CN111208291A - Kit for detecting content of helicobacter pylori antibody in serum and use method thereof - Google Patents
Kit for detecting content of helicobacter pylori antibody in serum and use method thereof Download PDFInfo
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Classifications
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- G—PHYSICS
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- 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
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
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- 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
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- 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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56922—Campylobacter
Abstract
The invention discloses a kit for detecting the content of helicobacter pylori antibody in blood serum and a use method thereof in the related technical field of biochemistry, the kit for detecting the content of the helicobacter pylori antibody in blood serum comprises an anti-reagent A, an anti-reagent B, a magnetic particle reagent, a calibrator, a quality control product, a cleaning solution and a luminescent substrate which are packaged independently; the invention adopts magnetic particles as a solid phase of immunoreaction, utilizes a chemiluminescence enzyme-linked immunoassay method to be matched with a chemiluminescence measuring instrument, is used for measuring the content of the helicobacter pylori antibody in human serum, can simultaneously measure a plurality of samples on a full-automatic chemiluminescence instrument, realizes high-flux rapid measurement of the helicobacter pylori antibody, and has high accuracy, strong specificity and greatly improved accuracy and detection efficiency.
Description
Technical Field
The invention relates to the related technical field of biochemistry, in particular to a kit for detecting the content of helicobacter pylori antibody in serum and a use method thereof.
Background
Helicobacter pylori is a gram-negative microaerophilic bacterium that is bent, coiled or S-shaped. In 1979, the first time that H.pylori was isolated and cultured from the gastric mucosa by Warren and Marshall, the leading cause of peptic ulcer and chronic gastritis was subsequently found to be H.pylori. Helicobacter pylori belongs to clinically common gram-negative bacilli, is mainly distributed in the stomach, duodenum and other areas of an organism, can cause chronic inflammation of gastric mucosa, even gastric and duodenal ulcers or gastric cancer of severe patients, directly threatens the life safety of patients, and is mainly concerned by the medical field. The helicobacter pylori can easily penetrate through gastric mucosa, so that the helicobacter pylori grows on gastric epithelial cells, simultaneously promotes the production of a large amount of urease, and can form alkaline ammonia cloud around the bacteria after the urease is decomposed, so that the acidic environment in the stomach is resisted, and the helicobacter pylori is prevented from being killed by gastric acid. Helicobacter pylori is one of the currently recognized pathogenic factors of chronic gastritis, peptic ulcer and gastric cancer, and has been classified as a class I carcinogenic source by the world health organization. The Helicobacter Pylori (HP) infection rate is extremely high, and accounts for 30-50% of adults in developed countries, 40-80% in developing countries, and 59% in average in 40-90% in Chinese epidemic investigation results. The helicobacter pylori comprises three types of CanA, Urease and VacA, wherein the CagA has no cytotoxicity and may have close correlation with the transcription capability of the VacA; whereas VacA can alter the vacuole-like appearance of epithelial cells and cause them to become damaged, necrotic and even ulcerated.
Currently known methods for assaying helicobacter pylori antibodies include enzyme-linked immunosorbent assay (ELISA), latex-enhanced turbidimetry, and the like. 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.
Disclosure of Invention
Aiming at the technical problems in the prior art, the invention provides a kit for detecting the content of helicobacter pylori antibody in serum and a using method thereof.
Based on the following technical principle: fluorescein Isothiocyanate (FITC) -labeled HP antigen was conjugated with Alkaline Phosphatase (AP) -labeled HP antigen and HP antibody in the samples, calibrators, or quality controls to form a "sandwich" complex. Subsequently, magnetic particles with anti-FITC antibodies attached thereto are added, and the antigen-antibody immune complex is bound to the magnetic particles by the specific binding of the anti-FITC antibodies to FITC. Separating the complex formed by immunoreaction from other unbound substances under the action of an external magnetic field, washing the complex, and adding an enzymatic chemiluminescent substrate. The substrate is catalytically cracked under the action of enzyme to form an unstable excited intermediate, and when the excited intermediate returns to the ground state, photons are emitted to form a luminescence reaction, namely, a chemiluminescence apparatus is used for detecting the luminescence intensity of the reaction. Within the detection range, the luminous intensity is in direct proportion to the content of the HP antibody in the sample, and the concentration of the HP antibody in the sample can be calculated by using improved four-parameter Logistic equation fitting.
The technical scheme of the invention is as follows:
the kit for detecting the content of the helicobacter pylori antibody in serum comprises an anti-reagent A, an anti-reagent B, a magnetic particle reagent, a calibrator, a quality control product, a cleaning solution and a luminescent substrate which are packaged independently;
the anti-reagent A is BSA buffer solution containing a fluorescein isothiocyanate labeled helicobacter pylori antibody conjugate; the anti-reagent A is prepared by the following steps:
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: adding the fluorescein isothiocyanate solution which is 1.1-1.5 times of the mass of the helicobacter pylori antibody into the helicobacter pylori antibody and fully mixing;
sa 3: adjusting the pH balance by using a carbonate buffer solution with the pH of 8-9, and separating and purifying by using gel chromatography to prepare a fluorescein isothiocyanate-labeled helicobacter pylori antibody-coated antibody;
the anti-reagent B is a buffer solution of BSA (bovine serum albumin) containing an alkaline phosphatase-labeled helicobacter pylori antigen conjugate; the anti-reagent B is prepared by the following operation steps:
sb 1: preparing alkaline phosphatase into an alkaline phosphatase solution with the concentration of 1.0-5.0 mg/mL by using an anti-reagent buffer solution;
sb 2: respectively activating the reactive groups by using helicobacter pylori antigens and alkaline phosphatase, and then respectively activating the reactive groups by using the alkaline phosphatase: the helicobacter pylori antigens are mixed fully and uniformly in a ratio of 1: 1-1: 3 under the catalysis of a catalyst to carry out coupling reaction;
sb 3: regulating the pH balance by using Tris buffer solution with the pH of 8-9, and separating and purifying by using a gel column to obtain an alkaline phosphatase-labeled helicobacter pylori antigen conjugate;
sb 4: screening out the molecular fragment with the optimal effect for testing;
the magnetic particle reagent passes through BSA buffer solution containing anti-fluorescein isothiocyanate goat anti-FITC and carboxyl magnetic bead conjugate;
the calibrator and the quality control product are both prepared by the following steps: adding 0.01-0.05 g of tetracycline and 0.1-0.5 g of neomycin sulfate into 1L of BSA-containing buffer solution, then adding helicobacter pylori antibody and fully dissolving;
the cleaning solution is prepared by dissolving 150-170 g of NaCl, 3-5 g of KCl, 24-24.4 g of tris (hydroxymethyl) aminomethane and 0.8-1.2 mL of Tween20 in 900mL of double distilled water, adjusting the pH value to 7.2-7.6 by using HCl, and finally fixing the volume to 1000mL by using the double distilled water;
the luminescent substrate is prepared by dissolving ALPS in a buffer.
Further, the anti-reagent buffer solution is prepared by the following operation steps: accurately weighing the following components: 10-20 gNa2PO3H·12H2O、1～2gNaPO3H2·12H2O, 3-10 g of newborn bovine serum; adding the weighed components into 1L of purified water, and fully stirring until the components are completely dissolved; and adjusting the pH value to 5-6 to obtain the product.
Further, the reagent for adjusting pH was 4M HCl.
Further, Sa2 was operated under light-shielding conditions.
Further, the calibrator and the quality control material are both treated by 0.22 μm filter membranes.
Further, the magnetic particle reagent is prepared by the operation comprising the following steps:
sc 1: placing the fully and uniformly mixed carboxyl magnetic bead concentrated solution in a magnetic field for 15-20 min, absorbing a supernatant after all the carboxyl magnetic beads are settled, adding a magnetic particle buffer solution with the volume 2-5 times that of the carboxyl magnetic beads into the carboxyl magnetic beads, shaking and cleaning for 20-30 min, placing in the magnetic field for 15-20 min, and absorbing the supernatant; repeatedly cleaning the carboxyl magnetic beads for 1-3 times; finally, fixing the volume of the mixed solution of the carboxyl magnetic beads to 10-50 mg/mL to prepare a carboxyl magnetic bead solution;
sc 2: adding fluorescein isothiocyanate-resistant goat anti-FITC with the mass of 0.8-1.2% of that of the carboxyl magnetic bead solution into the carboxyl magnetic bead solution, and reacting for 16-20 h at the temperature of 2-8 ℃ in a uniformly mixed state;
sc 3: and (3) placing the solution prepared by the Sc2 in a magnetic field for 12-18 min, washing the solution for 1-3 times by using a magnetic particle buffer solution after the carboxyl magnetic beads are settled, then fixing the volume to 8-12 mg/mL, and storing at 2-8 ℃.
Further, the magnetic particle buffer solution is prepared by adding 12.12-15.26 mg of Tris, 5.82-8.58 mg of NaCl and 50-60 g of methyl cellulose ether into 1L of purified water, and fully stirring until the mixture is completely dissolved.
The use method of the kit for detecting the content of the helicobacter pylori antibody in the serum comprises the following steps:
sd 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;
sd 2: adding 30 μ L of the anti-reagent A and 30 μ L of the anti-reagent B into each test tube, covering the test tubes with a plastic film, slightly oscillating the test tubes for 30s, and placing in a water bath at 37 ℃ for 5 min;
sd 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;
sd 4: precipitating all tubes on a magnetic separator for 3min, 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;
sd 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;
sd 6: 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.
Further, adding a surfactant which accounts for 0.01-0.5% of the weight of the solution in each test tube into each test tube in the operation of Sd 2; the surfactant is one or more of Tween20, TritonX-100 and Bronidox.
Further, the operation of Sd5 includes Sd 5.1: repeating the operation of Sd5 for 1-3 times.
The invention has the following advantages:
1. the invention combines the chemiluminescence technology with the immunomagnetic particles, provides a reaction system close to homogeneous phase, adopts a one-step reaction mode, greatly improves the detection performances including sensitivity, precision and detection range, greatly shortens the reaction time, and is obviously faster than similar kits, and the time from sample adding to detection result is less than 35 min;
2. the method has the advantages of high coupling efficiency, firm combination and stable process, and greatly reduces the product cost while improving the product performance.
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 shows the correlation between the detection results of the kit of the present invention and the commercially available H.pylori antibody kit.
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.
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
The kit for detecting the content of the helicobacter pylori antibody in serum comprises an anti-reagent A, an anti-reagent B, a magnetic particle reagent, a calibrator, a quality control product, a cleaning solution and a luminescent substrate which are packaged independently;
the anti-reagent A is BSA buffer solution containing a fluorescein isothiocyanate labeled helicobacter pylori antibody conjugate; the anti-reagent A is prepared by the following operation:
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 is bufferedThe liquid is prepared by the following steps: accurately weighing the following components: 10gNa2PO3H·12H2O、1gNaPO3H2·12H2O, 3g newborn bovine serum; adding the weighed components into 1L of purified water, and fully stirring until the components are completely dissolved; adjusting pH to 5 with 4M HCl;
sa 2: adding a fluorescein isothiocyanate solution with the mass 1.1 times that of the helicobacter pylori antibody into the helicobacter pylori antibody under the condition of keeping out of the sun, and fully mixing;
sa 3: adjusting pH balance with carbonate buffer solution with pH of 8, and separating and purifying by gel chromatography to obtain fluorescein isothiocyanate labeled helicobacter pylori antibody coated antibody;
the anti-reagent B is a buffer solution of BSA (bovine serum albumin) containing an alkaline phosphatase-labeled helicobacter pylori antigen conjugate; the anti-reagent B is prepared by the following operation:
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 reactive groups by using helicobacter pylori antigens and alkaline phosphatase, and then respectively activating the reactive groups by using the alkaline phosphatase: the helicobacter pylori antigen is in a ratio of 1:1, and is fully and uniformly mixed under the catalysis of a catalyst to carry out coupling reaction;
sb 3: regulating pH balance with Tris buffer solution with pH of 8, and separating and purifying with gel column to obtain alkaline phosphatase-labeled helicobacter pylori antigen conjugate;
the magnetic particle reagent passes through BSA buffer solution containing anti-fluorescein isothiocyanate goat anti-FITC and carboxyl magnetic bead conjugate; the magnetic particle reagent is prepared by the following operation steps:
sc 1: placing the fully and uniformly mixed carboxyl magnetic bead concentrated solution in a magnetic field for 15min, absorbing the supernatant after the carboxyl magnetic beads are completely settled, adding a magnetic particle buffer solution with the volume 2 times that of the carboxyl magnetic beads into the carboxyl magnetic beads, shaking and cleaning for 20min, placing in the magnetic field for 15min, and absorbing the supernatant; repeatedly cleaning the carboxyl magnetic beads for 1 time; finally, fixing the volume of the mixed solution of the carboxyl magnetic beads to 10mg/mL to prepare a carboxyl magnetic bead solution;
sc 2: adding fluorescein isothiocyanate-resistant goat anti-FITC with the mass of 0.8% of that of the carboxyl magnetic bead solution into the carboxyl magnetic bead solution, and reacting for 16h at the temperature of 2 ℃ in a uniformly mixed state;
sc 3: placing the solution prepared by Sc2 in a magnetic field for 12min, washing for 1 time by using magnetic particle buffer after the carboxyl magnetic beads are settled, then fixing the volume to 8mg/mL, and storing at 2 ℃;
wherein, the magnetic particle buffer solution is prepared by adding 12.12mg of Tris, 5.82mg of NaCl and 50g of methyl cellulose ether into 1L of purified water and fully stirring until the Tris, the NaCl and the methyl cellulose ether are completely dissolved;
the calibrator and the quality control product are both prepared by the following steps: adding 0.01g of tetracycline and 0.1g of neomycin sulfate into 1L of BSA-containing buffer solution, adding helicobacter pylori antibody, fully dissolving, and treating with 0.22 μm filter membrane;
the cleaning solution is prepared by dissolving 150g of NaCl, 3g of KCl, 24g of tris (hydroxymethyl) aminomethane and 0.8mL of Tween20 in 900mL of double distilled water, adjusting the pH value to 7.2 by using HCl, and finally fixing the volume to 1000mL by using the double distilled water;
the luminogenic substrate was prepared by dissolving ALPS in a buffer.
Example 2
The kit for detecting the content of the helicobacter pylori antibody in serum comprises an anti-reagent A, an anti-reagent B, a magnetic particle reagent, a calibrator, a quality control product, a cleaning solution and a luminescent substrate which are packaged independently;
the anti-reagent A is BSA buffer solution containing a fluorescein isothiocyanate labeled helicobacter pylori antibody conjugate; the anti-reagent A is prepared by the following operation:
sa 1: preparing fluorescein isothiocyanate into a fluorescein isothiocyanate solution with the concentration of 3.0mg/mL by using an anti-reagent buffer solution;
the anti-reagent buffer solution is prepared by the following steps: accurately weighing the following components: 15gNa2PO3H·12H2O、1.5gNaPO3H2·12H2O, 6g newborn bovine serum; adding the weighed components into1L of purified water is fully stirred until the purified water is completely dissolved; adjusting pH to 5.5 with 4M HCl;
sa 2: adding a fluorescein isothiocyanate solution with the mass 1.3 times that of the helicobacter pylori antibody into the helicobacter pylori antibody under the condition of keeping out of the sun, and fully mixing;
sa 3: adjusting pH balance with carbonate buffer solution with pH of 8.5, and separating and purifying by gel chromatography to obtain fluorescein isothiocyanate-labeled helicobacter pylori antibody-coated antibody;
the anti-reagent B is a buffer solution of BSA (bovine serum albumin) containing an alkaline phosphatase-labeled helicobacter pylori antigen conjugate; the anti-reagent B is prepared by the following operation:
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 reactive groups by using helicobacter pylori antigens and alkaline phosphatase, and then respectively activating the reactive groups by using the alkaline phosphatase: the helicobacter pylori antigen is in a ratio of 1:2, and is fully and uniformly mixed under the catalysis of a catalyst to carry out coupling reaction;
sb 3: regulating pH balance with Tris buffer solution with pH of 8.5, and separating and purifying with gel column to obtain alkaline phosphatase-labeled helicobacter pylori antigen conjugate;
the magnetic particle reagent passes through BSA buffer solution containing anti-fluorescein isothiocyanate goat anti-FITC and carboxyl magnetic bead conjugate; the magnetic particle reagent is prepared by the following operation steps:
sc 1: placing the fully and uniformly mixed carboxyl magnetic bead concentrated solution in a magnetic field for 18min, absorbing the supernatant after the carboxyl magnetic beads are completely settled, adding a magnetic particle buffer solution with the volume 4 times that of the carboxyl magnetic beads into the carboxyl magnetic beads, shaking and cleaning for 25min, placing in the magnetic field for 18min, and absorbing the supernatant; repeatedly washing the carboxyl magnetic beads for 2 times; finally, fixing the volume of the mixed solution of the carboxyl magnetic beads to 30mg/mL to prepare a carboxyl magnetic bead solution;
sc 2: adding fluorescein isothiocyanate-resistant goat anti-FITC (fluorescein isothiocyanate) with the mass of 1% of that of the carboxyl magnetic bead solution into the carboxyl magnetic bead solution, and reacting for 18h at the temperature of 4 ℃ in a uniformly mixed state;
sc 3: placing the solution prepared by Sc2 in a magnetic field for 15min, washing for 2 times by using magnetic particle buffer after the carboxyl magnetic beads are settled, then fixing the volume to 10mg/mL, and storing at 4 ℃;
wherein, the magnetic particle buffer solution is prepared by adding 14.26mg of Tris, 6.58mg of NaCl and 55g of methyl cellulose ether into 1L of purified water and fully stirring until the Tris and the NaCl are completely dissolved;
the calibrator and the quality control product are both prepared by the following steps: adding 0.03g of tetracycline and 0.3g of neomycin sulfate into 1L of BSA-containing buffer solution, adding helicobacter pylori antibody, fully dissolving, and treating with 0.22 μm filter membrane;
the cleaning solution is prepared by dissolving 160g of NaCl, 4g of KCl, 24.2g of tris (hydroxymethyl) aminomethane and 1mL of Tween20 in 900mL of double distilled water, adjusting the pH value to 7.4 by using HCl, and finally fixing the volume to 1000mL by using the double distilled water;
the luminogenic substrate was prepared by dissolving ALPS in a buffer.
Example 3
The kit for detecting the content of the helicobacter pylori antibody in serum comprises an anti-reagent A, an anti-reagent B, a magnetic particle reagent, a calibrator, a quality control product, a cleaning solution and a luminescent substrate which are packaged independently;
the anti-reagent A is BSA buffer solution containing a fluorescein isothiocyanate labeled helicobacter pylori antibody conjugate; the anti-reagent A is prepared by the following operation:
sa 1: preparing fluorescein isothiocyanate into a fluorescein isothiocyanate solution with the concentration of 5.0mg/mL by using an anti-reagent buffer solution;
the anti-reagent buffer solution is prepared by the following steps: accurately weighing the following components: 20gNa2PO3H·12H2O、2gNaPO3H2·12H2O, 10g newborn calf serum; adding the weighed components into 1L of purified water, and fully stirring until the components are completely dissolved; adjusting pH to 6 with 4M HCl;
sa 2: adding a fluorescein isothiocyanate solution with the mass 1.5 times that of the helicobacter pylori antibody into the helicobacter pylori antibody under the condition of keeping out of the sun, and fully mixing;
sa 3: adjusting pH balance with carbonate buffer solution with pH of 9, and separating and purifying by gel chromatography to obtain fluorescein isothiocyanate-labeled helicobacter pylori antibody-coated antibody;
the anti-reagent B is a buffer solution of BSA (bovine serum albumin) containing an alkaline phosphatase-labeled helicobacter pylori antigen conjugate; the anti-reagent B is prepared by the following operation:
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 reactive groups by using helicobacter pylori antigens and alkaline phosphatase, and then respectively activating the reactive groups by using the alkaline phosphatase: the helicobacter pylori antigen is in a ratio of 1:3, and is fully and uniformly mixed under the catalysis of a catalyst to carry out coupling reaction;
sb 3: regulating pH balance with Tris buffer solution with pH of 9, and separating and purifying with gel column to obtain alkaline phosphatase-labeled helicobacter pylori antigen conjugate;
the magnetic particle reagent passes through BSA buffer solution containing anti-fluorescein isothiocyanate goat anti-FITC and carboxyl magnetic bead conjugate; the magnetic particle reagent is prepared by the following operation steps:
sc 1: placing the fully and uniformly mixed carboxyl magnetic bead concentrated solution in a magnetic field for 20min, absorbing the supernatant after the carboxyl magnetic beads are completely settled, adding a magnetic particle buffer solution with the volume 5 times that of the carboxyl magnetic beads into the carboxyl magnetic beads, shaking and cleaning for 30min, placing in the magnetic field for 20min, and absorbing the supernatant; repeatedly cleaning the carboxyl magnetic beads for 3 times; finally, fixing the volume of the mixed solution of the carboxyl magnetic beads to 50mg/mL to prepare a carboxyl magnetic bead solution;
sc 2: adding fluorescein isothiocyanate-resistant goat anti-FITC (fluorescein isothiocyanate) with the mass of 1.2% of that of the carboxyl magnetic bead solution into the carboxyl magnetic bead solution, and reacting for 20 hours in a uniformly mixed state at the temperature of 8 ℃;
sc 3: placing the solution prepared by Sc2 in a magnetic field for 18min, washing for 3 times by using magnetic particle buffer after the carboxyl magnetic beads are settled, then fixing the volume to 12mg/mL, and storing at 8 ℃;
wherein, the magnetic particle buffer solution is prepared by adding 15.26mg of Tris, 8.58mg of NaCl and 60g of methyl cellulose ether into 1L of purified water and fully stirring until the Tris and the NaCl are completely dissolved;
the calibrator and the quality control product are both prepared by the following steps: adding 0.05g of tetracycline and 0.5g of neomycin sulfate into 1L of BSA-containing buffer solution, adding helicobacter pylori antibody, fully dissolving, and treating with 0.22 μm filter membrane;
the cleaning solution is prepared by dissolving 170g of NaCl, 5g of KCl, 24.4g of tris (hydroxymethyl) aminomethane and 1.2mL of Tween20 in 900mL of double distilled water, adjusting the pH value to 7.6 by using HCl, and finally fixing the volume to 1000mL by using the double distilled water;
the luminogenic substrate was prepared by dissolving ALPS in a buffer.
Example 4
The method for using the kit for detecting the content of the helicobacter pylori antibody in the blood serum of any one of embodiments 1 to 3, which is used for detecting the content of the helicobacter pylori antibody in the blood serum prepared in embodiment 2, and comprises the following steps:
1. sample collection
Serum was collected using the correct medical technique (severely hemolyzed or lipemic specimens could not be used for the assay), and the collected specimens were allowed to stand at room temperature for no more than 8 hours; if the detection is not carried out within 8 hours, the sample needs to be placed in a refrigerator at the temperature of 2-8 ℃; if the food needs to be stored or transported for more than 72 hours, the food should be frozen below-20 ℃ to avoid repeated freezing and thawing. Before use, return to room temperature and mix by gentle shaking.
2. Preparation before experiment
2.1 taking a bottle of concentrated washing liquor to dilute by 15 times by using distilled water;
2.2, adjusting the temperature of the constant temperature box or the water bath kettle to 37 ℃, and using the constant temperature box or the water bath kettle after the temperature is stable;
2.3 mix the magnetic particle suspension well until no visible precipitation.
3. Detection operation
Sd 1: numbering three test tubes, and respectively adding 30 mu L of calibrator, 30 mu L of quality control material and 30 mu L of sample to be tested;
sd 2: adding 30 μ L of the anti-reagent A, 30 μ L of the anti-reagent B and a surfactant into each test tube, covering the test tubes with a plastic film, slightly oscillating the test tubes for 30s, and placing in a water bath at 37 ℃ for 5 min;
wherein the surfactant is Tween20, and in specific implementation, TritonX-100 or Bronidox can be used for substitution, and the surfactant accounts for 0.2% of the weight of the solution in the corresponding test tube, and in specific implementation, the surfactant can be selected at will between 0.01% and 0.5%;
sd 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;
sd 4: precipitating all test tubes on a magnetic separator for 3min, slowly inverting the test tubes and 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;
sd 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; the operation of the part can be repeated for 1-3 times during specific implementation;
sd 6: 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.
And establishing a calibration curve by adopting a four-parameter fitting mode and taking the concentration value of the calibrator as an X axis and the luminous intensity value of the calibrator as a Y axis, and calculating the corresponding concentration value according to the luminous intensity value of the sample to be detected.
The kit disclosed by the invention can be identified according to methodology to achieve the following indexes:
the standard curve is linear: r is more than 0.9900;
the lowest detection limit is: less than or equal to 0.1U/ml;
accuracy: the adding recovery rate is 85-115%;
repeatability: coefficient of variation CV is less than or equal to 8 percent;
inter-batch difference: the coefficient of variation is less than or equal to 15 percent;
linear dilution: r is greater than 0.9900.
As shown in FIG. 1, the correlation between the detection results of the kit of the present invention and the detection results of the commercially available H.pylori antibody kit was good when the measurement values of 100 serum samples were compared with those of the commercially available H.pylori antibody kit A.
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 (10)
1. The kit for detecting the content of the helicobacter pylori antibody in serum is characterized in that: comprises an anti-reagent A, an anti-reagent B, a magnetic particle reagent, a calibrator, a quality control product, a cleaning solution and a luminescent substrate which are packaged independently;
the anti-reagent A is BSA buffer solution containing a fluorescein isothiocyanate labeled helicobacter pylori antibody conjugate; the anti-reagent A is prepared by the following steps:
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: adding the fluorescein isothiocyanate solution which is 1.1-1.5 times of the mass of the helicobacter pylori antibody into the helicobacter pylori antibody and fully mixing;
sa 3: adjusting the pH balance by using a carbonate buffer solution with the pH of 8-9, and separating and purifying by using gel chromatography to prepare a fluorescein isothiocyanate-labeled helicobacter pylori antibody-coated antibody;
the anti-reagent B is a buffer solution of BSA (bovine serum albumin) containing an alkaline phosphatase-labeled helicobacter pylori antigen conjugate; the anti-reagent B is prepared by the following operation steps:
sb 1: preparing alkaline phosphatase into an alkaline phosphatase solution with the concentration of 1.0-5.0 mg/mL by using an anti-reagent buffer solution;
sb 2: respectively activating the reactive groups by using helicobacter pylori antigens and alkaline phosphatase, and then respectively activating the reactive groups by using the alkaline phosphatase: the helicobacter pylori antigens are mixed fully and uniformly in a ratio of 1: 1-1: 3 under the catalysis of a catalyst to carry out coupling reaction;
sb 3: regulating the pH balance by using Tris buffer solution with the pH of 8-9, and separating and purifying by using a gel column to obtain an alkaline phosphatase-labeled helicobacter pylori antigen conjugate;
the magnetic particle reagent passes through BSA buffer solution containing anti-fluorescein isothiocyanate goat anti-FITC and carboxyl magnetic bead conjugate;
the calibrator and the quality control product are both prepared by the following steps: adding 0.01-0.05 g of tetracycline and 0.1-0.5 g of neomycin sulfate into 1L of BSA-containing buffer solution, then adding helicobacter pylori antibody and fully dissolving;
the cleaning solution is prepared by dissolving 150-170 g of NaCl, 3-5 g of KCl, 24-24.4 g of tris (hydroxymethyl) aminomethane and 0.8-1.2 mL of Tween20 in 900mL of double distilled water, adjusting the pH value to 7.2-7.6 by using HCl, and finally fixing the volume to 1000mL by using the double distilled water;
the luminescent substrate is prepared by dissolving ALPS in a buffer.
2. The kit for detecting the content of helicobacter pylori antibody in serum according to claim 1, wherein: the anti-reagent buffer solution is prepared by the following steps: accurately weighing the following components: 10-20 gNa2PO3H·12H2O、1～2gNaPO3H2·12H2O, 3-10 g of newborn bovine serum; adding the weighed components into 1L of purified water, and fully stirring until the components are completely dissolved; and adjusting the pH value to 5-6 to obtain the product.
3. The kit for detecting the content of helicobacter pylori antibody in serum according to claim 2, wherein: the pH adjusting reagent was 4M HCl.
4. The kit for detecting the content of helicobacter pylori antibody in serum according to claim 1, wherein: sa2 was operated under dark conditions.
5. The kit for detecting the content of helicobacter pylori antibody in serum according to claim 1, wherein: and the calibrator and the quality control product are both treated by a 0.22 mu m filter membrane.
6. The kit for detecting the content of helicobacter pylori antibody in serum according to claim 1, wherein: the magnetic particle reagent is prepared by the following operation steps:
sc 1: placing the fully and uniformly mixed carboxyl magnetic bead concentrated solution in a magnetic field for 15-20 min, absorbing a supernatant after all the carboxyl magnetic beads are settled, adding a magnetic particle buffer solution with the volume 2-5 times that of the carboxyl magnetic beads into the carboxyl magnetic beads, shaking and cleaning for 20-30 min, placing in the magnetic field for 15-20 min, and absorbing the supernatant; repeatedly cleaning the carboxyl magnetic beads for 1-3 times; finally, fixing the volume of the mixed solution of the carboxyl magnetic beads to 10-50 mg/mL to prepare a carboxyl magnetic bead solution;
sc 2: adding fluorescein isothiocyanate-resistant goat anti-FITC with the mass of 0.8-1.2% of that of the carboxyl magnetic bead solution into the carboxyl magnetic bead solution, and reacting for 16-20 h at the temperature of 2-8 ℃ in a uniformly mixed state;
sc 3: and (3) placing the solution prepared by the Sc2 in a magnetic field for 12-18 min, washing the solution for 1-3 times by using a magnetic particle buffer solution after the carboxyl magnetic beads are settled, then fixing the volume to 8-12 mg/mL, and storing at 2-8 ℃.
7. The kit for detecting the content of helicobacter pylori antibody in serum according to claim 6, wherein: the magnetic particle buffer solution is prepared by adding 12.12-15.26 mg of Tris, 5.82-8.58 mg of NaCl and 50-60 g of methyl cellulose ether into 1L of purified water, and fully stirring until the Tris, the NaCl and the methyl cellulose ether are completely dissolved.
8. The use method of the kit for detecting the content of helicobacter pylori antibody in serum according to any one of claims 1 to 6, wherein: the method comprises the following steps:
sd 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;
sd 2: adding 30 μ L of the anti-reagent A and 30 μ L of the anti-reagent B into each test tube, covering the test tubes with a plastic film, slightly oscillating the test tubes for 30s, and placing in a water bath at 37 ℃ for 5 min;
sd 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;
sd 4: precipitating all tubes on a magnetic separator for 3min, 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;
sd 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;
sd 6: 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.
9. The use method of the kit for detecting the content of the helicobacter pylori antibody in the serum according to claim 8, wherein: adding a surfactant which accounts for 0.01-0.5% of the weight of the solution in each test tube into each test tube in the operation of Sd 2; the surfactant is one or more of Tween20, TritonX-100 and Bronidox.
10. The use method of the kit for detecting the content of helicobacter pylori antibody in serum according to claim 8 or 9, characterized in that: operation of Sd5 also includes Sd 5.1: repeating the operation of Sd5 for 1-3 times.
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| US5932430A (en) * | 1996-05-09 | 1999-08-03 | Meridian Diagnostics, Inc. | Immunoassay for H. pylori in fecal specimens |
| CN108196043A (en) * | 2017-11-28 | 2018-06-22 | 泰州泽成生物技术有限公司 | Kit of microdose urine protein content and preparation method thereof in a kind of detection serum |
| CN108205059A (en) * | 2017-11-28 | 2018-06-26 | 泰州泽成生物技术有限公司 | A kind of kit and its test method for measuring calcitonin content |
| CN110244040A (en) * | 2019-06-04 | 2019-09-17 | 江苏泽成生物技术有限公司 | A kind of free estriol (fE3) detection kit and the preparation method and application thereof |
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2020
- 2020-02-27 CN CN202010124942.6A patent/CN111208291A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5932430A (en) * | 1996-05-09 | 1999-08-03 | Meridian Diagnostics, Inc. | Immunoassay for H. pylori in fecal specimens |
| CN108196043A (en) * | 2017-11-28 | 2018-06-22 | 泰州泽成生物技术有限公司 | Kit of microdose urine protein content and preparation method thereof in a kind of detection serum |
| CN108205059A (en) * | 2017-11-28 | 2018-06-26 | 泰州泽成生物技术有限公司 | A kind of kit and its test method for measuring calcitonin content |
| CN110244040A (en) * | 2019-06-04 | 2019-09-17 | 江苏泽成生物技术有限公司 | A kind of free estriol (fE3) detection kit and the preparation method and application thereof |
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