CN111781386A - Antibody-coated magnetic particle buffer solution, gastrin 17 determination kit and preparation method thereof - Google Patents
Antibody-coated magnetic particle buffer solution, gastrin 17 determination kit and preparation method thereof Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/575—Hormones
- G01N2333/595—Gastrins; Cholecystokinins [CCK]
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Abstract
The invention provides a magnetic particle buffer solution for coating an antibody, a gastrin 17 determination kit and a preparation method thereof, belonging to the technical field of immunodetection, wherein the formula of the magnetic particle buffer solution for coating the antibody is as follows: 20-100mmol/L of 3- (N-morpholinyl) -2-hydroxypropanesulfonic acid, 150-300mmol/L of sodium chloride, 0.2-1% of complexing agent, 0.5-2% of bovine serum albumin, 0.03-0.1% of ProClin300, and the pH value is 7.0-8.0, wherein the complexing agent is one or two of EGTA and BAPTA. The kit comprises: an anti-gastrin 17 antibody-alkaline phosphatase marker reagent R2 and a reagent M obtained by solubilizing the anti-gastrin 17 antibody-coated magnetic microparticles with the antibody-coated magnetic microparticle buffer. The invention can effectively eliminate the problem of test difference between new and old samples caused by unstable G17 samples.
Description
Technical Field
The invention belongs to the technical field of immunodetection, and relates to a coated antibody magnetic particle buffer solution, a gastrin 17 determination kit and a preparation method thereof.
Background
Gastrin 17(G-17) is amidated gastrin synthesized and secreted by G cells of antrum, and is a polypeptide consisting of 17 amino acid residues, mainly functions to stimulate gastric acid secretion, promote proliferation and differentiation of gastric mucosal cells, and its content in human body accounts for 80-90% of the total amount of bioactive gastrin. In healthy individuals, G17 serum concentrations were very low, only 1-7 pmol/L. G-17 is a sensitive index for reflecting the secretion function of the antrum of the stomach and has an important position in diagnosing and screening atrophic gastritis and gastric cancer. The G-17 assay helps to determine the presence, location and extent of atrophy. In patients with atrophic gastric body, the acid secretion glands are reduced, and the stomach is in a low gastric acid state, resulting in an increase in serum gastrin G17 level; in patients with antral atrophy, the number of G cells is reduced, and the level of serum gastrin G-17 is reduced; in patients with total gastric atrophy (multifocal atrophy), G-17 is reduced. Most scholars believe that fasting serum G-17 of less than 1pmol/L can be used as a threshold for atrophic gastritis in the antrum.
At present, enzyme-linked immunoassay is mainly used as a detection method of G17, such as G-17 assay kit (enzyme-linked immunoassay) manufactured by Biohit company, which takes long time and is tedious to operate, and has narrow linear range (0.5-40pmol/L), and samples higher than 40pmol/L need pre-dilution to be tested. G17 is extremely easy to degrade, the sample can hardly be stored under the condition that the plasma is not separated, and the concentration of G17 is still greatly reduced even if the stability of the plasma is prolonged by 2-3d at low temperature; the stability of the plasma separation group G17 is still low, and the stability of the plasma separation group G17 is only maintained for 5-6 days at the temperature of 4 ℃ and-20 ℃, so that the existing detection kit has the problem of test difference between a new sample and an old sample due to the instability of a G17 sample.
Therefore, how to prepare a buffer solution and a detection kit for magnetic particles coated with antibodies for eliminating the difference between the measured values of the new and old gastrin 17 samples is a technical problem to be solved.
Disclosure of Invention
In order to solve the technical problem of the test difference of new and old samples caused by the instability of the G17 sample, the invention provides a magnetic particle buffer solution, a gastrin 17 determination kit and a preparation method thereof, and the method can eliminate the test value difference of the new and old samples of the gastrin 17.
The invention is realized by the following technical scheme:
one of the objectives of the present invention is to provide an antibody-coated magnetic particle buffer solution, wherein the formulation of the antibody-coated magnetic particle buffer solution is as follows:
20-100mmol/L of 3- (N-morpholinyl) -2-hydroxypropanesulfonic acid, 150-300mmol/L of sodium chloride, 0.2-1% of complexing agent, 0.5-2% of bovine serum albumin, 0.03-0.1% of ProClin300, and the pH value is 7.0-8.0, wherein the complexing agent is one or two of EGTA and BAPTA.
Further, the mass ratio of the EGTA to the BAPTA is (0.8-1.2): (0.8-1.2).
Another object of the present invention is to provide a gastrin 17 assay kit, comprising: reagent M and anti-gastrin 17 antibody-alkaline phosphatase marker reagent R2;
and the reagent M is used for dissolving the magnetic particles coated with the anti-gastrin 17 antibody by using the magnetic particle buffer solution coated with the antibody.
Further, the preparation method of the magnetic particles coated with the anti-gastrin 17 antibody comprises the following steps:
washing and re-suspending the magnetic bead particles to obtain a magnetic particle solution;
activating the magnetic particle solution by using EDC to obtain an activated magnetic particle solution;
and coating the activated magnetic particle solution with the gastrin 17 antibody to obtain the magnetic particles coated with the anti-gastrin 17 antibody.
Further, the obtaining of the magnetic particle solution includes: and taking magnetic bead particles, washing the magnetic bead particles for multiple times by using a buffer solution, and then re-suspending the magnetic bead particles by using the buffer solution to obtain a magnetic particle solution with the concentration of 1-4mg/ml, wherein the buffer solution is one or more of a phosphate buffer solution, a HEPES buffer solution, a carbonate buffer solution, a MES buffer solution and a boric acid buffer solution.
Further, the mass ratio of the EDC to the magnetic particles is 1 (90-110); the mass ratio of the gastrin 17 antibody to the magnetic particles is 1 (480-520).
Further, the coating of the activated magnetic particle solution with the gastrin 17 antibody comprises: adding a gastrin 17 antibody into the activated magnetic particle solution, uniformly mixing, adding a sealing liquid for sealing, uniformly mixing, performing magnetic separation to remove supernatant liquid to obtain magnetic particles coated with the anti-gastrin 17 antibody, adding a magnetic particle buffer solution, and uniformly mixing to obtain a magnetic particle reagent M coated with the anti-gastrin 17 antibody.
Further, the anti-gastrin 17 antibody-alkaline phosphatase marker reagent R2 is an anti-gastrin 17 antibody-alkaline phosphatase marker diluted by an enzyme-labeled antibody buffer solution;
the formula of the enzyme-labeled antibody buffer solution is as follows: 25-50mmol/L phosphate buffer solution, 0.005-0.01mol/L magnesium chloride, 0.05-0.2mmol/L zinc chloride, 150-300mmol/L sodium chloride, 0.5-2% bovine serum albumin, 0.03-0.1% ProClin300, and the pH is 6.0-7.5;
the anti-gastrin 17 antibody-alkaline phosphatase marker is obtained by reacting alkaline phosphatase, a gastrin 17 antibody and glutaraldehyde.
Further, the anti-gastrin 17 antibody-alkaline phosphatase marker is obtained by reacting alkaline phosphatase, a gastrin 17 antibody and glutaraldehyde, and specifically comprises:
dissolving the gastrin 17 antibody in a PBS (phosphate buffer solution) to obtain a gastrin 17 antibody solution with the concentration of 0.5-2 mg/ml;
adding alkaline phosphatase into the gastrin 17 antibody solution, uniformly mixing, adding glutaraldehyde, reacting completely, dialyzing to remove unreacted glutaraldehyde, and obtaining an anti-gastrin 17 antibody-alkaline phosphatase marker; the mass ratio of the alkaline phosphatase to the anti-gastrin 17 antibody is (8-12):1, and the final concentration of the glutaraldehyde is 0.1-0.4%.
One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:
the invention relates to a buffer solution for coating antibody magnetic particles, a gastrin 17 determination kit and a preparation method thereof, wherein any ion complexing agent of ethylene glycol bis (2-aminoethyl ether) tetraacetic acid (EGTA)/1, 2-bis (2-aminophenoxy) ethane-N, N, N' -tetraacetic acid (BAPTA) is added into the buffer solution for coating the antibody magnetic particles, and the ion complexing agents can chelate calcium and magnesium ions in a sample and avoid complement activation, so that the problem of test difference of new and old samples caused by instability of a G17 sample can be effectively solved.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.
FIG. 1 is a graph showing the correlation between the measured value of the reagent G17 of the present invention and the G17 concentration value of the Biohit enzyme-linked immunosorbent assay.
Detailed Description
The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.
Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is a conflict, the present specification will control.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.
In order to solve the technical problems, the embodiment of the invention provides the following general ideas:
according to an exemplary embodiment of the present invention, there is provided an antibody-coated magnetic particle buffer, wherein the antibody-coated magnetic particle buffer is formulated as:
20-100mmol/L of 3- (N-morpholinyl) -2-hydroxypropanesulfonic acid, 150-300mmol/L of sodium chloride, 0.2-1% of complexing agent, 0.5-2% of bovine serum albumin, 0.03-0.1% of ProClin300, and the pH value is 7.0-8.0, wherein the complexing agent is one or two of EGTA and BAPTA.
G-17 is a polypeptide consisting of 17 amino acid residues, and its half-life is usually only 6min, so that the G17 sample is easily degraded. The complement in the sample is activated by calcium and magnesium ions in the sample to form protein with enzyme activity, so that the G17 sample may be degraded to some extent. The buffer solution of the magnetic particles coated with the antibody usually comprises a buffer system, sodium chloride, a non-related protein, a preservative and the like, which cannot effectively solve the problem of test difference between a new sample and an old sample caused by instability of the G17 sample.
The comparison document CN109307776A shows that EDTA added to the sample diluent can chelate calcium and magnesium ions in blood and block the classical pathway of complement activation, and EDTA needs to be added to the sample diluent, so that the sample needs to be pretreated by the diluent, which is not favorable for full-automatic operation.
According to the invention, any one ion complexing agent of ethylene glycol bis (2-aminoethyl ether) tetraacetic acid (EGTA)/1, 2-bis (2-aminophenoxy) ethane-N, N, N' -tetraacetic acid (BAPTA) is added into a buffer solution of magnetic particles coated with antibodies, and the ion complexing agent can chelate calcium and magnesium ions in a sample, so that complement is prevented from being activated, and the problem of test difference of a new sample and an old sample caused by instability of a G17 sample can be effectively solved.
As a preferred embodiment, the mass ratio of EGTA and BAPTA is (0.8-1.2): (0.8-1.2). The applicant finds that both complexing agents EGTA and BAPTA simultaneously have the mass ratio of (0.8-1.2): (0.8-1.2), the effect is better than that of only 1 of the above-mentioned materials. The too high or too low mass ratio is not favorable for the chelation of calcium and magnesium ions in the sample, so that the problem of test difference of a new sample and an old sample caused by the instability of the G17 sample cannot be effectively solved.
According to another exemplary embodiment of the present invention, there is provided a gastrin 17 assay kit, comprising: reagent M and anti-gastrin 17 antibody-alkaline phosphatase marker reagent R2;
and the reagent M is used for dissolving the magnetic particles coated with the anti-gastrin 17 antibody by using the magnetic particle buffer solution coated with the antibody.
In a preferred embodiment, the method for producing the magnetic fine particles coated with the anti-gastrin 17 antibody comprises:
washing and re-suspending the magnetic bead particles to obtain a magnetic particle solution;
activating the magnetic particle solution by using EDC to obtain an activated magnetic particle solution;
and coating the activated magnetic particle solution with the gastrin 17 antibody to obtain the magnetic particles coated with the anti-gastrin 17 antibody.
Wherein the obtaining of the magnetic particle solution comprises: and taking magnetic bead particles, washing the magnetic bead particles for multiple times by using a buffer solution, and then re-suspending the magnetic bead particles by using the buffer solution to obtain a magnetic particle solution with the concentration of 1-4mg/ml, wherein the buffer solution is one or more of a phosphate buffer solution, a HEPES buffer solution, a carbonate buffer solution, a MES buffer solution and a boric acid buffer solution.
The mass ratio of the EDC to the magnetic particles is 1 (90-110), preferably 1: 100; when the mass ratio is increased and the use amount of EDC is excessive, the activation rate of the magnetic particles is increased, and the concentration of the activated magnetic particles is increased to easily cause the coagulation of the magnetic particles; when the mass ratio is decreased and the number of magnetic particles is too large, the activation rate of the magnetic particles is decreased, resulting in a decrease in the antibody coating rate.
The mass ratio of the gastrin 17 antibody to the magnetic particles is 1 (480-520), and preferably 1: 500. When the mass ratio is increased and the amount of the antibody is excessive, the antibody not coated on the magnetic particles forms free antibody, so that the long-term stability of the M reagent is easily reduced; when the mass ratio is decreased and the number of magnetic particles is too large, nonspecific adsorption between the magnetic particles is likely to occur, which in turn increases the background value of the test system.
The coating of the activated magnetic particle solution with the gastrin 17 antibody specifically comprises the following steps: adding a gastrin 17 antibody into the activated magnetic particle solution, uniformly mixing, adding a sealing liquid for sealing, uniformly mixing, performing magnetic separation to remove supernatant liquid to obtain magnetic particles coated with the anti-gastrin 17 antibody, adding a magnetic particle buffer solution, and uniformly mixing to obtain a magnetic particle reagent M coated with the anti-gastrin 17 antibody.
The anti-gastrin 17 antibody-alkaline phosphatase marker reagent R2 is an anti-gastrin 17 antibody-alkaline phosphatase marker diluted by adopting an enzyme-labeled antibody buffer solution;
the formula of the enzyme-labeled antibody buffer solution is as follows: 25-50mmol/L phosphate buffer solution, 0.005-0.01mol/L magnesium chloride, 0.05-0.2mmol/L zinc chloride, 150-300mmol/L sodium chloride, 0.5-2% bovine serum albumin, 0.03-0.1% ProClin300, and the pH is 6.0-7.5;
the anti-gastrin 17 antibody-alkaline phosphatase marker is obtained by reacting alkaline phosphatase, a gastrin 17 antibody and glutaraldehyde, and specifically comprises the following steps:
dissolving the gastrin 17 antibody in a PBS (phosphate buffer solution) to obtain a gastrin 17 antibody solution with the concentration of 0.5-2 mg/ml;
adding alkaline phosphatase into the gastrin 17 antibody solution, uniformly mixing, adding glutaraldehyde, reacting completely, dialyzing to remove unreacted glutaraldehyde, and obtaining an anti-gastrin 17 antibody-alkaline phosphatase marker;
the mass ratio of the alkaline phosphatase to the anti-gastrin 17 antibody is (8-12):1, when the mass ratio is increased and the dosage of the alkaline phosphatase is excessive, the background value of the system is increased, and the photon value of the high-value sample exceeds the upper limit of the measured value of the instrument, so that the testing precision of the high-value sample is influenced; when the mass ratio is reduced and there are too many antibodies, unlabeled free antibody competes with the alkaline phosphatase-anti-gastrin 17 antibody complex for the antigenic site, reducing the sensitivity of the low-end sample.
The final concentration of glutaraldehyde is 0.1-0.4%. Too high a concentration of glutaraldehyde may increase non-specific reactions, while too low a concentration may affect sensitivity.
A gastrin 17 assay kit of the present application will be described in detail below with reference to examples, comparative examples, and experimental data.
The present embodiment provides a gastrin 17 quantitative determination kit, which comprises a calibrator, a quality control material, and an anti-gastrin 17 antibody-alkaline phosphatase marker reagent R2And a magnetic particulate agent M coating the anti-gastrin 17 antibody.
1. The preparation method of the gastrin 17 calibrator and the gastrin 17 quality control product comprises the following steps:
and dissolving the gastrin 17 antigen by using a calibrator buffer, and configuring a gastrin 17 calibrator and a quality control product, wherein the calibrator comprises a known concentration of gastrin 17, and the concentration of gastrin 17 is in a range of 0-200 pmol/L. In specific embodiments, the concentration of gastrin 17 in the calibrator is 0, 5, 10, 50, 100, 200pmol/L, and the concentration of gastrin 17 in the quality control is 5pmol/L, 100pmol/L, respectively; wherein the calibrator buffer is prepared by adding 0.5-3% sorbitol, 0.5-3% trehalose, 0.5-1% bovine serum albumin, 150-300mmol/L sodium chloride and 0.03-0.1% ProClin into 4- (2-hydroxyethyl) piperazine-1-2-hydroxypropanesulfonic acid (HEPPSO) bufferTM300, the pH value is 6.0-8.0, and the product is prepared by stirring and dissolving and then treating with a 0.22 mu m filter membrane;
2. anti-gastrin 17 antibody-alkaline phosphatase marker reagent R2
The gastrin 17 antibody-alkaline phosphatase marker was dissolved in a buffer labeled with an enzyme antibody. Wherein the gastrin 17 antibody-alkaline phosphatase marker is prepared by dissolving 10mg of alkaline phosphatase in 1ml of PBS (0.01mol/L pH7.0) containing 1mg of anti-gastrin 17 antibody, adding 4ml of 1% glutaraldehyde solution to a final concentration of 0.2% after complete dissolution, dialyzing to remove excess glutaraldehyde, and storing at 4 ℃.
Wherein the buffer solution of the enzyme-labeled antibody contains 25-50mmol/L phosphate buffer solution, 0.005-0.01mol/L magnesium chloride, 0.05-0.2mmol/L zinc chloride, 150 mmol/L sodium chloride, 0.5-2% bovine serum albumin, 0.03-0.1% ProClinTM300, the pH value is 6.0-7.5, and the mixture is fully stirred until the mixture is completely dissolved to obtain the compound;
3. magnetic microparticle reagent M coated with anti-gastrin 17 antibody
(1) Preparation of magnetic microparticles coated with anti-gastrin 17 antibody:
cleaning: taking a proper amount of magnetic bead particles, washing the magnetic bead particles for 3 times by using 0.1mol/L MES, and then re-suspending the magnetic bead particles by using 0.1mol/L MES to ensure that the concentration of the magnetic bead particles is 2 mg/ml;
activation: taking the prepared EDC solution, adding the EDC solution into the resuspended magnetic particles according to the mass ratio of EDC to the magnetic particles of 1:100, and horizontally mixing for 20min at room temperature after uniformly mixing in a vortex manner;
coating: adding the gastrin 17 antibody to be coated into the activated magnetic particles according to the coating proportion of the gastrin 17 antibody to the magnetic particles in a mass ratio of 1:500, and horizontally and uniformly mixing for 12h at room temperature after uniformly mixing in a vortex manner;
sealing: adding 5% BSA solution into the magnetic particles, and horizontally and uniformly mixing for 2h at room temperature;
preservation: removing supernatant by magnetic separation, adding into magnetic bead preservation solution, and mixing.
(2) The preparation method of the buffer solution without complexing agent/containing complexing agent magnetic particles comprises the following steps:
the magnetic particles coated with the anti-gastrin 17 antibody were dissolved in a buffer solution of the antibody-coated magnetic particles.
The buffer solution containing the complexing agent magnetic particles is prepared by mixing 20-100mmol/L of 3- (N-morpholinyl) -2-hydroxypropanesulfonic acid (MOPSO), 150-300mmol/L of sodium chloride, 0.2-1% of the complexing agent, 0.5-2% of bovine serum albumin, 0.03-0.1% of ProClinTM300, fully stirring until the mixture is completely dissolved, and adjusting the pH value to 7.0-8.0 to obtain the product. In this example, the complexing agent is specifically 0.5% EGTA.
Example 2
The complexing agent for the buffer solution of the magnetic particles coated with the antibody in this example was 0.2% EGTA, and the other processes and components were the same as in example 1.
Example 3
The complexing agent of the buffer solution for magnetic particles coated with the antibody in this example was 1% EGTA, and the other processes and components were the same as in example 1.
Example 4
In this example, the mass ratio of the complexing agent in the buffer solution for coating the magnetic particles of the antibody was 1:1 EGTA and BAPTA, other processes and components were the same as in example 1.
Example 5
In this example, the mass ratio of the complexing agent in the buffer solution for coating the magnetic particles of the antibody was 0.8: 1.2 EGTA and BAPTA, the other processes and components being the same as in example 1.
Example 6
In this example, the mass ratio of the complexing agent in the buffer solution for coating the magnetic particles of the antibody was 1.2: 0.8 EGTA and BAPTA, other processes and components were the same as in example 1.
Comparative example 1
The complexing agent for the buffer of the magnetic particles coated with the antibody in this comparative example was 0.1% EGTA, and the other processes and components were the same as in example 1.
Comparative example 2
The complexing agent for the buffer of the magnetic particles coated with the antibody in this comparative example was 1.5% EGTA, and the other processes and components were the same as in example 1.
Comparative example 3
The complexing agent for the buffer of the magnetic particles coated with the antibody in this comparative example was 2.0% EGTA, and the other processes and components were the same as in example 1.
Comparative example 4
The buffer of the magnetic particles coated with the antibody in this comparative example was a buffer of magnetic particles without a complexing agent: 20-100mmol/L of 3- (N-morpholinyl) -2-hydroxypropanesulfonic acid (MOPSO), 150-300mmol/L of sodium chloride, 0.5-2% of bovine serum albumin, 0.03-0.1% of ProClinTM300, fully stirring until the mixture is completely dissolved, and adjusting the pH value to 7.0-8.0 to obtain the product; the other processes and components were the same as in example 1.
Test example 1
The performance evaluation was performed for examples 1 to 6 and comparative examples 1 to 4, and the specific evaluation method was as follows:
changes in the concentration values of single samples (4pmol/L) stored for 0-72h at room temperature were evaluated using buffer kits containing different concentrations of EGTA magnetic particles. The results are shown in tables 1 to 3.
TABLE 1 concentration change of G17 samples stored at room temperature for 0-72h measured by EGTA magnetic particle buffer kit containing different concentrations
Note: p <0.05 compared to group 0h
TABLE 2 EGTA magnetic particle buffer kit with different concentrations to determine the concentration change of G17 sample preserved at 2-8 deg.C for 0d-7d
Note: p <0.05 compared to group 0h
TABLE 3 change of concentration of G17 samples stored at-20 deg.C for 0d-28d measured by EGTA magnetic particle buffer kit with different concentrations
Note: p <0.05 compared to group 0h
The data in tables 1-3 show that:
the buffer solution of the magnetic particles in the comparative example 4 does not contain EGTA, when the concentration of the EGTA in the comparative example 1 is less than 0.2%, the P of the G17 sample is less than 0.05 when compared with the 0h under the conditions of room temperature of 12h/2-8 ℃, 3d/-20 ℃, 14d, and the concentration value is remarkably changed;
in examples 1-3, when the concentration of EGTA in the magnetic particle buffer solution is 0.2% -1.0%, the G17 sample has P >0.05 compared with 0h under the conditions of room temperature of 72h/2-8 ℃ and 7d/-20 ℃ and 28d, and the concentration value is not significantly changed;
in comparative examples 2-3, when the concentration of EDTA in the magnetic particle buffer is > 1.0%, the concentration of the G17 sample is not significantly changed compared to that in 0h under the conditions of room temperature of 72h/2-8 ℃ of 7d/-20 ℃ of 28d, and the concentration is >0.05, but the value is lower, the relative deviation is more than 10%, and it is likely that high concentrations of EGTA have an effect on magnesium chloride and zinc chloride in the enzyme diluent. Therefore, when the content of EGTA in the magnetic particle buffer solution is in the range of 0.2-1%, the sample difference can be eliminated, and the accuracy of the measured value is not influenced.
In examples 4 to 6, both complexing agents EGTA and BAPTA were used with the mass ratio of (0.8 to 1.2): (0.8-1.2) and the relative deviation of the measured values of the samples is within 1 percent, thereby improving the test accuracy and having better effect than that of only adding 1 of the samples (examples 1-3).
2. The buffer solution kit without complexing agent/containing 0.2-1% of complexing agent magnetic particles respectively evaluates the change of the concentration measured value of the clinical samples under different storage conditions and storage time of a plurality of samples:
8 clinical samples in a linear range of 0.5-200pmol/L are selected, and buffer reagent kits without complexing agent and containing 0.2-1% of magnetic particles of the complexing agent are respectively used for evaluating the change of the measured concentration value of the clinical samples under different storage conditions and storage time.
(1) The evaluation results of the kit of comparative example 4 are shown in tables 4 to 6.
TABLE 4-magnetic particle buffer kit without complexing agent to determine the change in concentration of the G17 samples stored for 0h to 72h at room temperature
| Time of day | Sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample 5 | Sample 6 | Sample 7 | Sample 8 |
| 0h | 0.98±0.06 | 1.81±0.09 | 3.45±0.14 | 11.99±0.60 | 35.66±1.78 | 52.40±2.10 | 81.27±4.06 | 127.52±5.10 |
| 1h | 0.85±0.03 | 1.78±0.09 | 3.56±0.14 | 11.86±0.59 | 33.48±2.34 | 56.18±3.37 | 92.36±6.47 | 121.94±6.10 |
| 4h | 0.92±0.05 | 1.61±0.06 | 4.70±0.33 | 11.00±0.66 | 43.85±2.19 | 57.57±2.30 | 88.13±6.17 | 148.20±5.93 |
| 8h | 0.71±0.04 | 1.41±0.07* | 3.58±0.14 | 12.08±0.48 | 35.26±2.47 | 53.95±2.70 | 96.34±6.74 | 150.95±10.57 |
| 12h | 0.68±0.03* | 1.69±0.08 | 3.72±0.22 | 10.56±0.42 | 48.36±2.42 | 66.26±3.31 | 102.37±7.17* | 167.51±8.38* |
| 24h | 0.69±0.03* | 1.64±0.10 | 3.41±0.17 | 8.95±0.63* | 47.01±3.29 | 65.97±2.64* | 105.56±6.33* | 166.51±6.66* |
| 48h | 0.67±0.03 | 1.52±0.09 | 4.96±0.30 | 9.87±0.49 | 49.58±2.97 | 78.12±3.12* | 118.72±5.94* | 156.43±10.95 |
| 72h | 0.66±0.03* | 1.38±0.08* | 4.95±0.25* | 8.73±0.44* | 51.05±2.04* | 80.03±4.80* | 118.89±7.13* | 164.64±11.52 |
Note: p <0.05 compared to group 0h
TABLE 5 magnetic particle buffer kit without complexing agent to determine the concentration change of G17 samples preserved for 0d-7d at 2-8 deg.C
| Time of day | Sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample(s)5 | Sample 6 | Sample 7 | Sample 8 |
| 0d | 0.71±0.04 | 1.32±0.05 | 3.52±0.25 | 12.31±0.86 | 49.34±3.45 | 78.04±4.68 | 122.29±7.34 | 127.21±6.36 |
| 1d | 0.69±0.03 | 1.52±0.08 | 3.38±0.20 | 11.15±0.45 | 42.20±2.11 | 79.44±3.18 | 109.55±6.57 | 147.60±5.90 |
| 2d | 0.74±0.05 | 1.49±0.07 | 4.53±0.18 | 11.33±0.45 | 45.74±1.83 | 62.92±2.52 | 112.15±6.73 | 130.32±5.21 |
| 3d | 0.69±0.04 | 1.73±0.12* | 3.84±0.27 | 11.24±0.67 | 46.74±1.87 | 76.39±3.06 | 85.57±5.99 | 148.14±10.37 |
| 4d | 0.75±0.03 | 1.65±0.10* | 3.97±0.16 | 10.44±0.42* | 38.19±1.91 | 71.89±3.59 | 96.68±4.83* | 135.63±9.49 |
| 5d | 0.89±0.04* | 1.89±0.13* | 4.71±0.19 | 10.21±0.51* | 33.74±2.36* | 73.77±5.16 | 84.77±4.24* | 162.29±6.49 |
| 6d | 0.97±0.04 | 1.86±0.07* | 4.98±0.35 | 8.50±0.34* | 37.88±2.65 | 57.14±2.29 | 90.83±5.45 | 152.44±7.62 |
| 7d | 0.97±0.07* | 1.92±0.08* | 4.91±0.25* | 8.28±0.33* | 33.62±1.68* | 56.22±3.94* | 84.03±5.88* | 187.75±13.14* |
Note: p <0.05 compared to group 0d
TABLE 6-magnetic particle buffer kit without complexing agent measurement of concentration change of G17 samples preserved at-20 deg.C for 0d-28d
Note: p <0.05 compared to group 0d
From the data in tables 4-6, it can be seen that: the G17 sample is stored for 8h at room temperature, compared with 0h, P is less than 0.05, and the concentration value is changed remarkably; the G17 sample is stored at 2-8 ℃ for 3d, compared with 0d, P is less than 0.05, and the concentration value is changed remarkably; the G17 sample is preserved at the temperature of minus 20 ℃ for 7d, compared with the 0d, the P is less than 0.05, and the concentration value is changed remarkably; therefore, when no complexing agent is added to the magnetic particle buffer, the difference of the measured value of the sample is observed when the sample is stored for 8h under the room temperature condition, the difference of the measured value of the sample is observed when the sample is stored for 3d under the condition of 2-8 ℃, and the difference of the measured value of the sample is observed when the sample is stored for 7d under the condition of-20 ℃.
(2) The evaluation results of the kit of example 1 are shown in tables 7 to 9.
TABLE 7-magnetic particle buffer kit of example 1 measurement of the change in concentration of G17 samples stored at room temperature for 0h to 72h
| Time of day | Sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample 5 | Sample 6 | Sample 7 | Sample 8 |
| 0h | 0.77±0.04 | 1.58±0.05 | 4.18±0.21 | 10.12±0.3 | 40.34±2.02 | 62.41±2.50 | 94.00±3.76 | 146.7±5.87 |
| 1h | 0.82±0.04 | 1.61±0.05 | 3.81±0.11 | 10.44±0.42 | 39.70±1.99 | 67.98±2.04 | 94.48±3.78 | 145.83±4.37 |
| 4h | 0.83±0.04 | 1.66±0.05 | 4.02±0.12 | 10.13±0.51 | 41.03±1.64 | 66.66±3.33 | 103.22±3.1 | 153.16±7.66 |
| 8h | 0.84±0.03 | 1.65±0.08 | 4.08±0.12 | 10.37±0.31 | 42.85±2.14 | 63.07±3.15 | 95.33±2.86 | 158.22±6.33 |
| 12h | 0.82±0.03 | 1.52±0.06 | 3.87±0.15 | 10.36±0.31 | 39.49±1.97 | 65.38±3.27 | 94.05±2.82 | 155.20±6.21 |
| 24h | 0.80±0.04 | 1.61±0.05 | 4.00±0.2 | 10.66±0.43 | 40.14±2.01 | 66.32±2.65 | 95.12±3.8 | 158.22±4.75 |
| 48h | 0.76±0.02 | 1.54±0.08 | 4.07±0.12 | 10.31±0.31 | 41.78±1.25 | 67.15±2.01 | 98.46±3.94 | 152.3±6.09 |
| 72h | 0.79±0.03 | 1.61±0.05 | 3.86±0.15 | 10.60±0.32 | 43.29±2.16 | 66.33±2.65 | 95.11±2.85 | 144.04±7.2 |
Note: p <0.05 compared to group 0h
TABLE 8-magnetic particle buffer kit of example 1 measurement of the change in concentration of G17 samples stored at 2-8 deg.C for 0d-7d
| Time of day | Sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample 5 | Sample 6 | Sample 7 | Sample 8 |
| 0d | 0.82±0.03 | 1.60±0.05 | 4.07±0.12 | 10.36±0.52 | 43.20±1.30 | 64.52±3.23 | 98.97±3.96 | 157.06±7.85 |
| 1d | 0.78±0.04 | 1.68±0.05 | 4.19±0.17 | 10.04±0.50 | 42.57±1.28 | 63.56±3.18 | 97.68±2.93 | 150.78±6.03 |
| 2d | 0.84±0.04 | 1.67±0.05 | 4.13±0.21 | 10.70±0.32 | 42.72±2.14 | 67.49±2.70 | 97.31±2.92 | 148.12±4.44 |
| 3d | 0.79±0.02 | 1.67±0.07 | 3.86±0.15 | 9.70±0.39 | 42.16±1.26 | 66.52±2.00 | 94.97±2.85 | 158.39±6.34 |
| 4d | 0.80±0.02 | 1.54±0.05 | 3.91±0.20 | 10.26±0.41 | 42.79±1.71 | 68.51±3.43 | 102.11±5.11 | 149.61±7.48 |
| 5d | 0.77±0.03 | 1.57±0.05 | 4.13±0.17 | 10.64±0.43 | 42.16±1.26 | 65.40±1.96 | 103.24±5.16 | 157.86±7.89 |
| 6d | 0.84±0.03 | 1.55±0.08 | 4.15±0.21 | 10.54±0.42 | 41.61±1.66 | 68.08±3.40 | 101.36±3.04 | 143.80±5.75 |
| 7d | 0.80±0.02 | 1.61±0.06 | 3.89±0.12 | 10.20±0.41 | 42.10±1.68 | 65.16±3.26 | 102.34±4.09 | 145.91±7.30 |
Note: p <0.05 compared to group 0d
TABLE 9 magnetic particle buffer kit of example 1 measurement of the change in concentration of G17 samples from 0d to 28d when stored at-20 deg.C
| Time of day | Sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample 5 | Sample 6 | Sample 7 | Sample 8 |
| 0d | 0.79±0.03 | 1.67±0.08 | 4.00±0.16 | 10.30±0.52 | 41.97±1.26 | 62.14±3.11 | 99.20±2.98 | 149.98±4.50 |
| 1d | 0.80±0.03 | 1.68±0.08 | 4.05±0.20 | 9.95±0.40 | 43.08±2.15 | 66.27±3.31 | 96.69±2.90 | 149.28±4.48 |
| 3d | 0.81±0.04 | 1.67±0.05 | 3.85±0.15 | 10.00±0.40 | 40.58±1.22 | 66.46±1.99 | 100.48±3.01 | 156.45±6.26 |
| 5d | 0.77±0.02 | 1.64±0.08 | 4.10±0.16 | 10.16±0.30 | 42.78±1.71 | 67.26±2.69 | 94.98±2.85 | 146.44±4.39 |
| 7d | 0.81±0.04 | 1.62±0.05 | 3.95±0.20 | 10.58±0.32 | 39.87±1.20 | 65.79±3.29 | 102.27±3.07 | 150.93±4.53 |
| 14d | 0.79±0.03 | 1.56±0.05 | 4.07±0.16 | 10.35±0.41 | 40.20±1.21 | 62.50±2.50 | 101.26±3.04 | 144.91±4.35 |
| 21d | 0.84±0.03 | 1.54±0.06 | 4.05±0.16 | 10.50±0.32 | 41.20±1.24 | 67.10±2.68 | 100.60±4.02 | 148.35±4.45 |
| 28d | 0.83±0.03 | 1.61±0.08 | 4.01±0.16 | 10.26±0.51 | 39.95±1.60 | 66.45±3.32 | 94.87±3.79 | 152.53±6.10 |
Note: p <0.05 compared to group 0d
From the data in tables 7 to 9, it can be seen that: the magnetic particle buffer kit of example 1 evaluates the change in concentration of clinical samples under different storage conditions and storage times:
the G17 sample is stored for 72h at room temperature, compared with 0h, P is greater than 0.05, and the concentration value of the sample has no significant change;
the G17 sample is preserved at 2-8 ℃ for 7d, compared with 0d, P is greater than 0.05, and the concentration value of the sample has no significant change;
the G17 sample is stored at the temperature of minus 20 ℃ for 28d, compared with 0d, P is greater than 0.05, and the concentration value of the sample has no significant change;
therefore, after 0.2-1% complexing agent is added into the magnetic particle buffer solution, the sample is respectively stored for 72h at room temperature, stored for 7d at 2-8 ℃ and stored for 28d at-20 ℃ without significant difference. The kit prepared by the magnetic particle buffer solution containing the complexing agent can eliminate the problem of difference of measured values of new and old gastrin 17 samples.
3. And (3) repeatability evaluation:
the kit of the comparative example 4 and the kit of the example 1 are respectively used for measuring a human-derived sample with high concentration and a human-derived sample with low concentration, the measurement is repeated for 10 times, and the Coefficient of Variation (CV) of the measurement result is less than or equal to 5.0 percent. The results are shown in Table 10.
TABLE 10-results of the measurement of the kit of comparative example 4 and example 1
From the data in table 10, it can be seen that: the magnetic particle buffer solution kit without the complexing agent in the comparative example 4 and the magnetic particle buffer solution kit containing the complexing agent in the example 1 are respectively tested repeatedly for 10 times, and the measured CV is less than 5 percent, which shows that the complexing agent is added to have no influence on the repeatability test
4. Sample correlation evaluation:
50 fresh serum samples were selected in a linear range of 0.5-200pmol/L and the correlation of the assay results were compared with the Biohit enzyme-linked immunoassay kit, which diluted and assayed over 40pmol/L samples using the methods described in the specification. The results are shown in Table 11 and FIG. 1.
TABLE 11 correlation of the reagents of the invention with Biohit enzyme-linked immunosorbent assay samples
The correlation analysis of the measured value of the inventive reagent G17 in Table 11 and the G17 concentration value of Biohit enzyme-linked immunosorbent assay is shown in the attached FIG. 1, and the results are as follows:
the regression linear equation is Y-1.0441X-2.4391;
coefficient of correlation R2＝0.9733；
Therefore, the G17 concentration value measured by the kit prepared by the invention has good correlation with the G17 concentration value measured by the Biohit enzyme-linked immunosorbent assay.
In conclusion, by adding any one or two complexing agents of 0.2-1% of EGTA/BAPT into the buffer solution of the magnetic particles coated with the antibody, the problem of test difference of new and old samples caused by instability of G17 samples can be effectively solved, and the addition of the complexing agents does not influence the repeatability performance of the reagents. The kit prepared by the magnetic particle buffer solution has good correlation with the measured value of the reagent of an import manufacturer.
Finally, it should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (10)
1. The magnetic particle buffer solution for coating the antibody is characterized by comprising the following formula:
20-100mmol/L of 3- (N-morpholinyl) -2-hydroxypropanesulfonic acid, 150-300mmol/L of sodium chloride, 0.2-1% of complexing agent, 0.5-2% of bovine serum albumin, 0.03-0.1% of ProClin300, and the pH value is 7.0-8.0, wherein the complexing agent is one or two of EGTA and BAPTA.
2. The antibody-coated magnetic particle buffer of claim 1, wherein the mass ratio of EGTA to BAPTA is (0.8-1.2): (0.8-1.2).
3. A gastrin 17 assay kit, comprising: reagent M and anti-gastrin 17 antibody-alkaline phosphatase marker reagent R2;
the reagent M is a magnetic microparticle coated with an anti-gastrin 17 antibody dissolved in the antibody-coated magnetic microparticle buffer according to any one of claims 1 to 2.
4. The gastrin 17 assay kit according to claim 3, wherein the magnetic fine particles coated with the anti-gastrin 17 antibody are prepared by:
washing and re-suspending the magnetic bead particles to obtain a magnetic particle solution;
activating the magnetic particle solution by using EDC to obtain an activated magnetic particle solution;
and coating the activated magnetic particle solution with the gastrin 17 antibody to obtain the magnetic particles coated with the anti-gastrin 17 antibody.
5. The gastrin 17 assay kit of claim 4, wherein the obtaining the solution of magnetic particles comprises: and taking magnetic bead particles, washing the magnetic bead particles for multiple times by using a buffer solution, and then re-suspending the magnetic bead particles by using the buffer solution to obtain a magnetic particle solution with the concentration of 1-4mg/ml, wherein the buffer solution is one or more of phosphate buffer solution, HEPES buffer solution, carbonate buffer solution, MES buffer solution and boric acid buffer solution.
6. The gastrin 17 assay kit of claim 4, wherein the mass ratio of EDC to the magnetic particles is 1 (90-110); the mass ratio of the gastrin 17 antibody to the magnetic particles is 1 (480-520).
7. The method for preparing the gastrin 17 assay kit according to claim 4, wherein the step of coating the activated magnetic particle solution with the gastrin 17 antibody specifically comprises: adding a gastrin 17 antibody into the activated magnetic particle solution, uniformly mixing, adding a sealing liquid for sealing, uniformly mixing, performing magnetic separation to remove supernatant liquid to obtain magnetic particles coated with the anti-gastrin 17 antibody, adding a magnetic particle buffer solution, and uniformly mixing to obtain a magnetic particle reagent M coated with the anti-gastrin 17 antibody.
8. The gastrin 17 assay kit according to claim 3, wherein the anti-gastrin 17 antibody-alkaline phosphatase marker reagent R2 is an anti-gastrin 17 antibody-alkaline phosphatase marker diluted with an enzyme-labeled antibody buffer;
the formula of the enzyme-labeled antibody buffer solution is as follows: 25-50mmol/L phosphate buffer solution, 0.005-0.01mol/L magnesium chloride, 0.05-0.2mmol/L zinc chloride, 150-300mmol/L sodium chloride, 0.5-2% bovine serum albumin, 0.03-0.1% ProClin300, and the pH is 6.0-7.5;
the anti-gastrin 17 antibody-alkaline phosphatase marker is obtained by reacting alkaline phosphatase, a gastrin 17 antibody and glutaraldehyde.
9. The gastrin 17 assay kit according to claim 8, wherein the anti-gastrin 17 antibody-alkaline phosphatase marker is obtained by reacting alkaline phosphatase, a gastrin 17 antibody and glutaraldehyde, and specifically comprises:
dissolving the gastrin 17 antibody in a PBS (phosphate buffer solution) to obtain a gastrin 17 antibody solution with the concentration of 0.5-2 mg/ml;
adding alkaline phosphatase into the gastrin 17 antibody solution, uniformly mixing, adding glutaraldehyde, reacting completely, dialyzing to remove unreacted glutaraldehyde, and obtaining an anti-gastrin 17 antibody-alkaline phosphatase marker; the mass ratio of the alkaline phosphatase to the anti-gastrin 17 antibody is (8-12):1, and the final concentration of the glutaraldehyde is 0.1-0.4%.
10. A method of making a gastrin 17 assay kit, comprising the formulation of any one of claims 1 to 9.
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