, and when the mass percentage of β -cyclodextrin is higher than 5%, resources are wasted.
In summary, the embodiment of the invention performs phosphorylation modification on the detection antibody labeled by alkaline phosphatase, performs hydrophilization modification on magnetic beads coated by streptavidin, and adds β -cyclodextrin into the biotin-labeled VB12 solution, thereby greatly improving the stability of the VB12 assay kit in the embodiment of the invention, integrally reducing the signal-to-noise ratio of detection, improving the sensitivity, reducing the batch difference and improving the detection accuracy.
The embodiment of the invention also provides a vitamin B12 determination kit, which comprises: magnetic separation reagent, antibody reagent and enzyme labeling reagent;
the magnetic separation reagent comprises magnetic beads coated with streptavidin;
the enzyme labeling reagent comprises an enzyme labeling antibody compound, and the enzyme labeling antibody compound is a product of alkaline phosphatase-labeled detection antibody which is subjected to phosphorylation modification by an organic phosphate compound;
the antigen reagent comprises biotin-labeled VB12 and a biotin-labeled working solution.
In a preferred embodiment, the surface of the streptavidin-coated magnetic bead is further modified with a hydrophilic group, and the hydrophilic group is a polyethyleneimine group or an ethylenediamine ethanesulfonic group.
As a preferred embodiment, the biotin marker working solution also contains β -cyclodextrin.
Furthermore, each liter of biotin marker working solution comprises 0.03-0.06 mol of Tris, 8-12 g of β -cyclodextrin, 8-12 g of BSA, 200.8-1.2 g of Tween-2, 7-11 g of NaCl and 3000.3-0.7 g of proclin.
Furthermore, the working concentration of the biotin-labeled VB12 in the antigen reagent is preferably 50 ng/mL-500 ng/mL. When the working concentration of the biotin-labeled VB12 is less than 50ng/mL, the RLU value of the determination kit is too low, and the linear relation is poor; when the working concentration of the biotin-labeled VB12 is more than 500ng/mL, the sensitivity of the assay kit is lower.
In the VB12 assay kit, the magnetic separation reagent further comprises a magnetic bead working solution, and the working concentration of the magnetic beads in the magnetic separation reagent is preferably 0.1-0.5 mg/mL. When the working concentration of the magnetic beads is less than 0.1mg/mL, the RLU value of the determination kit is too low, and the linear relation is poor; when the working concentration of the magnetic beads is more than 0.5mg/mL, the sensitivity of the determination kit is low.
Further, each liter of magnetic bead working solution comprises: 0.03-0.06 mol of Tris; 8-12 g of gelatin; 8-12 g of BSA; 200.8-1.2 g of tween-1; 7-11 g of NaCl; proclin 3000.3-0.7 g.
In the VB12 determination kit, the enzyme labeling reagent further comprises an enzyme label working solution, and the working concentration of a detection antibody marked by alkaline phosphatase in the enzyme labeling reagent is preferably 1-10 ug/mL. When the working concentration of the detection antibody marked by alkaline phosphatase is less than 1ug/mL, the RLU value of the detection kit is too low, and the linear relation is poor; when the working concentration of the detection antibody marked by alkaline phosphatase is higher than 10ug/mL, the sensitivity of the determination kit is lower.
Further, each liter of enzyme marker working solution comprises: 0.03-0.06 mol of MES; 8-12 g of BSA; 200.8-1.2 g of tween-1; 7-11 g of NaCl; MgCl20.8～1.2mmol；ZnCl23～7mmol；Proclin3000.3～0.7g。
In the VB measuring kit, the organic phosphate compound is one or more of diethyl phosphate, dimethyl phosphate, triethyl phosphate, dimethyl phosphate and derivatives thereof;
in the detection antibodies marked by alkaline phosphatase, the detection antibodies are endoglin protein, a vitamin B12 monoclonal antibody or a vitamin B12 polyclonal antibody;
the magnetic beads are carboxyl magnetic beads, amino magnetic beads, sulfonic magnetic beads or hydroxyl magnetic beads.
When the kit provided by the embodiment of the invention is used for measuring VB12 in a sample to be measured, biotin-labeled vitamin B12 is connected with streptavidin-coated magnetic beads, a sample to be measured and an enzyme-labeled antibody compound are added, and the mixture is uniformly mixed and incubated to obtain a compound; and carrying out chemiluminescence reaction on the compound, detecting the luminous intensity of the compound by adopting a luminous detector, and calculating the content of procalcitonin in the sample to be detected.
In order to make the technical problems, technical solutions and advantageous effects to be solved by the present invention more apparent, the present invention is further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
The embodiment provides a vitamin B12 determination kit, which is prepared by the following steps:
1. avidin coated magnetic bead
Taking 10mg carboxyl magnetic beads (the particle size is 2.80 mu m), adding 2mL MES (0.02mol/L, pH6.0) buffer solution for resuspension, adding 20 mu L newly configured 10mg/mL EDC aqueous solution, and activating the carboxyl on the surfaces of the magnetic beads for 30 min; then, 0.5mg of Streptavidin (SA) and 0.1mg of ethylenediamine ethanesulfonic acid sodium (AAS) are added, suspended for 3 hours at room temperature, magnetically separated and supernatant removed; subsequently, Tris buffer (0.1M, pH7.4) containing 1% BSA was further added thereto, and the reaction was continued at room temperature for 3 hours, followed by magnetic separation and removal of the supernatant to obtain streptavidin-coated magnetic beads.
When the magnetic separation reagent is used, the magnetic beads coated with the streptavidin are added into the magnetic bead working solution, the magnetic beads are uniformly mixed, the working concentration of the magnetic beads is adjusted to be 0.2mg/mL, and the magnetic separation reagent R1 is obtained.
The formula of the magnetic bead working solution is as follows: 100mL of Tris (0.05mol/L, pH7.4); 1g of gelatin; BSA1 g; tween-200.1 g; NaCl 0.9 g; proclin 3000.05g.
2. Alkaline phosphatase labeled detection antibody
Taking the intrinsic factor protein as a detection antibody, uniformly mixing the intrinsic factor protein (100 mu L, 1.0mg/mL) and a PBS buffer solution (250 mu L,0.1 mol/L, pH 7.0), adding a newly-prepared EDC aqueous solution (20 mu L, 10mg/mL), and activating for 30 min; then, adding alkaline phosphatase solution (200 mu L, 5.0mg/mL), mixing uniformly, and reacting for 2h at room temperature in a dark place; then, Tris buffer (250. mu.L, 0.1M, pH7.4) containing 1% BSA was added for blocking reaction at room temperature for 30 min; then, adding 250 microliter of diethyl phosphate with the mass percentage concentration of 0.5 percent, and continuing the reaction for 30min at room temperature; finally, centrifugal purification was performed using a 30KD ultrafiltration column, half the volume of glycerol was added and stored at-20 ℃.
When in use, the detection antibody marked by alkaline phosphatase is added into the working solution of the enzyme marker, and the concentration of the working solution of the AP enzyme is kept at 5ug/ml, thus obtaining the enzyme-labeled reagent R2.
The formula of the enzyme marker working solution is as follows: 100mL of MES (0.05mol/L, pH 6.5); BSA1 g; tween-200.1 g; NaCl 0.9 g; MgCl2(1mmol/L)；ZnCl2(5mmol/L)；Proclin300 0.05g。
3. Biotin marker VB12
Activating biotin (100 mu L, 5.0mg/mL), adding PBS buffer (200 mu L,0.1 mol/L, pH8.5), mixing, adding newly-prepared VB12 aqueous solution (10 mu L, 5.0mg/mL), and reacting at room temperature in a dark place for 2 h; then, centrifugal purification is carried out by adopting a desalting column, liquid of the first peak is collected, half of glycerol is added, and the mixture is stored at the temperature of minus 20 ℃ for standby.
When the antigen reagent is used, the VB12 marked by the biotin is added into a biotin marker working solution, and the working concentration of VB12 is kept at 100ng/mL, so that the antigen reagent R3 is obtained.
Wherein the formula of the biotin marker working solution is 100mL of Tris (0.05mol/L, pH7.4), 1g of β -cyclodextrin, 1g of BSA, 200.1g of Tween, 0.9g of NaCl and 3000.05g of Proclin.
4. Preparation of vitamin B12 calibration product
VB12 was prepared in VB12 solutions at concentrations of 0pg/mL, 75pg/mL, 150pg/mL, 300pg/mL, 750pg/mL and 1500pg/mL using calibrator buffer, and the solutions were dispensed into bottles of 0.5mL and stored at 4 ℃ until use.
The formula of the calibrator buffer solution comprises 100mL of Tris (0.05mol/L, pH7.4), 1g of β -cyclodextrin, 1g of BSA, 1g, 200.1g of Tween, 0.9g of NaCl and 3000.05g of Proclin.
5. Preparation of sample treatment solution
The formula of the sample treatment fluid is as follows: MES (50mmol/L)100 mL; dithiothreitol (DTT, 10 mmol/L); KCN0.1g; proclin 3000.05g; pH 5.5.
When the kit is used, a sample to be detected is mixed with a sample treatment solution, then a magnetic separation reagent R1 and an antigen reagent R3 are added, and the mixture is mixed, and then an enzyme-labeled reagent R2 is added.
Comparative example 1
The present comparative example provides a vitamin B12 assay kit, the preparation of which comprises the steps of:
1. avidin coated magnetic bead
Taking 10mg carboxyl magnetic beads (the particle size is 2.80 mu M), adding 0.02M MES buffer solution with the pH value of 6.0 for resuspension, adding 20 mu L of newly configured 10mg/mL EDC aqueous solution, and activating the carboxyl on the surfaces of the magnetic beads for 30 min; then, 0.5mg of Streptavidin (SA) is added, and the mixture is suspended for 3 hours at room temperature, magnetically separated and supernatant is removed; subsequently, Tris buffer (0.1M, pH 7.4.4) containing 1% BSA was further added thereto and the reaction was continued at room temperature for 3 hours, followed by magnetic separation and removal of the supernatant to obtain streptavidin-coated magnetic beads.
When the magnetic separation reagent is used, the magnetic beads coated with the streptavidin are added into the magnetic bead working solution, the magnetic beads are uniformly mixed, the working concentration of the magnetic beads is adjusted to be 0.2mg/mL, and the magnetic separation reagent r1 is obtained.
The formula of the magnetic bead working solution is as follows: 100mL of Tris (0.05mol/L, pH7.4); 1mg of gelatin; BSA1 mg; tween-200.1 mg; NaCl 0.9 mg; proclin 3000.05mg.
2. Alkaline phosphatase labeled detection antibody
Taking the intrinsic factor protein as a detection antibody, uniformly mixing the intrinsic factor protein (100 mu L, 1.0mg/mL) and a PBS buffer solution (250 mu L,0.1 mol/L, pH 7.0), adding a newly-prepared EDC aqueous solution (20 mu L, 10mg/mL), and activating for 30 min; then, adding alkaline phosphatase solution (200 mu L, 5.0mg/mL), mixing uniformly, and reacting for 2h at room temperature in a dark place; then, Tris buffer (0.1M, pH7.4) containing 1% BSA was added for blocking reaction at room temperature for 30 min; finally, centrifugal purification was performed using a 30KD ultrafiltration column, half the volume of glycerol was added and stored at-20 ℃.
When in use, the detection antibody marked by alkaline phosphatase is added into the working solution of the enzyme marker and diluted according to the dilution ratio of 1:800 to obtain the enzyme-labeled reagent r 2.
The formula of the enzyme marker working solution is as follows: 100mL of MES (0.05mol/L, pH 6.5); BSA1 g; tween-200.1 g; NaCl 0.9 g; MgCl2(1mmol/L)；ZnCl2(5mmol/L)；Proclin300(0.05g)。
3. Biotin marker VB12
Activating biotin (100 mu L, 5.0mg/mL), adding PBS buffer (200 mu L,0.1 mol/L, pH8.5), mixing, adding newly-prepared VB12 aqueous solution (10 mu L, 5.0mg/mL), and reacting at room temperature in a dark place for 2 h; then, centrifugal purification is carried out by adopting a desalting column, liquid of the first peak is collected, half of glycerol is added, and the mixture is stored at the temperature of minus 20 ℃ for standby.
When in use, the VB12 marked by the biotin is added into the biotin marker working solution, and the mixture is added according to the VB12 working concentration of 100ng/ml, so that the antigen reagent r3 is obtained.
The formula of the biotin marker working solution is as follows: tris (0.05mol/L, pH7.4); BSA1 g; tween-200.1 g; NaCl 0.9 g; proclin 3000.05g.
The rest of the process is basically the same as that of embodiment 1, and the description thereof is omitted.
Example 2
The present comparative example differs from comparative example 1 in that: in step 1, 0.5mg of streptavidin was added along with 0.1mg of AAS during the coating of the beads with avidin.
The rest of the process is substantially the same as that of comparative example 1, and the description thereof is omitted.
Example 3
The present comparative example differs from comparative example 1 in that: in step 2, during the process of labeling the detection antibody with alkaline phosphatase, 0.5% diethyl phosphate is added between the blocking reaction and the centrifugal purification, and the reaction is continued for 30min at room temperature.
The rest of the process is substantially the same as that of comparative example 1, and the description thereof is omitted.
Example 4
The difference between the comparative example and the comparative example 1 is that in the process of marking VB12 by biotin in the step 3, the biotin-marked working solution also contains 1 percent of β -cyclodextrin.
The rest of the process is substantially the same as that of comparative example 1, and the description thereof is omitted.
Test example
1. The vitamin B12 assay kits of comparative example 1 and example 2 were used for detection by chemiluminescence, and Table 1 shows the results. As shown in a detection result, the surface of the magnetic bead is subjected to hydrophilic modification by adopting AAS, so that the signal to noise ratio of a detection signal value can be remarkably improved, and the sensitivity of the VB12 determination kit in the embodiment of the invention is higher.
TABLE 1
2. And (3) taking the vitamin B12 determination kits of the comparative example 1 and the example 3, detecting by a chemiluminescence method, wherein the determination temperature is 2-8 ℃, and recording the low-temperature detection value. Then, the VB12 measurement kits of comparative example 1 and example 3 were subjected to a test after being accelerated at 37 ℃ for 7 days, the detection values after the high-temperature treatment were recorded, and the deviation of the detection values after the high-temperature acceleration from the original detection values was calculated, and table 2 shows the detection results.
As shown in the results, the VB12 assay kit provided by the embodiment of the invention has the advantage that the stability is remarkably improved after the enzyme-labeled detection antibody is modified by adopting an organic phosphate compound.
TABLE 2
3. The vitamin B12 assay kits of comparative example 1 and example 4 were used for detection by chemiluminescence in the dark, and the detection values were recorded under dark conditions. Then, the vitamin B12 measurement kits of comparative example 1 and example 4 were placed under an LED lamp and directly irradiated for 24 hours, followed by detection by chemiluminescence, and the detection values after light treatment were recorded, and the deviation of the detection values after light treatment from the detection values under a dark condition was calculated, and table 3 shows the detection results.
As shown in the detection result, the β -cyclodextrin is added to the biotin-labeled working solution in the embodiment of the invention, so that the photolysis effect of the detection kit can be reduced, and the stability of the VB12 detection kit in the embodiment of the invention under illumination can be improved.
TABLE 3
4. The vitamin B12 assay kit of example 1 was used to test its sensitivity, precision, stability and clinical sample compliance by chemiluminescence.
1) Sensitivity detection
The sensitivity of the assay kit of VB12, an example of the invention, was measured to be 20pg/mL, with reference to the protocol recommended by the CLSI EP17-A document.
2) Detection of precision
Two VB12 samples with the concentrations of 100pg/mL and 1000pg/mL are taken, 3 batches of VB12 determination kits prepared at different times are adopted, the detection is repeated for three times each time, the average value is taken, then the intra-batch and inter-batch differences of the VB12 determination kit in the embodiment of the invention are calculated, and the result shows that the intra-batch and inter-batch differences of the kit are both less than 8%.
3) Stability detection
The VB12 determination kit provided by the embodiment of the invention is used for quality control test by adopting a full-automatic chemiluminescence apparatus, the quality control test is carried out once a day and continuously for 40 times, the quality control results all conform to the range again, and the mechanical stability is improved to 40 days, so that the VB12 determination kit provided by the embodiment of the invention is excellent in storage stability.
The VB12 determination kit provided by the embodiment of the invention is placed at 37 ℃ for acceleration for 7 days and then tested, the numerical value is reduced within 5%, the thermal stability is very good, and the influence of some unexpected factors on the performance of the reagent in the transportation and use processes can be avoided.
4) Clinical sample compliance testing
200 samples provided by a medical institution in the city are taken and tested by adopting the VB12 determination kit and the Beckman VB12 determination kit, and FIG. 1 is a correlation fitting curve of the VB12 determination kit and the Beckman VB12 determination kit, and the correlation is as follows: 0.9867, K value 0.9734, the correlation was very good.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (10)
1. A preparation method of a vitamin B12 determination kit is characterized by comprising the following steps:
reacting the magnetic beads with streptavidin to prepare streptavidin-coated magnetic beads;
coupling the detection antibody with alkaline phosphatase to prepare a detection antibody marked by the alkaline phosphatase; then, carrying out phosphorylation modification on the detection antibody marked by the alkaline phosphatase by adopting an organic phosphate compound to obtain an enzyme-marked antibody compound;
biotin was coupled to vitamin B12 to prepare biotin-labeled vitamin B12.
2. The method according to claim 1, wherein the mass ratio of the alkaline phosphatase to the organophosphate compound is 100 (3-5).
3. The method according to claim 1 or 2, wherein the time for the phosphorylation modification is 30min or more; and/or
The organic phosphate compound is one or more of diethyl phosphate, dimethyl phosphate, triethyl phosphate, dimethyl phosphate and derivatives thereof; and/or
The detection antibody is an intrinsic factor protein, a vitamin B12 monoclonal antibody or a vitamin B12 polyclonal antibody.
4. The preparation method according to any one of claims 1 to 3, wherein, in the preparation of streptavidin-coated magnetic beads, a hydrophilic reagent is added to the reaction system for mixing reaction, so that the surfaces of the streptavidin-coated magnetic beads are modified with hydrophilic groups;
the hydrophilic agent is at least one of polyethyleneimine, polyethyleneimine derivatives, ethylenediamine ethanesulfonate and ethylenediamine ethanesulfonate derivatives.
5. The method according to claim 4, wherein the mass ratio of the streptavidin to the hydrophilic agent is (0.2-0.5): (0.02-0.1); and/or
The magnetic beads are carboxyl magnetic beads, amino magnetic beads, sulfonic magnetic beads or hydroxyl magnetic beads; and/or
The reaction conditions for reacting the magnetic beads with streptavidin are as follows: reacting for 1-3 h at room temperature.
6. The method of any one of claims 1 to 3, wherein β -cyclodextrin is added to said biotin-labeled vitamin B12.
7. A vitamin B12 assay kit, comprising: magnetic separation reagent, antibody reagent and enzyme labeling reagent;
the magnetic separation reagent comprises magnetic beads coated with streptavidin;
the enzyme-labeled reagent comprises an enzyme-labeled antibody compound, and the enzyme-labeled antibody compound is a product obtained by carrying out phosphorylation modification on an alkaline phosphatase-labeled detection antibody by an organic phosphate compound;
the antigen reagent comprises biotin-labeled vitamin B12 and a biotin-labeled working solution.
8. The vitamin B12 assay kit of claim 7, wherein the streptavidin-coated magnetic beads are modified with hydrophilic groups, and the hydrophilic groups are polyethyleneimine groups or ethylenediamine-based ethanesulfonic acid groups; and/or
The biotin marker working solution contains β -cyclodextrin.
9. The vitamin B12 assay kit according to claim 8, wherein each liter of the biotin-marker working solution comprises 0.03-0.06 mol of Tris, 8-12 g of β -cyclodextrin, 8-12 g of BSA, 200.8-1.2 g of Tween-2, 7-11 g of NaCl, and 3000.3-0.7 g of Proclin.
10. A method for detecting vitamin B12, comprising:
providing a vitamin B12 assay kit according to any one of claims 7 to 9, connecting biotin-labeled vitamin B12 to streptavidin-coated magnetic beads, adding a sample to be tested and an enzyme-labeled antibody complex, mixing uniformly, and incubating to obtain a complex;
and carrying out chemiluminescence reaction on the compound, detecting the luminous intensity of the compound by adopting a luminescence detector, and calculating the content of VB12 in a sample to be detected.
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