CN114252595A - Magnetic bead diluent and immunoassay kit for reducing sample matrix interference - Google Patents

Abstract

The invention discloses a magnetic bead diluent for reducing sample matrix interference and an immunoassay kit. This magnetic bead diluent includes: 20-100mM buffer solution, 0.1-1% nonionic surfactant, 0.5-3% protein protective agent, 0.02-0.5% preservative, 100-1000mM inorganic salt and water. According to the invention, a large amount of salt is added into the magnetic bead diluent to increase the ionic strength of the diluent so as to reduce the binding capacity of the antibody and the protein, so that the foreign proteins in blood plasma cannot interact with the antibody through weak interaction, and possible nonspecific antigen-antibody binding is reduced, thereby reducing the influence brought by the matrix effect of the sample, and further improving the accuracy of the detection result; the detection method does not need to carry out pretreatment on the sample, does not need to carry out extra long-time closed treatment on the magnetic beads coated with the antigen or the antibody, and has simple operation and short consumed time.

Description

Magnetic bead diluent and immunoassay kit for reducing sample matrix interference

Technical Field

The invention relates to the technical field of biological detection, in particular to a magnetic bead diluent for reducing sample matrix interference and a preparation method thereof.

Background

Immunodiagnosis is a detection technology for detecting substances in a human body and acquiring clinical diagnosis information based on antigen-antibody specific reaction so as to judge human diseases, and is one of basic technologies of medical inspection. The immunodiagnosis mainly comprises three technical platforms of colloidal gold, enzyme-linked immunosorbent assay (ELISA) and chemiluminescence assay (CLIA). Chemiluminescence (CLIA) is a detection and analysis technique that combines a chemiluminescence assay technique with high sensitivity with a highly specific antigen-antibody reaction, and is used for detecting antigens, haptens, antibodies, hormones, enzymes, fatty acids, vitamins, drugs, and the like, and is a latest immunoassay technique developed after radioimmunoassay, enzyme immunoassay, fluorescence immunoassay, and time-resolved fluorescence immunoassay. Compared with colloidal gold and enzyme-linked immunosorbent assay, chemiluminescence is more sensitive, rapid, stable, strong in specificity and good in reproducibility, and high-throughput and automation are easier to realize, so that chemiluminescence is expected to be the trend in the field of immunodiagnosis. At present, chemiluminiscence manufacturers with higher market acceptance are generally foreign manufacturers such as Roche, Yapeh, Beckman and the like, and the acceptance of the domestic chemiluminiscence is not high enough. Therefore, the quality of the domestic chemiluminescence project is well-aimed.

The matrix refers to components in the sample except for the analyte, and often has significant interference on the analysis process of the analyte, and influences the accuracy of the analysis result. Sample matrix interference is easily encountered during the development of chemiluminescent projects. Patent CN113391060A discloses a blood sample treatment agent, which reduces the matrix interference of plasma sample by pre-treating the sample, and makes the immunoassay result consistent with the comparison method. The method needs to carry out pretreatment on the blood sample or use a specially treated blood collection tube, the operation is complicated, and the determination time is increased. In patent CN201811579298.0, polyoxyethylene-containing high molecular compound is used to seal the magnetic beads coated with antigen or antibody for 1-10 hours, so as to reduce the matrix effect between different blood collection tubes in immunoassay, which requires post-treatment of the coated magnetic beads, thus increasing the labeling time and complexity of the magnetic beads and providing higher operation requirements for the personnel labeled with the magnetic beads.

Disclosure of Invention

The invention aims to overcome the technical defects, provides a magnetic bead diluent and an immunoassay kit for reducing sample matrix interference, and solves the technical problems of complex operation and long time consumption of a method for reducing sample matrix interference in the prior art.

The first aspect of the present invention provides a magnetic bead diluent, comprising: 20-100mM buffer solution, 0.1-1% nonionic surfactant, 0.5-3% protein protective agent, 0.02-0.5% preservative, 100-1000mM inorganic salt and water.

A second aspect of the invention provides a chemiluminescent immunoassay kit comprising: the kit comprises an M reagent, an R reagent, a luminescent substrate solution and a calibrator; the reagent M is a magnetic bead reagent coated with an antibody M, the reagent R is an enzyme labeling reagent connected with an antibody R, and the reagent M is obtained by diluting magnetic beads coated with the antibody M with the magnetic bead diluent provided by the first aspect of the invention.

The third aspect of the invention provides a preparation method of a chemiluminescence immunoassay kit, which comprises the following steps:

preparation of M reagent: cleaning and activating magnetic beads, adding an antibody M and a coupling buffer solution for coupling reaction, then adding a sealing buffer solution for sealing, and diluting with a magnetic bead diluent to obtain an M reagent;

preparation of the R reagent: respectively activating the antibody R and enzyme, mixing, carrying out enzyme-linked reaction to obtain an enzyme-labeled antibody compound, and diluting with an R diluent to obtain an R reagent.

Preparing a luminescent substrate solution;

preparation of a calibrator: and preparing the antigen into the calibrator with different concentrations by using the calibrator diluent.

Compared with the prior art, the invention has the beneficial effects that:

according to the invention, a large amount of salt is added into the magnetic bead diluent to increase the ionic strength of the diluent so as to reduce the binding capacity of the antibody and the protein, so that the foreign proteins in blood plasma cannot interact with the antibody through weak interaction, and possible nonspecific antigen-antibody binding is reduced, thereby reducing the influence brought by the matrix effect of the sample, and further improving the accuracy of the detection result; the detection method provided by the invention does not need to carry out pretreatment on the sample, does not need to carry out additional long-time sealing treatment on the magnetic beads coated with the antigen or the antibody, only adds the additive in the magnetic bead diluent to reduce the interference of the sample matrix, improves the accuracy of the detection result, and is simple to operate and short in time consumption.

Drawings

FIG. 1 is a linear graph comparing the detection results of MYO detection item groups 1 and 16 of the present invention with the detection results of an imported Roche electrochemiluminescence MYO kit;

FIG. 2 is a linear graph comparing the test results of the CYFRA21-1 test item groups 1 and 16 with the test result of the imported Roche electrochemiluminescence CYFRA21-1 kit.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and 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.

The first aspect of the present invention provides a magnetic bead diluent, comprising: 20-100mM buffer solution, 0.1-1% nonionic surfactant, 0.5-3% protein protective agent, 0.02-0.5% preservative, 100-1000mM inorganic salt and water.

According to the invention, a large amount of salt is added into the magnetic bead diluent to increase the ionic strength of the diluent so as to reduce the binding capacity of the antibody and the protein, so that the foreign protein in blood plasma cannot interact with the antibody through weak interaction, and possible nonspecific antigen-antibody binding is reduced, thereby reducing the influence brought by the sample matrix effect and improving the accuracy of the detection result.

In the present invention, the inorganic salt is Na₂CO₃、CH₃COONa、(NH₄)₂SO₄、K₂SO₄、Na₂SO₄、MgSO₄、NaCl、KCl、CaCl₂、MgCl₂、KNO₃、NaNO₃、Mg(NO₃)₂、Ca(NO₃)₂At least one of (1). In some preferred embodiments of the invention, the concentration of the inorganic salt in the magnetic bead dilution is 200-800mM, further 400-600mM, and further 400 mM.

In the invention, the buffer solution is one or more of Tris buffer solution, HEPES buffer solution, HEPPSO buffer solution, MES buffer solution and phosphate buffer solution.

In the invention, the non-ionic surfactant is one or more of Tween20 (Tween-20), Tween-80 (Tween-80), EMULGEN A-60, EMULGEN A-90, Brij-35, Triton X-405, Triton X-114 and Triton X-100.

In the invention, the protein protective agent is one or more of sucrose, pullulan, trehalose, Bovine Serum Albumin (BSA), casein, acetaminophen and glycine. By adding the protein protective agent, the antibody protein is protected, and the stability of the reagent is improved.

In the invention, the preservative is one or more of sodium azide, potassium sorbate, sodium benzoate and Proclin series.

In the invention, macromolecular polymers are also added into the magnetic bead diluent for wrapping or polymerizing interference proteins, so that the interference of macromolecular proteins such as fibrin in serum and plasma is eliminated, and the accuracy of a detection result is improved. The macromolecular polymer is at least one of a cationic polymer or a neutral polymer. Further, the cationic polymer is at least one of Polyacrylamide (PAM), Polyethyleneimine (PEI), Polyisobutylene (PIB), vinyl alkyl ether, protamine, polybrene and aminodextran; the neutral polymer is at least one of polyethylene oxide (PEO) or polyvinyl alcohol. In some preferred embodiments of the invention, the concentration of the macromolecular polymer in the dilution of magnetic beads is between 0.01% and 0.5%, further between 0.01% and 0.1%, and still further between 0.05%.

In the invention, the magnetic bead diluent is also added with an amphoteric surfactant for adsorbing sites which are not completely sealed on the magnetic beads, so that the steric hindrance on the surfaces of the magnetic beads is improved, the non-specific adsorption of the antibody-enzyme conjugate or naked enzyme on the magnetic beads is reduced, and the accuracy of a detection result is improved. Further, the amphoteric surfactant is at least one of cetyltrimethylammonium bromide (CTAB), Dodecylethoxysulfobetaine (DESB), and octadecyldihydroxyethyl amine oxide (OAE-12). In some preferred embodiments of the present invention, the concentration of amphoteric surfactant in the diluted solution of magnetic beads is 0.05% to 0.5%, further 0.05% to 0.2%, and further 0.2%.

In the present invention, the pH of the magnetic bead diluent is 7 to 8, further 7.2 to 7.8, further 7.4 to 7.6, and further 7.5.

A second aspect of the invention provides a chemiluminescent immunoassay kit comprising: the kit comprises an M reagent, an R reagent, a luminescent substrate solution and a calibrator; the reagent M is a magnetic bead reagent coated with an antibody M, the reagent R is an enzyme labeling reagent connected with an antibody R, and the reagent M is obtained by diluting magnetic beads coated with the antibody M with the magnetic bead diluent provided by the first aspect of the invention.

The specific type of the chemiluminescent immunoassay kit is not limited by the present invention and can be selected by one skilled in the art according to the actual situation. In some embodiments of the present invention, the chemiluminescence immunoassay kit is a MYO chemiluminescence immunoassay kit or a CYFRA21-1 chemiluminescence immunoassay kit. A chemiluminescence immunoassay double-antibody sandwich method is adopted, and the MYO quantitative detection kit has the main principle that: the magnetic particle coated mouse anti-human monoclonal antibody is combined with MYO in human serum or plasma, and then forms a double-antibody sandwich with the mouse anti-human MYO monoclonal antibody marked by alkaline phosphatase, the alkaline phosphatase catalyzes a substrate to emit light under the condition of the existence of the substrate, and quantitative detection is carried out through a chemiluminescence detection system. The kit for quantitatively detecting CYFRA21-1 mainly comprises the following components in percentage by weight: magnetic particles are coated with a mouse anti-human monoclonal antibody, the magnetic particles are combined with CYFRA21-1 in human serum or plasma, and then the magnetic particles and an alkaline phosphatase-labeled mouse anti-human CYFRA21-1 monoclonal antibody form a double-antibody sandwich, the alkaline phosphatase catalyzes a substrate to emit light under the condition of the existence of the substrate, and quantitative detection is carried out through a chemiluminescence detection system.

The third aspect of the invention provides a preparation method of a chemiluminescence immunoassay kit, which comprises the following steps:

s1, preparation of M reagent: cleaning and activating magnetic beads, adding an antibody M and a coupling buffer solution for coupling reaction, then adding a sealing buffer solution for sealing, and diluting with a magnetic bead diluent to obtain an M reagent;

preparation of S2 and R reagent: respectively activating the antibody R and enzyme, mixing, carrying out enzyme-linked reaction to obtain an enzyme-labeled antibody compound, and diluting with an R diluent to obtain an R reagent.

S3, preparing a luminescent substrate solution;

s4, preparing a calibration product: preparing the antigen into different concentrations by using a calibrator diluent to obtain calibrators;

s5, independently placing the M reagent, the R reagent, the luminescent substrate liquid and the calibrator in a container to obtain the chemiluminescent immunoassay kit.

In the present invention, step S1 is specifically:

s11, taking 0.1-0.3mL of carboxyl magnetic beads with the concentration of 10-100mg/mL, removing supernate, carrying out magnetic separation and washing by using 0.3-3mL of magnetic bead washing liquid, and removing supernate; wherein, the magnetic bead washing liquid includes: 20-100mM MES, 0.01-0.2% Tween20, pH 5.8-6.2;

s12, taking 0.3-3mL of magnetic bead cleaning solution as a dispersing agent, adding 0.05-0.5mL of 5-20mg/mL EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) solution and 0.05-0.5mL of 5-20mg/mL NHS (N-hydroxysuccinimide) solution, adding the cleaned magnetic beads, activating at 20-30 ℃ for 20-30min, completing the activation of carboxyl on the surfaces of the magnetic beads, and removing supernatant;

s13, adding 0.05-0.1mg of antibody M and 0.1-1mL of coupling buffer solution, coupling for 2-3h at 20-30 ℃, completing coupling, and removing supernatant; wherein the coupling buffer comprises: 10-100mM boric acid solution, pH 8.0-8.5;

s14, adding 0.3-3mL of blocking buffer solution, reacting for 0.5-2h at 20-30 ℃, blocking unreacted activated carboxyl groups on the surfaces of the magnetic beads, removing supernatant after completion of blocking, and cleaning with a cleaning solution; wherein the blocking buffer comprises: 10-100mM PBS, 0.5% -2% BSA, pH 7-7.5; the washing liquid comprises: 10-100mM PBS buffer solution, pH 7-7.5;

s15, diluting the antibody M coated by the magnetic beads with a magnetic bead diluent according to the proportion of 1 (10-50) to obtain an M reagent.

In the present invention, the step S1 further includes: preserving the closed magnetic bead coated antibody M through a preserving fluid, specifically: adding 0.5-5mL of preservation solution for preservation at 2-8 deg.C; wherein, the preservation solution includes: 20-100mM Tris, 0.05% -0.2% PC-300, pH 7.8-8.2.

In the present invention, step S2 is specifically:

taking 0.1-1mg of antibody R, adding 0.1-1mL of antibody activator solution, activating at room temperature for 10-30min, and purifying after the reaction is finished to obtain an activated antibody; the antibody activator solution comprises: 20-100mM Tris, 0.1-0.5% NaCl, 1-10mM EDTA, 5-30mg/mL 2IT (2-imino tetrahydrothiophene) cross-linking agent;

taking 0.1-1mg of alkaline phosphatase, adding 0.1-1mL of enzyme activator, reacting at room temperature for 10-20min, and purifying after the reaction is finished to obtain activated alkaline phosphatase; wherein the enzyme activator comprises: 10-100mM Tris, 0.1% -0.5% NaCl, 1-10mM EDTA, 1-10mg/mL SMCC (4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester) cross-linker (dissolved with DMF);

mixing the activated antibody and the activated alkaline phosphatase according to the mass ratio of 1: (0.5-3) mixing, adding 0.005-0.5mL of 0.5-2M MgCl₂Reacting for 8-24h at 2-8 ℃, and purifying after the reaction is finished to obtain an enzyme-labeled antibody compound;

the enzyme-labeled antibody R was diluted with an enzyme-labeled diluent in the following ratio of 1: (200-1000) to obtain an R reagent; wherein, the enzyme labeling diluent comprises: 20-100mM Tris, 0.6-1.5% NaCl, 2-6mM MgCl₂、0.2-0.6mM ZnCl₂0.05-0.2% of PC-300, 1-10% of glycerol, 0.05-2% of BSA, 1-10% of fetal bovine serum and pH 6-7.

In the present invention, the step S2 further includes: the complex of antibody labeled by enzyme is preserved by glycerol. The method specifically comprises the following steps: adding glycerol into the enzyme-labeled antibody complex according to the volume of 1:1, and storing at the temperature of-15 to-30 ℃.

In the present invention, the luminescent substrate solution includes: 10-30g/L Tris, 100-200g/L NaCl, 1-10g/L KCl, 10-20mg/L HCl, 50-500mg/L luminescent substrate, 0.1-5mg/L PC-300. In some embodiments of the invention, the luminescent substrate is a 1, 2-dioxetane compound (AMPPD).

In the present invention, the MYO calibrator diluent comprises: 10-50mM MES, 0.6-1.5% NaCl, 0.1-0.3% Tween20, 0.1-0.3% casein, 0.5-2% BSA, 0.05-0.2% PC-300, pH 7.8-8.2; CYFRA21-1 calibrator dilutions comprising: 20-100mM Tris, 0.6-1.5% NaCl, 0.1-0.3% Tween20, 0.1-0.3% casein, 0.5-2% BSA, 0.05-0.2% PC-300, pH 7.2-7.8.

Example 1 preparation of magnetic bead diluent:

group 1:50 mM Tris, 0.2% Tween-20, 1% BSA, 0.05% PC-300, pH 7.5.

Group 2: under the conditions of group 1, 200mM Na was added₂CO₃；

Group 3: under the conditions of group 1, 200mM of (NH) was added₄)₂SO₄；

Group 4: under the conditions of group 1, 200mM KCl was added;

group 5: under the conditions of group 1, 0.05% protamine was added;

group 6: under the conditions of group 1, 0.05% Polyacrylamide (PAM) was added;

group 7: under the conditions of group 1, 0.1% cetyltrimethylammonium bromide (CTAB) was added;

group 8: under the conditions of group 1, 0.1% dodecylethoxy sulfobetaine (DESB) was added;

group 9: under the conditions of group 1, 100mM KCl was added;

group 10: under the conditions of group 1, 400mM KCl was added;

group 11: under the conditions of group 1, 600mM KCl was added;

group 12: 0.01% protamine was added under the conditions of group 1;

group 13: 0.1% protamine was added under group 1 conditions;

group 14: 0.05% cetyltrimethylammonium bromide (CTAB) was added under the conditions of group 1;

group 15: 0.2% cetyltrimethylammonium bromide (CTAB) was added under the conditions of group 1;

group 16: under the conditions of group 1, 400mM KCl, 0.05% protamine, 0.2% cetyltrimethylammonium bromide (CTAB) were added.

Example 2 preparation of a chemiluminescent immunoassay kit for MYO or CYFRA21-1

(1) Test materials:

carboxyl magnetic beads: purchased from JSR corporation of japan;

② alkaline phosphatase: purchased from roche diagnostics biotechnology;

③ a MYO antibody: purchased from Nanjing Kinsrui Biotech, Inc., MYO magnetic bead antibody cat #: M8D, MYO enzyme-labeled antibody cat no: M8A;

CYFRA21-1 antibody: magnetic bead antibody cat No. purchased from Medix, CYFRA 21-1: MAB1604, CYFRA21-1 enzyme-labeled antibody cat number: MAB 1605.

(2) Preparation of magnetic bead-coated MYO or CYFRA21-1 antibodies:

first, 0.2mL of carboxyl magnetic beads (100mg/mL) was put in a centrifuge tube, and the supernatant was removed, followed by magnetic separation and washing 3 times with 0.5mL of a magnetic bead washing solution (100mM MES, 0.05% Tween20, pH6.0), and the supernatant was removed.

Adding 0.5mL of magnetic bead cleaning solution as a dispersing agent, adding 0.1mL of 10mg/mLEDC solution and 0.1mL of 10mg/mLNHS solution (activating agent, which is used as the preparation at present) into a centrifuge tube with magnetic beads, uniformly mixing by vortex to fully suspend the magnetic beads, activating at 25 ℃ for 30min, keeping the suspension state of the magnetic beads during the period, completing the activation of carboxyl on the surfaces of the magnetic beads, and removing supernatant.

③ adding 0.1mg of antibody and 0.5mL of coupling buffer (100mM boric acid solution, pH8.0-8.5), coupling at 25 ℃ for 2h, keeping the suspension state of the magnetic beads during the coupling, completing the coupling, and removing the supernatant.

Adding 0.5mL of blocking buffer (100mM PBS, 1% BSA, pH7.2), reacting at 25 ℃ for 1h, blocking unreacted activated carboxyl groups on the surfaces of the magnetic beads, keeping the magnetic beads in a suspended state, removing the supernatant, and washing with a washing solution (100mM PBS buffer, pH7.2) for 3 times.

Fifthly, adding 1mL of preservation solution (100mM Tris, 0.1% PC-300, pH8.0) for preservation to obtain the antibody M coated by the magnetic beads, and preserving at the temperature of 2-8 ℃.

(3) Preparation of enzyme-labeled antibodies for MYO or CYFRA 21-1:

taking 0.5mg of antibody, adding 0.2mL of antibody activator solution (100mM Tris, 0.3% NaCl, 5mM EDTA, 10mg/mL2IT cross-linking agent), activating at room temperature for 20min, and purifying by molecular sieve chromatography to obtain the activated antibody.

② 0.4mg of alkaline phosphatase is taken, 0.2mL of enzyme activator (100mM Tris, 0.3% NaCl, 5mM EDTA, 5mg/mLSMCC cross-linking agent (dissolved by DMF)) is added, reaction is carried out for 15min at room temperature, and purification is carried out by molecular sieve chromatography, thus obtaining the activated alkaline phosphatase.

③ mixing the activated antibody and the activated alkaline phosphatase according to the mass ratio of 1:0.8, adding 0.01mL of 1M MgCl₂Reacting for 12h at the temperature of 2-8 ℃, and purifying by using a molecular sieve after the reaction is finished to obtain the enzyme-labeled antibody compound.

Adding glycerol according to the volume of 1:1 for storage to obtain an enzyme-labeled antibody R, and storing at-20 ℃.

(4) M and R reagent preparation for MYO and CYFRA21-1

M reagent: and respectively diluting the MYO antibody or CYFRA21-1 antibody coated by the magnetic beads with a magnetic bead diluent according to the ratio of 1:20 to obtain the M reagent.

(ii) reagent R: enzyme-labeled MYO antibody or CYFRA21-1 antibody was diluted with enzyme (50mM Tris, 0.9% NaCl, 4mM MgCl) to obtain an enzyme-labeled antibody₂，0.4mM ZnCl₂0.1% PC-300, 5% glycerol, 1% BSA, 5% fetal bovine serum, pH 6.5) was diluted at a ratio of 1:500 to obtain reagent R.

(5) Preparing a luminescent substrate solution: 24g Tris, 160g NaCl, 6g KCl, 15mL HCl, 200mL AMPPD (1, 2-dioxetane compound) and 1mL PC-300 were dissolved in purified water and the volume was adjusted to 1L to obtain a luminescence substrate solution.

(6) Preparation of a calibrator:

MYO antigen is prepared into concentrations of 0ng/mL, 70ng/mL, 150ng/mL, 750ng/mL, 1500ng/mL and 3000ng/mL by using MYO calibrator diluent (25mM MES, 0.9% NaCl, 0.2% Tween20, 0.2% casein, 1% BSA, 0.1% PC-300 and pH of 8.0).

② CYFRA21-1 antigen is prepared into the concentration of 0ng/mL, 1ng/mL, 10ng/mL, 100ng/mL, 250ng/mL and 500ng/mL by CYFRA21-1 calibrator diluent (50mM Tris, 0.9% NaCl, 0.2% Tween20, 0.2% casein, 1% BSA, 0.1% PC-300, pH 7.5).

Evaluation protocol

Selecting experimental instruments: and (3) selecting a calibrator of a corresponding item for calibration by a full-automatic chemiluminescence immunoassay analyzer, and detecting linearity.

(1) Evaluation of MYO detection reagent:

the results of the MYO kit detection prepared from the M reagent prepared from the magnetic bead diluents of groups 1-16 are shown in FIG. 1 and tables 1-2, compared with the results of the detection in the imported Roche electrochemiluminescence MYO kit.

Correlation coefficient: testing samples with different concentrations and covering linear range, comparing with Roche concentration value, and observing correlation coefficient R²。

Table 1: MYO project group 1-8 Linear results

As a result: as can be seen from Table 1, of group 1Sample correlation coefficient R₂The worst is 0.8625, groups 2-4, and after the salt is added, the correlation coefficient is obviously improved to be more than 0.9600; groups 5-6, correlation coefficient increased to above 0.9200 after addition of macromolecular polymer; in groups 7-8, after the amphoteric surfactant is added, the correlation coefficient is improved to be more than 0.9200, which shows that the sample correlation can be improved after the salt, the macromolecular polymer or the amphoteric surfactant is added, but the effect of the salt addition is obviously better than that of the macromolecular polymer and the amphoteric surfactant.

Table 2: MYO project set 9-16 linear results

As a result: as can be seen from Table 2, the sample correlations gradually increased with the increase in the salt-KCL concentration in groups 4 and 9-11, while the sample correlations in groups 10 and 11 were not substantially changed, indicating that a KCl concentration of 400mM was sufficient; group 5, 12, 13, the change in the concentration of the macromolecular polymer-protamine had little effect on the correlation, optimally 0.05% protamine; group 7, 14, 15, amphoteric surfactant cetyltrimethylammonium bromide (CTAB) concentration changes do little to the correlation, optimally 0.2% cetyltrimethylammonium bromide (CTAB); group 16 sample correlation after addition of salt, macromolecular polymer and amphoteric surfactant was optimally 0.9912. As can be seen from fig. 1, the linear regression equation between the detection result (y) of the kit prepared in the MYO group 16 and the roche detection result (x) of the import manufacturer is that y is 1.0163x +2.105, and the correlation coefficient can reach R²0.9912, it is shown that the addition of salts, macromolecular polymers and amphoteric surfactants to the magnetic bead diluent can improve the accuracy of the assay results.

(2) CYFRA21-1 assay reagent evaluation

The results of the detection with the CYFRA21-1 kit prepared by preparing the M reagent from groups 1, 4, 5, 7 and 16 are shown in Table 3 and FIG. 2, compared with the detection results with the imported Roche electrochemiluminescence CYFRA21-1 kit.

Correlation coefficient: testing samples with different concentrations and covering linear range, comparing with Roche concentration value, and observing correlation coefficient R²。

Table 3: CYFRA21-1 project linear results

As a result: as can be seen from Table 3, after the salt, the macromolecular polymer and the amphoteric surfactant are respectively added into the CYFRA21-1 magnetic bead diluent, the correlation coefficients of the samples are all improved, but the effect of the single salt is better than that of the macromolecular polymer and the neutral surfactant; the optimal result is set 16, the sample correlation coefficient is 0.9955, as can be seen from fig. 2, the linear regression equation of the test result (y) of the kit prepared by CYFRA21-1 set 16 and the imported manufacturer roche test result (x) is 1.0086x +1.5453, and the correlation coefficient can reach R²0.9955, the addition of salt, macromolecular polymer and amphoteric surfactant to the magnetic bead diluent can improve the accuracy of the detection result. Consistent with the MYO evaluation results.

Compared with the prior art, the invention has the beneficial effects that:

according to the invention, a large amount of salt is added into the magnetic bead diluent to increase the ionic strength of the diluent so as to reduce the binding capacity of the antibody and the protein, so that the hetero-protein in blood plasma cannot be bound with the antibody through weak interaction, and possible nonspecific antigen-antibody binding is reduced, thereby reducing the influence brought by the sample matrix effect; the polymer added with macromolecules is used for wrapping or polymerizing interference protein, and the interference of macromolecular proteins such as fibrin in serum and plasma is eliminated; adding amphoteric surfactant to adsorb onto incompletely closed sites on magnetic bead to improve steric hindrance on magnetic bead surface, so as to reduce nonspecific adsorption of antibody-enzyme conjugate or naked enzyme on magnetic bead, thereby improving accuracy of detection result.

The above-described embodiments of the present invention should not be construed as limiting the scope of the present invention. Any other corresponding changes and modifications made according to the technical idea of the present invention should be included in the protection scope of the claims of the present invention.

Claims (10)

1. A magnetic bead diluent, comprising: 20-100mM buffer solution, 0.1-1% nonionic surfactant, 0.5-3% protein protective agent, 0.02-0.5% preservative, 100-1000mM inorganic salt and water.

2. A diluent for magnetic beads according to claim 1, wherein the inorganic salt is Na2CO₃、CH₃COONa、(NH₄)₂SO₄、K₂SO₄、Na₂SO₄、MgSO₄、NaCl、KCl、CaCl₂、MgCl₂、KNO₃、NaNO₃、Mg(NO₃)₂、Ca(NO₃)₂At least one of (1).

3. The magnetic bead diluent of claim 1, wherein the concentration of the inorganic salt in the magnetic bead diluent is 400-600 mM.

4. The diluted solution of magnetic beads according to claim 1, wherein a polymer of macromolecule is further added to the diluted solution of magnetic beads; the macromolecular polymer is at least one of a cationic polymer or a neutral polymer; the concentration of the macromolecular polymer in the magnetic bead diluent is 0.01-0.5%.

5. A magnetic bead diluent as claimed in claim 4, wherein said cationic polymer is at least one of polyacrylamide, polyethyleneimine, polyisobutylene, vinyl alkyl ether, protamine, polybrene, and aminodextran; the neutral polymer is at least one of polyethylene oxide or polyvinyl alcohol.

6. The diluted magnetic bead solution of claim 1, further comprising an amphoteric surfactant, wherein the concentration of the amphoteric surfactant in the diluted magnetic bead solution is 0.05% to 0.5%.

7. A magnetic bead diluent as claimed in claim 6, wherein the amphoteric surfactant is at least one of cetyltrimethylammonium bromide, dodecylethoxysulphobetaine, and octadecyldihydroxyethyl amine oxide.

8. A chemiluminescent immunoassay kit comprising: the kit comprises an M reagent, an R reagent, a luminescent substrate solution and a calibrator; the reagent M is a magnetic bead reagent coated with an antibody M, the reagent R is an enzyme labeling reagent connected with an antibody R, and the reagent M is obtained by diluting magnetic beads coated with the antibody M with the magnetic bead diluent disclosed by any one of claims 1-7.

9. The chemiluminescent immunoassay kit of claim 8, wherein the chemiluminescent immunoassay kit is a MYO chemiluminescent immunoassay kit or a CYFRA21-1 chemiluminescent immunoassay kit.

10. A method of making a chemiluminescent immunoassay kit of claim 8 comprising the steps of:

preparation of M reagent: cleaning and activating magnetic beads, adding an antibody M and a coupling buffer solution for coupling reaction, then adding a sealing buffer solution for sealing, and diluting with a magnetic bead diluent to obtain an M reagent;

preparation of the R reagent: respectively activating the antibody R and enzyme, mixing, carrying out enzyme-linked reaction to obtain an enzyme-labeled antibody compound, and diluting with an R diluent to obtain an R reagent;

preparing a luminescent substrate solution;

preparation of a calibrator: and preparing the antigen into the calibrator with different concentrations by using the calibrator diluent.

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