CN111175494A - Thyroglobulin antibody detection kit and use method thereof - Google Patents
Thyroglobulin antibody detection kit and use method thereof Download PDFInfo
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Classifications
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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
- G01N33/54326—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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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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
- G01N33/531—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
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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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
Abstract
The invention discloses the medical appliance biological immune in-vitro diagnosis related technical field, in particular to a thyroglobulin antibody detection kit and a use method thereof; the kit comprises a calibrator, a quality control product, an anti-reagent, a magnetic particle reagent, a luminescent substrate and a cleaning solution which are packaged independently; wherein the antitrochane fluorescein labeled thyroglobulin coating antigen and the alkaline phosphatase labeled antithyroid globulin labeled antibody; the magnetic particle reagent is prepared by coupling magnetic particles and goat anti-fluorescein isothiocyanate antibodies; the chemiluminescence technology is combined with the immunomagnetic particles, so that the detection sensitivity and precision are greatly improved, the detection range is expanded, and the reaction time is short; and a plurality of samples can be simultaneously measured on a full-automatic chemiluminescence apparatus, high-flux rapid measurement of the thyroglobulin antibody is realized, the performance is reliable, the sensitivity is high, the linear range is wide, and the thyroglobulin antibody can be matched with a semi-automatic and full-automatic apparatus for use.
Description
Technical Field
The invention relates to the technical field related to biological immune in-vitro diagnosis of medical instruments, in particular to a thyroglobulin antibody detection kit and a using method thereof.
Background
Thyroglobulin (TG) is a glycoprotein in thyroid follicular columnar cells, has a molecular weight of 660KD, and plays an important role in synthesis and storage of thyroid hormone. In vivo, thyroglobulin, which contains T3 and T4 in the form of a colloid, is stored in the follicular lumen, and under the action of enzymes, T3 and T4 are liberated into the cytosol and enter the blood circulation. Meanwhile, a small amount of TG is released into blood, and normal people can detect the TG with the concentration of 3-40 ug/L. However, when the thyroid gland is damaged by pathological causes, a large amount of TG is released into the blood, and TG acts as an autoantigen to induce the production of anti-TG antibodies.
TGAb is used as an autoantibody of thyroglobulin, has a high positive rate in autoimmune thyroid diseases (such as Hashimoto's thyroiditis and Graves disease), has a thyroiditis appearance frequency of about 80 percent, has a positive rate of a TG antibody of the Graves disease of about 60 percent, can be detected in sera of other thyroid diseases and healthy people, but has a low titer. Therefore, the quantitative detection of the thyroglobulin antibody has important significance for diagnosing and treating thyroid diseases clinically. In addition, thyroid cancer has a certain correlation with TGAb, and the increase of TGAb value is a sign of tumor deterioration. The thyroid peroxidase antibody is also used as an autoimmune antibody and is an important index for diagnosing autoimmune thyroid diseases, so that the joint detection of the TGAb and TPOAb antibodies is beneficial to accurate diagnosis and treatment of early thyroid autoimmune diseases.
Presently known methods for assaying thyroglobulin antibodies include Radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), latex-enhanced turbidimetry, and the like. The radioimmunoassay has complicated steps, expensive reagents, and the use of a matched instrument and radioactive pollution. The ELISA has the defects of long detection time, complex operation and poor repeatability, and is not suitable for the needs of emergency treatment and timely diagnosis of clinical patients. The latex enhanced immunoturbidimetry is simple and rapid to operate, but has low sensitivity and poor low-value repeatability.
Therefore, a detection technique that has high sensitivity and reliability for detecting thyroglobulin antibodies (TGAb) and can reduce the detection cost is yet to be developed.
Disclosure of Invention
Aiming at the technical problems of high detection sensitivity and reliability of a thyroglobulin antibody (TGAb) and capability of reducing detection cost in the prior art, the invention provides a thyroglobulin antibody detection kit and a use method thereof.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a thyroglobulin antibody detection kit comprises a calibrator, a quality control product, an anti-reagent, a magnetic particle reagent, a luminescent substrate and a cleaning solution which are packaged independently;
the calibration material and the quality control material have the same components and are prepared by dissolving a thyroglobulin antibody in a buffer solution containing protein and diluting the thyroglobulin antibody in a buffer solution containing newborn bovine serum:
the anti-reagent is prepared by the following steps:
and SA: preparing a fluorescein isothiocyanate labeled thyroglobulin coated antigen:
sa 1: preparing fluorescein isothiocyanate into a fluorescein isothiocyanate solution with the concentration of 1.0-5.0 mg/mL by using an anti-reagent buffer solution;
sa 2: mixing thyroglobulin antigen and the fluorescein isothiocyanate solution according to the mass ratio of 1: 1.1-1: 1.5 under a shading condition;
sa 3: balancing the solution prepared by Sa2 with a carbonate buffer solution with the pH of 8-9, and then separating and purifying by gel chromatography to obtain a fluorescein isothiocyanate labeled thyroglobulin coated antigen;
SB: preparing an alkaline phosphatase-labeled thyroglobulin labeled antibody:
sb 1: preparing alkaline phosphatase into an alkaline phosphatase solution with the concentration of 1.0-5.0 mg/mL by using the anti-reagent buffer solution;
sb 2: respectively activating reaction groups of alkaline phosphatase and a thyroglobulin antibody, and then fully and uniformly mixing the activated reaction groups with the thyroglobulin antibody in a molar ratio of 1: 1-1: 3 under the catalysis of a catalyst to perform a coupling reaction;
sb 3: balancing the solution prepared by Sb2 by using a carbonate buffer solution with the pH value of 8-9, and then performing separation and purification of different molecular sizes by using a gel column to obtain an alkaline phosphatase-labeled thyroglobulin-labeled antibody;
SC: adding the fluorescein isothiocyanate labeled thyroglobulin coating antigen and the alkaline phosphatase labeled thyroglobulin labeled antibody into the anti-reagent buffer solution containing the surfactant to obtain the composition;
the magnetic particle reagent is prepared by the following operation steps:
sd 1: fully and uniformly mixing the concentrated solution of the carboxyl magnetic beads, placing the mixture in a magnetic field for 15-20 min, and removing the supernatant after the carboxyl magnetic beads are completely settled;
sd 2: adding a magnetic particle buffer solution with the volume 2-5 times that of the carboxyl magnetic beads into the carboxyl magnetic beads, and washing for 20-30 min in a shaking way; placing the mixture in a magnetic field for 15-20 min, and then sucking the supernatant;
sd 3: repeating the operation of Sd2 for 0-3 times;
sd 4: fixing the volume of the carboxyl magnetic beads to 10-50 mg/mL by using the magnetic particle buffer solution, and uniformly mixing to obtain a carboxyl magnetic bead buffer solution;
sd 5: according to the mass ratio of the carboxyl magnetic beads to the fluorescein isothiocyanate antibodies (90-110): 1, adding the anti-fluorescein isothiocyanate antibody into the carboxyl magnetic bead buffer solution, and reacting for 16-20 h at 2-8 ℃ in a uniformly mixed state;
sd 6: washing a product prepared from Sd5 for 0-3 times by using a phosphate buffer solution, and then fixing the volume to 9-11 mg/mL to obtain the product;
the luminescent substrate is prepared by adding a reagent ALPS into a luminescent substrate buffer solution with the volume 4-10 times that of the reagent ALPS.
Furthermore, the buffer solution containing the newborn bovine serum comprises 0.01-0.05% of preservative tetracycline and 0.1-0.5% of neomycin sulfate, and the buffer solution containing the newborn bovine serum is filtered by a 0.22-micron filter membrane.
Further, the anti-reagent buffer solution comprises 0.1-0.4 mM of Tris salt in purified water, and also comprises the following components in percentage by volume: 0.01-0.05% of tetracycline, 1-5% of sheep serum, 3-10% of newborn bovine serum and 1-5% of horse serum; the pH value of the anti-reagent buffer solution is 7-8.
Further, the surfactant is one or more of Tween20, TritonX-100 and Bronidox, and the volume percentage of the surfactant in the anti-reagent buffer is 0.01-0.5%.
Further, the luminescence bottom buffer solution comprises Tris0.1-1M, and also comprises the following components in concentration: 0.1% of sodium sulfite, 0.3% of SDS 1%, 0.3% of lucigenin and 0.15% of bovine serum albumin; the pH of the luminescence bottom buffer solution is 9.5.
Further, the magnetic particle buffer solution is a solution prepared by dissolving 12.12-15.26 mg of Tris, 5.82-8.58 mg of sodium chloride and 50-60 g of methyl cellulose ether in per liter of purified water.
Furthermore, 160g of NaCl, 4g of KCl, 24.2g of tris (hydroxymethyl) aminomethane and 1mL of Tween20 are dissolved in each liter of double distilled water; the pH of the cleaning solution is 7.4.
A method for using the thyroglobulin antibody detection kit of any one of the above includes the following steps:
se 1: taking three test tubes, and respectively adding 30 mu L of the calibrator, 30 mu L of the quality control material and 30 mu L of a sample to be detected;
se 2: adding 60 μ L of the anti-reagent into each test tube, covering the test tube with a plastic film, gently shaking the test tube for 30s, and placing in a water bath at 37 ℃ for 5 min;
se 3: adding 30 μ L of the magnetic particle reagent into each test tube, covering the test tube with a plastic film, slightly oscillating the test tube for 30s, and placing in a water bath at 37 ℃ for 5 min;
se 4: precipitating all tubes on a magnetic separator for 2min, slowly inverting the tubes and the magnetic separator, and decanting the supernatant; placing the inverted test tube together with the magnetic separator on a filter paper, and patting the bottom of the magnetic separator to remove all droplets adhering to the tube wall;
se 5: adding 300 μ L of the cleaning solution to each test tube, covering the test tube with a plastic film, gently shaking the test tube for 30s, slowly inverting the test tube and the magnetic separator after mixing, pouring out the supernatant, placing the inverted test tube together with the magnetic separator on a filter paper, and strongly slapping the bottom of the separator to remove all droplets adhering to the tube wall;
se 6: repeating the operation of Se5 for 0-3 times;
se 7: adding 200 mu L of the luminescent substrate into each test tube, shaking and uniformly mixing for 3s, and detecting the luminous intensity by using a chemiluminescence apparatus.
The key points of the invention comprise:
firstly, Alkaline Phosphatase (AP) is used as a labeled enzyme, an antibody is labeled through a chemical reaction, and unreacted enzyme, antibody or antigen are separated through gel chromatography, so that the reaction sensitivity is improved;
secondly, the immunomagnetic particles are used as a solid phase, and goat-anti-fluorescein isothiocyanate antibodies are coupled with the magnetic microspheres and used as a general separation reagent, so that the immunoreaction is easier to mix and separate, and the reaction speed is greatly improved;
furthermore, a novel chemiluminescence substrate ALPS is used as a substrate, the substrate is a glow substrate and quickly reaches a plateau phase, signal detection is facilitated, and the sensitivity and specificity performance of the final kit are improved; furthermore, a chemiluminescence enhancement system is further optimized, and high signal sensitivity, good stability and small variation of a final product are ensured;
in addition, the ALPS substrates have the advantages of high sensitivity and long platform stability;
the technical principle of the reaction is as follows: TGAb in a sample to be detected, a calibrator or a quality control product and TG antibody marked by Alkaline Phosphatase (AP) compete to be combined with TGAb antigen coated by Fluorescein Isothiocyanate (FITC), then magnetic particles connected with anti-fluorescein antibody are added, an antigen-antibody compound is connected on the magnetic particles through the specific combination of the anti-fluorescein antibody and fluorescein, the compound formed by immunoreaction is directly precipitated in an external magnetic field, and the compound is separated from other uncombined substances. Cleaning the precipitated compound, adding an enzymatic chemiluminescent substrate, catalytically cracking the substrate under the action of enzyme to form an unstable excited state intermediate, emitting photons when the excited state intermediate returns to a ground state to form a luminescent reaction, and measuring the luminous intensity of the reaction by using a chemiluminescence tester. Within the measurement range, the luminescence intensity is inversely proportional to the TGAb concentration in the sample, and the TGAb concentration in the sample to be measured can be quantitatively calculated by using improved four-parameter Logistic equation fitting.
The invention has the following advantages:
1. the components of each reagent of the competitive method-thyroglobulin antibody (TGAb) detection kit (magnetic particle separation chemiluminescence method) comprise a calibrator, a quality control product, an anti-reagent, a magnetic particle reagent, a luminescent substrate and the like, the stability is good, the effective period can reach more than one year, and the cost is effectively reduced;
2. the detection sensitivity is high, the specificity performance is good, and the variation is small;
3. in the invention, a perfect and unified process is obtained through process optimization of a large number of experiments, and production is carried out strictly according to standard production operation regulations and quality control regulations;
4. the user only needs to carry out standard operation according to the operation instruction, and reliable results can be obtained, and the operation is simple and convenient.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a graph of a competition method-thyroglobulin antibody (TGAb) detection kit (magnetic particle isolation chemiluminescence method) standard curve;
FIG. 2 is a comparison of the performance of a competition method-thyroglobulin antibody (TGAb) detection kit (magnetic particle isolation chemiluminescence method) and imported reagents.
Detailed Description
The following further describes embodiments of the present invention with reference to the drawings. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. Moreover, in the following description, descriptions of well-known structures, techniques, and operations are omitted so as to not unnecessarily obscure the concepts of the present invention. In addition, the technical features related to the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The reagent components (such as cleaning solution, some necessary buffer solution and the like) which are not mentioned in detail in the kit of the invention, the external package of the kit, the independent packaging container of each reagent component and the like can be carried out according to the conventional operation in the field, and the kit meets the relevant industry regulations. The operation steps not mentioned in detail in the method of the present invention can also be performed by referring to the conventional operations in the field, for example, before detection, each reagent can be placed at room temperature (18-25 ℃), and before sample addition, the reagents are fully mixed; the detection instrument such as a chemiluminescence determinator is used according to the instruction operation;
in the present invention, the proportion and content of the unit are not particularly specified, the solid component is the mass proportion and content, and the liquid component is the volume proportion and content.
Example 1
A thyroglobulin antibody detection kit comprises a calibrator, a quality control product, an anti-reagent, a magnetic particle reagent, a luminescent substrate and a cleaning solution which are packaged independently;
the calibration material and the quality control material have the same components and are prepared by the operation of dissolving thyroglobulin antibodies by using buffer solution containing protein and diluting the thyroglobulin antibodies into buffer solution containing newborn bovine serum: wherein, the buffer solution containing protein can be replaced by buffer solution containing newborn calf serum, the latter comprises preservatives tetracycline 0.01% and neomycin sulfate 0.1%, and the buffer solution containing newborn calf serum is filtered by a 0.22 μm filter membrane;
the anti-reagent is prepared by the following steps:
and SA: preparing a fluorescein isothiocyanate labeled thyroglobulin coated antigen:
sa 1: preparing fluorescein isothiocyanate into a fluorescein isothiocyanate solution with the concentration of 1.0mg/mL by using an anti-reagent buffer solution; wherein the anti-reagent buffer solution comprises 0.1mM of Tris salt in purified water and also comprises the following components in percentage by volume: 0.01% of tetracycline, 1% of sheep serum, 3% of newborn bovine serum and 1% of horse serum; the pH of the anti-reagent buffer is 7;
sa 2: mixing thyroglobulin antigen and fluorescein isothiocyanate solution according to the mass ratio of 1:1.1 under a shading condition;
sa 3: balancing the solution prepared by Sa2 with a carbonate buffer solution with the pH value of 8, and then separating and purifying by gel chromatography to obtain fluorescein isothiocyanate labeled thyroglobulin coated antigen;
SB: preparing an alkaline phosphatase-labeled thyroglobulin labeled antibody:
sb 1: preparing alkaline phosphatase into an alkaline phosphatase solution with the concentration of 1.0mg/mL by using an anti-reagent buffer solution;
sb 2: respectively activating the reaction groups of alkaline phosphatase and thyroglobulin antibody, and then fully and uniformly mixing the activated reaction groups with the thyroglobulin antibody in a molar ratio of 1:1 under the catalysis of a catalyst to perform coupling reaction;
sb 3: balancing the solution prepared by Sb2 by using a carbonate buffer solution with the pH value of 8, and then carrying out separation and purification of different molecular sizes by using a gel column to obtain an alkaline phosphatase-labeled thyroglobulin-labeled antibody;
SC: adding a fluorescein isothiocyanate labeled thyroglobulin coating antigen and an alkaline phosphatase labeled thyroglobulin labeled antibody into an anti-reagent buffer solution containing a surfactant to obtain the composition; wherein the surfactant is Tween20, and the volume percentage of the surfactant in the anti-reagent buffer solution is 0.01%;
the magnetic particle reagent is prepared by the following operation steps:
sd 1: fully and uniformly mixing the concentrated solution of the carboxyl magnetic beads, placing the concentrated solution in a magnetic field for 15min, and removing a supernatant after the carboxyl magnetic beads are completely settled;
sd 2: adding a magnetic particle buffer solution with the volume 2 times that of the carboxyl magnetic beads into the carboxyl magnetic beads, and washing for 20min by shaking; placing in magnetic field for 15min, and removing supernatant;
sd 4: fixing the volume of the carboxyl magnetic beads to 10mg/mL by using a magnetic particle buffer solution, and uniformly mixing to obtain a carboxyl magnetic bead buffer solution; wherein, the magnetic particle buffer solution is a solution dissolved with 12.12mg of Tris, 5.82mg of sodium chloride and 50g of methyl cellulose ether in each liter of purified water;
sd 5: according to the mass ratio of the carboxyl magnetic beads to the fluorescein isothiocyanate antibodies of 90: 1, adding an anti-fluorescein isothiocyanate antibody into a carboxyl magnetic bead buffer solution, and reacting for 16 hours at 2 ℃ in a uniformly mixed state;
sd 6: fixing the volume of a product prepared from Sd5 to 9mg/mL by using a phosphate buffer solution to obtain the product;
the luminescent substrate is prepared by adding a reagent ALPS into a luminescent substrate buffer solution with the volume 4 times that of the reagent ALPS; the luminescence bottom buffer solution comprises Tris0.1M and also comprises the following components in concentration: 0.1% of sodium sulfite, 0.3% of SDS 1%, 0.3% of lucigenin and 0.15% of bovine serum albumin; the pH of the luminescence bottom buffer solution is 9.5;
the cleaning solution is prepared by dissolving 160g of NaCl, 4g of KCl, 24.2g of tris (hydroxymethyl) aminomethane and 1mL of Tween20 in per liter of double distilled water; the pH of the wash was 7.4.
Example 2
A thyroglobulin antibody detection kit comprises a calibrator, a quality control product, an anti-reagent, a magnetic particle reagent, a luminescent substrate and a cleaning solution which are packaged independently;
the calibration material and the quality control material have the same components and are prepared by the operation of dissolving thyroglobulin antibodies by using buffer solution containing protein and diluting the thyroglobulin antibodies into buffer solution containing newborn bovine serum: wherein, the buffer solution containing protein can be replaced by buffer solution containing newborn calf serum, the latter comprises preservatives tetracycline 0.03% and neomycin sulfate 0.3%, and the buffer solution containing newborn calf serum is filtered by a 0.22 μm filter membrane;
the anti-reagent is prepared by the following steps:
and SA: preparing a fluorescein isothiocyanate labeled thyroglobulin coated antigen:
sa 1: preparing fluorescein isothiocyanate into a fluorescein isothiocyanate solution with the concentration of 3.0mg/mL by using an anti-reagent buffer solution; wherein the anti-reagent buffer solution comprises 0.3mM of Tris salt in purified water and also comprises the following components in percentage by volume: 0.03% of tetracycline, 3% of sheep serum, 6% of newborn bovine serum and 3% of horse serum; the pH of the anti-reagent buffer was 7.5;
sa 2: mixing thyroglobulin antigen and fluorescein isothiocyanate solution according to the mass ratio of 1:1.3 under a shading condition;
sa 3: balancing the solution prepared by Sa2 with a carbonate buffer solution with the pH value of 8.5, and then separating and purifying by gel chromatography to obtain fluorescein isothiocyanate labeled thyroglobulin coated antigen;
SB: preparing an alkaline phosphatase-labeled thyroglobulin labeled antibody:
sb 1: preparing alkaline phosphatase into alkaline phosphatase solution with the concentration of 3.0mg/mL by using an anti-reagent buffer solution;
sb 2: respectively activating the reaction groups of alkaline phosphatase and thyroglobulin antibody, and then fully and uniformly mixing the activated reaction groups with the thyroglobulin antibody in a molar ratio of 1:2 under the catalysis of a catalyst to perform coupling reaction;
sb 3: balancing the solution prepared by Sb2 by using a carbonate buffer solution with the pH value of 8.5, and then separating and purifying different molecular sizes by using a gel column to obtain an alkaline phosphatase-labeled thyroglobulin labeled antibody;
SC: adding a fluorescein isothiocyanate labeled thyroglobulin coating antigen and an alkaline phosphatase labeled thyroglobulin labeled antibody into an anti-reagent buffer solution containing a surfactant to obtain the composition; wherein the surfactant is TritonX-100, and the volume percentage of the surfactant in the anti-reagent buffer solution is 0.25%;
the magnetic particle reagent is prepared by the following operation steps:
sd 1: fully mixing the concentrated solution of the carboxyl magnetic beads in a magnetic field for 18min, and removing the supernatant after the carboxyl magnetic beads are completely settled;
sd 2: adding a magnetic particle buffer solution with the volume 4 times that of the carboxyl magnetic beads into the carboxyl magnetic beads, and washing for 25min by shaking; placing in magnetic field for 18min, and removing supernatant;
sd 3: repeating the operation of Sd2 for 2 times;
sd 4: fixing the volume of the carboxyl magnetic beads to 30mg/mL by using a magnetic particle buffer solution, and uniformly mixing to obtain a carboxyl magnetic bead buffer solution; wherein, the magnetic particle buffer solution is a solution dissolved with 14.26mg of Tris, 6.58mg of sodium chloride and 55g of methyl cellulose ether in each liter of purified water;
sd 5: according to the mass ratio of the carboxyl magnetic beads to the fluorescein isothiocyanate antibodies of 100: 1, adding an anti-fluorescein isothiocyanate antibody into a carboxyl magnetic bead buffer solution, and reacting for 18 hours at 4 ℃ in a uniformly mixed state;
sd 6: washing a product prepared from Sd5 for 2 times by using a phosphate buffer solution, and then fixing the volume to 10mg/mL to obtain the product;
the luminescent substrate is prepared by adding a reagent ALPS into a luminescent substrate buffer solution with the volume 7 times that of the reagent ALPS; the luminescence bottom buffer solution comprises Tris0.5M and further comprises the following components in concentration: 0.1% of sodium sulfite, 0.3% of SDS 1%, 0.3% of lucigenin and 0.15% of bovine serum albumin; the pH of the luminescence bottom buffer solution is 9.5;
the cleaning solution is prepared by dissolving 160g of NaCl, 4g of KCl, 24.2g of tris (hydroxymethyl) aminomethane and 1mL of Tween20 in per liter of double distilled water; the pH of the wash was 7.4.
Example 3
A thyroglobulin antibody detection kit comprises a calibrator, a quality control product, an anti-reagent, a magnetic particle reagent, a luminescent substrate and a cleaning solution which are packaged independently;
the calibration material and the quality control material have the same components and are prepared by the operation of dissolving thyroglobulin antibodies by using buffer solution containing protein and diluting the thyroglobulin antibodies into buffer solution containing newborn bovine serum: wherein, the buffer solution containing protein can be replaced by buffer solution containing newborn bovine serum, the latter comprises preservatives tetracycline 0.05% and neomycin sulfate 0.5%, and the buffer solution containing newborn bovine serum is filtered by a 0.22 μm filter membrane;
the anti-reagent is prepared by the following steps:
and SA: preparing a fluorescein isothiocyanate labeled thyroglobulin coated antigen:
sa 1: preparing fluorescein isothiocyanate into a fluorescein isothiocyanate solution with the concentration of 5.0mg/mL by using an anti-reagent buffer solution; wherein the anti-reagent buffer solution comprises 0.4mM of Tris salt in purified water and also comprises the following components in percentage by volume: 0.05% of tetracycline, 5% of sheep serum, 10% of newborn bovine serum and 5% of horse serum; the pH of the anti-reagent buffer is 8;
sa 2: mixing thyroglobulin antigen and fluorescein isothiocyanate solution according to the mass ratio of 1:1.5 under a shading condition;
sa 3: balancing the solution prepared by Sa2 with a carbonate buffer solution with the pH value of 9, and then separating and purifying by gel chromatography to obtain fluorescein isothiocyanate labeled thyroglobulin coated antigen;
SB: preparing an alkaline phosphatase-labeled thyroglobulin labeled antibody:
sb 1: preparing alkaline phosphatase into an alkaline phosphatase solution with the concentration of 5.0mg/mL by using an anti-reagent buffer solution;
sb 2: respectively activating the reaction groups of alkaline phosphatase and thyroglobulin antibody, and then fully and uniformly mixing the activated reaction groups with the thyroglobulin antibody in a molar ratio of 1:3 under the catalysis of a catalyst to perform coupling reaction;
sb 3: balancing the solution prepared by Sb2 by using a carbonate buffer solution with the pH value of 9, and then carrying out separation and purification of different molecular sizes by using a gel column to obtain an alkaline phosphatase-labeled thyroglobulin-labeled antibody;
SC: adding a fluorescein isothiocyanate labeled thyroglobulin coating antigen and an alkaline phosphatase labeled thyroglobulin labeled antibody into an anti-reagent buffer solution containing a surfactant to obtain the composition; wherein the surfactant is Bronidox, and the volume percentage of the surfactant in the anti-reagent buffer solution is 0.5%;
the magnetic particle reagent is prepared by the following operation steps:
sd 1: fully and uniformly mixing the concentrated solution of the carboxyl magnetic beads, placing the mixture in a magnetic field for 20min, and removing the supernatant after the carboxyl magnetic beads are completely settled;
sd 2: adding a magnetic particle buffer solution with the volume 5 times that of the carboxyl magnetic beads into the carboxyl magnetic beads, and washing for 30min by shaking; placing in magnetic field for 20min, and removing supernatant;
sd 3: repeating the operation of Sd2 for 3 times;
sd 4: fixing the volume of the carboxyl magnetic beads to 50mg/mL by using a magnetic particle buffer solution, and uniformly mixing to obtain a carboxyl magnetic bead buffer solution; wherein, the magnetic particle buffer solution is a solution dissolved with 15.26mg of Tris, 8.58mg of sodium chloride and 60g of methyl cellulose ether in each liter of purified water;
sd 5: according to the mass ratio of the carboxyl magnetic beads to the fluorescein isothiocyanate antibodies of 110: 1, adding an anti-fluorescein isothiocyanate antibody into a carboxyl magnetic bead buffer solution, and reacting for 20 hours at 8 ℃ in a uniformly mixed state;
sd 6: washing a product prepared from Sd5 for 3 times by using a phosphate buffer solution, and then fixing the volume to 11mg/mL to obtain the product;
the luminescent substrate is prepared by adding a reagent ALPS into a luminescent substrate buffer solution with the volume 10 times that of the reagent ALPS; the luminescence bottom buffer solution comprises Tris1M and also comprises the following components in concentration: 0.1% of sodium sulfite, 0.3% of SDS 1%, 0.3% of lucigenin and 0.15% of bovine serum albumin; the pH of the luminescence bottom buffer solution is 9.5;
the cleaning solution is prepared by dissolving 160g of NaCl, 4g of KCl, 24.2g of tris (hydroxymethyl) aminomethane and 1mL of Tween20 in per liter of double distilled water; the pH of the wash was 7.4.
In the present invention, the coating antigen is an antigen capable of specifically binding to a human thyroglobulin antibody (TGAb), and the labeled antibody is a monoclonal antibody capable of specifically binding to a thyroglobulin antibody (TGAb). The labeled antibody competes with thyroglobulin antibody (TGAb) for specific binding to the coating antigen.
Example 4
To further illustrate the use of the kits prepared in examples 1-3, the relevant details are further detailed as follows:
1. thyroglobulin antibody (TGAb) antigen used in the examples was purchased from Fitzgerald, a well-known manufacturer at home and abroad, and thyroglobulin antibody (TGAb) coating antigen and labeled antibody used in the examples were purchased from Fitzgerald, a well-known manufacturer at home and abroad. The formulation of each buffer in this example is as follows:
[ anti-reagent buffer (Tris salt pH7.5 buffer) ]
| Name of reagent | Manufacturer of the product | Concentration of | 1L buffer solution dosage |
| Tris | Sigma | 0.1M | 12.12 |
| Sodium chloride | Sigma | 0.9％ | 5.82 |
| Sheep serum | Guangzhou pistit | 5％ | 50mL |
| Newborn bovine serum | Guangzhou pistit | 10％ | 100mL |
| Horse serum | Guangzhou pistit | 5％ | 50mL |
| 4M hydrochloric acid | Sigma | pH7.5 | About 20mL |
[ CALIBRATE BUFFER ]
| Name of reagent | Manufacturer of the product | Concentration of | 1L buffer solution dosage |
| Newborn bovine serum | Sigma | 100％ | 1000mL |
| Tetracycline derivatives | Sigma | 0.01％ | 10mg |
| Neomycin sulfate | Sigma | 0.1％ | 100mg |
[ magnetic particle buffer ]
| Name of reagent | Manufacturer of the product | Concentration of | 1L buffer solution dosage |
| Tris | Sigma | 0.1M | 12.12 |
| Sodium chloride | Sigma | 0.9％ | 5.82 |
| Methyl cellulose ether | Sigma | 5％ | 50g |
[ luminogenic substrate buffer ]
| Name of reagent | Manufacturer of the product | Concentration of | 1L buffer solution dosage |
| Tris | Sigma | 0.1M | 12.12 |
| Sodium chloride | Sigma | 0.9％ | 5.82 |
| Glossing essence | Sigma | 0.3％ | 30mg |
The main reagent components of the competitive method-thyroglobulin antibody (TGAb) detection kit (magnetic particle isolation chemiluminescence method) of the examples include:
1) a calibrator for thyroglobulin antibody (TGAb) antigen (a, B.., F) was diluted with calibrator buffer.
2) Thyroglobulin antibody (TGAb) antigen quality control (Q1, Q2) was diluted with calibrator buffer. And calibrating the concentration range of the quality control product in each batch.
3) Fluorescein Isothiocyanate (FITC) -labeled thyroglobulin-coated antigen and Alkaline Phosphatase (AP) -labeled thyroglobulin-labeled antibody were diluted with an anti-reagent buffer.
4) And (3) diluting the anti-fluorescein isothiocyanate antibody magnetic particles by using a magnetic particle buffer solution.
5) The washing solution was concentrated and diluted appropriately for use.
6) ALPS luminogenic substrate diluted with luminogenic buffer.
3. The preparation process of each reagent component of the competitive method-thyroglobulin antibody (TGAb) detection kit (magnetic particle separation chemiluminescence method) of this example is as follows:
preparing a calibrator and a quality control product: thyroglobulin antibody (TGAb) antigen was dissolved in a buffer containing protein to 0.5mg/mL solution for 30 min.
The above reaction was diluted to different concentration points with [ calibrator buffer ] at different concentrations and calibrated.
The target concentrations of the calibrator and the quality control material are as follows:
| name of reagent | Target concentration (IU/mL) |
| |
0 |
| Calibrator B | 7 |
| Calibrator C | 25 |
| Calibrator D | 100 |
| |
500 |
| |
1500 |
| Quality control product Q1 | 25 |
| Quality |
500 |
The preparation of the cleaning concentrated solution comprises the following formula:
| name of reagent | Manufacturer of the product | Specification of | 1L buffer solution dosage |
| Tris | Sigma | 0.1M | 12.12 |
| Sodium chloride | Sigma | 0.9％ | 5.82 |
| Tween-20 | Sigma | 5％ | 50mL |
| Triton-100 | Sigma | 5％ | 50mL |
The components of the calibrator, the quality control material, the anti-reagent, the magnetic particle reagent, the luminescent substrate, and the concentrated cleaning solution are prepared and then independently placed in a packaging container to form the competitive-thyroglobulin antibody (TGAb) assay kit (magnetic particle isolation chemiluminescence method) set of this example.
The specific operation method for detecting thyroglobulin antibody (TGAb) by using the magnetic particle chemiluminescence detection kit of this example is described in the kit Specification
The operation of carrying out the thyroglobulin antibody detection test by using the thyroglobulin antibody detection kit comprises the following steps:
setting a water bath kettle at 37 ℃;
preparing a chemiluminescence determinator;
se 1: taking three test tubes of the calibrator and the quality control material with each group of concentration, and respectively adding 30 mu L of calibrator, 30 mu L of quality control material and 30 mu L of sample to be detected;
se 2: adding 60 μ L of the anti-reagent into each test tube, covering the test tube with a plastic film, gently shaking the test tube for 30s, and placing in a water bath at 37 ℃ for 5 min;
se 3: adding 30 μ L magnetic particle reagent into each test tube, covering the test tube with plastic film, lightly shaking the test tube for 30s, and placing in water bath at 37 deg.C for 5 min;
se 4: precipitating all test tubes on a magnetic separator for 2min, slowly inverting the test tubes and the magnetic separator, and pouring out the supernatant; placing the inverted test tube together with the magnetic separator on a filter paper, and patting the bottom of the magnetic separator to remove all droplets adhered to the tube wall;
se 5: adding 300 μ L of cleaning solution into each test tube, covering the test tube with plastic film, slightly shaking the test tube for 30s, slowly inverting the test tube and magnetic separator after mixing, pouring out supernatant, putting the inverted test tube and magnetic separator together on filter paper, and strongly slapping the bottom of the separator to remove all droplets adhered to the tube wall;
se 6: repeating the operation of Se5 for 0-3 times;
se 7: add 200. mu.L of luminescent substrate to each tube, mix them evenly for 3s by shaking, and detect the luminescence intensity with a chemiluminescence apparatus.
The result calculation refers to the relevant description of the instrument; if a fully automatic detection instrument is used, the manual operation steps are replaced by automatic operation of the instrument, and the detection is carried out according to the strict instruction of the instrument.
And (3) processing data: and obtaining a standard curve through four-parameter nonlinear fitting according to the concentration value of the calibrator and the detected luminescence value, and substituting the luminescence value of the sample into the standard curve to obtain a corresponding concentration value.
FIG. 1 shows a competitive-thyroglobulin antibody (TGAb) assay kit (magnetic particle separation chemiluminescence) standard curve prepared according to the method of this example.
Table 1 shows the analytical performance and stability of the competitive-thyroglobulin antibody (TGAb) detection kit (magnetic particle isolation chemiluminescence) of this example.
FIG. 2 shows the comparison of the detection performance of the detection kit (magnetic particle separation chemiluminescence method) using the competitive method-thyroglobulin antibody (TGAb) of this example with that using the imported detection reagents. Separating low, medium and high value samples from 100 samples, performing independent linear regression analysis on the low, medium and high value samples, and analyzing the result, wherein the linear regression equation is 0.8299x +2.2591, R2＝0.9879。
It can be seen that the kit of the invention has reliable performance, high sensitivity and wide linear range, and can be matched with a full-automatic instrument for use.
Table 1: competition method-thyroglobulin antibody (TGAb) detection kit (magnetic particle separation chemiluminescence method) analytical performance and stability table.
Table 2 competition method-thyroglobulin antibody (TGAb) detection kit (magnetic particle separation chemiluminescence method) and imported TGAb detection kit (magnetic particle separation chemiluminescence method) detection reagents were compared for their detection performance.
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in the embodiments without departing from the principles and spirit of the invention, and these embodiments are within the scope of the invention.
Claims (8)
1. A thyroglobulin antibody detection kit is characterized in that: the kit comprises a calibrator, a quality control product, an anti-reagent, a magnetic particle reagent, a luminescent substrate and a cleaning solution which are packaged independently;
the calibration material and the quality control material have the same components and are prepared by dissolving a thyroglobulin antibody in a buffer solution containing protein and diluting the thyroglobulin antibody in a buffer solution containing newborn bovine serum:
the anti-reagent is prepared by the following steps:
and SA: preparing a fluorescein isothiocyanate labeled thyroglobulin coated antigen:
sa 1: preparing fluorescein isothiocyanate into a fluorescein isothiocyanate solution with the concentration of 1.0-5.0 mg/mL by using an anti-reagent buffer solution;
sa 2: mixing thyroglobulin antigen and the fluorescein isothiocyanate solution according to the mass ratio of 1: 1.1-1: 1.5 under a shading condition;
sa 3: balancing the solution prepared by Sa2 with a carbonate buffer solution with the pH of 8-9, and then separating and purifying by gel chromatography to obtain a fluorescein isothiocyanate labeled thyroglobulin coated antigen;
SB: preparing an alkaline phosphatase-labeled thyroglobulin labeled antibody:
sb 1: preparing alkaline phosphatase into an alkaline phosphatase solution with the concentration of 1.0-5.0 mg/mL by using the anti-reagent buffer solution;
sb 2: respectively activating reaction groups of alkaline phosphatase and a thyroglobulin antibody, and then fully and uniformly mixing the activated reaction groups with the thyroglobulin antibody in a molar ratio of 1: 1-1: 3 under the catalysis of a catalyst to perform a coupling reaction;
sb 3: balancing the solution prepared by Sb2 by using a carbonate buffer solution with the pH value of 8-9, and then performing separation and purification of different molecular sizes by using a gel column to obtain an alkaline phosphatase-labeled thyroglobulin-labeled antibody;
SC: adding the fluorescein isothiocyanate labeled thyroglobulin coating antigen and the alkaline phosphatase labeled thyroglobulin labeled antibody into the anti-reagent buffer solution containing the surfactant to obtain the composition;
the magnetic particle reagent is prepared by the following operation steps:
sd 1: fully and uniformly mixing the concentrated solution of the carboxyl magnetic beads, placing the mixture in a magnetic field for 15-20 min, and removing the supernatant after the carboxyl magnetic beads are completely settled;
sd 2: adding a magnetic particle buffer solution with the volume 2-5 times that of the carboxyl magnetic beads into the carboxyl magnetic beads, and washing for 20-30 min in a shaking way; placing the mixture in a magnetic field for 15-20 min, and then sucking the supernatant;
sd 3: repeating the operation of Sd2 for 0-3 times;
sd 4: fixing the volume of the carboxyl magnetic beads to 10-50 mg/mL by using the magnetic particle buffer solution, and uniformly mixing to obtain a carboxyl magnetic bead buffer solution;
sd 5: according to the mass ratio of the carboxyl magnetic beads to the fluorescein isothiocyanate antibodies (90-110): 1, adding the anti-fluorescein isothiocyanate antibody into the carboxyl magnetic bead buffer solution, and reacting for 16-20 h at 2-8 ℃ in a uniformly mixed state;
sd 6: washing a product prepared from Sd5 for 0-3 times by using a phosphate buffer solution, and then fixing the volume to 9-11 mg/mL to obtain the product;
the luminescent substrate is prepared by adding a reagent ALPS into a luminescent substrate buffer solution with the volume 4-10 times that of the reagent ALPS.
2. The thyroglobulin antibody detection kit of claim 1, characterized in that: the buffer solution containing the newborn bovine serum comprises preservatives tetracycline 0.01-0.05% and neomycin sulfate 0.1-0.5%, and the buffer solution containing the newborn bovine serum is filtered by a 0.22 mu m filter membrane.
3. The thyroglobulin antibody detection kit of claim 1, characterized in that: the anti-reagent buffer solution comprises 0.1-0.4 mM of Tris salt in purified water, and further comprises the following components in percentage by volume: 0.01-0.05% of tetracycline, 1-5% of sheep serum, 3-10% of newborn bovine serum and 1-5% of horse serum; the pH value of the anti-reagent buffer solution is 7-8.
4. The thyroglobulin antibody detection kit of claim 1, characterized in that: the surfactant is one or more of Tween20, TritonX-100 and Bronidox, and the volume percentage of the surfactant in the anti-reagent buffer is 0.01-0.5%.
5. The thyroglobulin antibody detection kit of claim 1, characterized in that: the luminescence bottom buffer solution comprises Tris0.1-1M and also comprises the following components in concentration: 0.1% of sodium sulfite, 0.3% of SDS 1%, 0.3% of lucigenin and 0.15% of bovine serum albumin; the pH of the luminescence bottom buffer solution is 9.5.
6. The thyroglobulin antibody detection kit of claim 1, characterized in that: the magnetic particle buffer solution is a solution prepared by dissolving 12.12-15.26 mg of Tris, 5.82-8.58 mg of sodium chloride and 50-60 g of methyl cellulose ether in per liter of purified water.
7. The thyroglobulin antibody detection kit of claim 1, characterized in that: the cleaning solution is prepared by dissolving 160g of NaCl, 4g of KCl, 24.2g of tris (hydroxymethyl) aminomethane and 1mL of Tween20 in per liter of double distilled water; the pH of the cleaning solution is 7.4.
8. A method of using the thyroglobulin antibody assay kit of any one of claims 1 to 7 wherein: the method comprises the following steps:
se 1: taking three test tubes, and respectively adding 30 mu L of the calibrator, 30 mu L of the quality control material and 30 mu L of a sample to be detected;
se 2: adding 60 μ L of the anti-reagent into each test tube, covering the test tube with a plastic film, gently shaking the test tube for 30s, and placing in a water bath at 37 ℃ for 5 min;
se 3: adding 30 μ L of the magnetic particle reagent into each test tube, covering the test tube with a plastic film, slightly oscillating the test tube for 30s, and placing in a water bath at 37 ℃ for 5 min;
se 4: precipitating all tubes on a magnetic separator for 2min, slowly inverting the tubes and the magnetic separator, and decanting the supernatant; placing the inverted test tube together with the magnetic separator on a filter paper, and patting the bottom of the magnetic separator to remove all droplets adhering to the tube wall;
se 5: adding 300 μ L of the cleaning solution to each test tube, covering the test tube with a plastic film, gently shaking the test tube for 30s, slowly inverting the test tube and the magnetic separator after mixing, pouring out the supernatant, placing the inverted test tube together with the magnetic separator on a filter paper, and strongly slapping the bottom of the separator to remove all droplets adhering to the tube wall;
se 6: repeating the operation of Se5 for 0-3 times;
se 7: adding 200 mu L of the luminescent substrate into each test tube, shaking and uniformly mixing for 3s, and detecting the luminous intensity by using a chemiluminescence apparatus.
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| CN112540174A (en) * | 2020-12-02 | 2021-03-23 | 泰州泽成生物技术有限公司 | Novel coronavirus IgG detection kit and preparation method thereof |
| CN113655217A (en) * | 2021-08-19 | 2021-11-16 | 四川沃文特生物技术有限公司 | Magnetic bead, kit, preparation method and application |
| CN114200132A (en) * | 2021-11-05 | 2022-03-18 | 江苏省人民医院（南京医科大学第一附属医院） | Kit for detecting thyroglobulin antibody and subtype thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112540175A (en) * | 2020-12-02 | 2021-03-23 | 泰州泽成生物技术有限公司 | Novel coronavirus IgM detection kit and preparation method and application thereof |
| CN112540174A (en) * | 2020-12-02 | 2021-03-23 | 泰州泽成生物技术有限公司 | Novel coronavirus IgG detection kit and preparation method thereof |
| CN113655217A (en) * | 2021-08-19 | 2021-11-16 | 四川沃文特生物技术有限公司 | Magnetic bead, kit, preparation method and application |
| CN114200132A (en) * | 2021-11-05 | 2022-03-18 | 江苏省人民医院（南京医科大学第一附属医院） | Kit for detecting thyroglobulin antibody and subtype thereof |
| CN114200132B (en) * | 2021-11-05 | 2022-12-09 | 江苏省人民医院（南京医科大学第一附属医院） | Kit for detecting thyroglobulin antibody and subtype thereof |
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Application publication date: 20200519 |