CN112147341A - Time-resolved fluorescence immunoassay kit for detecting gastrin G-17, and preparation method and detection method thereof - Google Patents
Time-resolved fluorescence immunoassay kit for detecting gastrin G-17, and preparation method and detection method thereof Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
- G01N33/534—Production of labelled immunochemicals with radioactive label
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- G—PHYSICS
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- 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
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- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/575—Hormones
- G01N2333/595—Gastrins; Cholecystokinins [CCK]
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/06—Gastro-intestinal diseases
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Abstract
The invention discloses a time-resolved fluorescence immunoassay kit for detecting gastrin G-17, a preparation method thereof and a detection method, belonging to the fields of immunoassay analysis technology and nano-biotechnology, wherein the kit comprises a reaction buffer solution, a cleaning solution, an enhancement solution, a magnetic particle solution coating a G-17 antibody, a europium-labeled G-17 antibody solution and 2 bottles of G-17 calibrator which are integrated on a reagent strip. The kit can provide a reaction system close to homogeneous phase, detect the content of G-17, shorten the detection time, improve the efficiency and provide convenience for clinical use.
Description
Technical Field
The invention belongs to the fields of immunoassay and nano-biotechnology, and particularly relates to a gastrin 17-based time-resolved fluorescence immunoassay kit, and a preparation method and a detection method thereof.
Background
Gastrin (gastrin) is an important gastrointestinal hormone, also known as gastrin, and is mainly secreted by G cells, and can promote the secretory function of the gastrointestinal tract, promote the contraction of the antrum and the stomach body, increase the motility of the gastrointestinal tract, and promote the contraction of the pyloric sphincter. The overall combined action is to slow down the gastric emptying, promote the division and proliferation of gastric and upper intestinal tract mucosa cells and promote the release of insulin and calcitonin. Gastrin 17(G-17) is mainly secreted by G cells at the antrum of the stomach, 80% -90% of gastrin can play a biological role, is an important index for reflecting the conditions of antrum atrophy and gastric mucosa injury, and is a novel gastric mucosa serological marker. The G-17 index can better reflect the severity of gastric mucosa atrophy and intestinal metaplasia, is closely related to gastric mucosa pathological changes, and can be used as auxiliary screening and diagnosis of gastric diseases
The detection of G-17 is carried out by adopting time-resolved fluoroimmunoassay (TRFIA). TRFIA is a new technology taking lanthanide chelate as a marker, has the characteristics of zero background, wide linear range, good stability and the like, and has the sensitivity of 10-18mol/L, 10 compared with ELISA, CLIA, ECLIA, etc-9～10-15The mol/L is higher, the main innovation of TRFIA lies in that lanthanide atoms with large fluorescence intensity, long decay time and small molecular weight are used for marking immune molecules instead of common macromolecular enzyme or radioactive isotope, so that the sensitivity and the measuring range of the established immune method are multiplied, and no radioactive pollution is caused. The fluorescence of the labeled ions by the time-resolved fluorescence immunoassay technology is a quasi-linear spectrum, and is characterized in that the wavelength range of exciting light is wider, and the peak range of an emission spectrum is narrower, so that the background fluorescence intensity is favorably reduced, and the detection resolution is improved; a larger Stokes shift exists between the excitation light and the emission light of the time-resolved fluorescence immune component, which is beneficial to eliminating the interference of non-specific fluorescence, thereby enhancing the specificity of measurement; the fluorescence intensity generated by the labeled ion chelate is high, and the service life is long, so that the influence of non-specific fluorescent substances in a sample and the environment on a detection result can be eliminated; each test is repeated 1000 times, and the average is takenThe fluorescence count value is taken as a result, thereby improving the accuracy of the detection. TRFIA is one of the main development directions of immunoassay at home and abroad.
Disclosure of Invention
Aiming at the defects in the prior art, the method integrates all reagents on one reagent strip by combining TRFIA and magnetic particle technology, establishes a G-17-TRFIA method, can be operated by full-automatic equipment, is accurate and high, can be detected by a single person, is convenient to use, is applied to the research of patients with stomach lesions, can more accurately identify and diagnose the stomach mucosa lesions, gastric ulcer, A-type atrophic gastritis and B-type atrophic gastritis, evaluates the curative effect of the stomach lesions by convenient serological examination, and provides a very convenient and fast new means for the diagnosis and treatment of the stomach lesions.
The technical scheme adopted by the invention for solving the technical problem is as follows: a time-resolved fluorescence immunoassay kit for detecting gastrin G-17 is disclosed, wherein the kit comprises at least one reagent rack, each reagent rack comprises at least five reagent strips, and the kit also comprises at least two bottles of G-17 calibrator; the reagent strip comprises a magnetic bead hole, a reaction buffer liquid hole, a europium label freeze-drying hole, a detection hole, a cleaning liquid hole, a reinforcing liquid hole and a plurality of reserved holes; magnetic particle solution coated with G-17 antibody is filled in the magnetic bead holes, reaction buffer solution is filled in the reaction buffer solution holes, europium-labeled G-17 antibody solution is filled in the europium-labeled freeze-dried holes, samples to be detected are filled in the detection holes, cleaning solution is filled in the cleaning solution holes, and enhancement solution is filled in the enhancement solution holes.
Furthermore, the reagent box comprises 5-20 reagent racks, and each reagent rack is provided with 5 reagent strips; the kit also comprises 2 bottles of G-17 calibrator.
Further, the G-17 antibody-coated magnetic particle solution is prepared by connecting activated magnetic particles with the diameter of 50-5000 nanometers with the G-17 antibody, washing and sealing; the europium-labeled G-17 antibody solution is Eu3+-N2- [ P-Isocyanato-benzyl group]The antibody G-17 is marked by sodium diethylenetriamine tetracetate, and the G-17 is preparedAntibody and the Eu3+-N2- [ P-Isocyanato-benzyl group]The mass ratio of the-sodium diethylenetriamine tetracetate is 5: 1.
Further, the reaction buffer is 50mmol/L Tris-HCl containing 8mmol/L NaCl, 0.1% BSA, 50. mu. mol/L DTPA, 0.1ml/L Tween-80 and 0.1% NaN at pH7.83(ii) a The cleaning solution is a buffer solution containing 0.48g/L of Tris, 12.49g/L of NaCl and 1.11g/L of Tween-20, and the pH value is adjusted to 7.8 by hydrochloric acid; the enhancement solution is a mixed solution containing glacial acetic acid with the volume concentration of 3.6 per thousand, sodium acetate with the volume concentration of 0.5g/L, beta-naphthoyl trifluoroacetone with the volume concentration of 0.05g/L, trioctylphosphine oxide with the volume concentration of 0.03g/L and Triton X-100 with the volume concentration of 1 per thousand; the G-17 calibrator was prepared by using 2G/L BSA and 1G/L NaN350mmol/L of Tris-HCl pH7.8 reaction buffer, and preparing G-17 into calibrator solutions with different concentrations.
A preparation method of a time-resolved fluoroimmunoassay kit for detecting gastrin G-17 comprises the following steps:
preparation of G-17 calibrator solution: taking high concentration G-17 antigen solution, adding 2G/L BSA and 1G/L NaN3Preparing 50mmol/L Tris-HCl reaction buffer solution with pH of 7.8 into calibrator solutions with different concentrations, subpackaging, and storing at 2-8 ℃;
preparation of magnetic particle solution coated with G-17 antibody: activating ferroferric oxide microspheres with the diameter of 50-5000 nm by using N-hydroxysuccinimide (NHS) and 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) with the mass concentration of 0.5-2%, uniformly mixing for 4 hours at room temperature, washing for 1-3 times by using 0.05mol/L MES buffer solution with the pH value of 5.0, and suspending by using the buffer solution to prepare magnetic particle suspension with the concentration of 100 mg/mL; taking 100 mu L of the magnetic particle suspension, adding 1mL of 0.05mol/L MES buffer solution with pH 5.0 and 50 mu G of G-17 antibody, mixing uniformly at 25 ℃, incubating for 2 hours, then performing magnetic separation, retaining the magnetic particles, washing with 5% BSA with pH 7.2 and 0.05mol/L Tris-HCl buffer solution for 1-3 times, blocking with 5% BSA with pH 7.2 and 0.05mol/L Tris-HCl buffer solution at 25 ℃ for 30 minutes, and blocking with a solution containing 0.5% BSA by mass and 0.1% NaN by mass30.05mol/L Tris-HCl buffer solution with pH 7.2 is washed for 1-3 times, and the solution is washed by the solution containing BSA with mass concentration of 0.5% and NaN with mass concentration of 0.1%3Resuspending in 0.05mol/L Tris-HCl buffer solution with pH of 7.2, subpackaging and storing at 2-8 ℃;
preparation of europium-labeled G-17 antibody solution: collecting 1mg of G-17 antibody, and subjecting to PD-10 conversion buffer condition to obtain eluent containing 0.155mol/L NaCl and 50mmol/L Na with pH of 8.52CO3-NaHCO3Buffering to obtain G-17 antibody solution concentrated to 2G/L; adding 0.2mg of benzyldiethylenetriamine tetraacetic acid europium sodium isothiocyanate into 500 mu L of the G-17 antibody solution, carrying out oscillation reaction at 25 ℃ for 20 hours, transferring the reaction solution to a Sephadex G-50 column equilibrated by a Tris buffer solution with the pH of 7.8 and the concentration of 80mmol/L for chromatography, collecting protein peaks, diluting, carrying out split charging, carrying out vacuum freeze drying, and storing at 2-8 ℃;
preparation of the reinforcing liquid: glacial acetic acid with the volume concentration of 3.6 per thousand, sodium acetate with the volume concentration of 0.5g/L, beta-naphthoyl trifluoroacetone with the volume concentration of 0.05g/L, trioctylphosphine oxide with the volume concentration of 0.03g/L and Triton X-100 with the volume concentration of 1 per thousand.
The reaction buffer is 50mmol/L Tris-HCl with pH7.8, 8mmol/L NaCl, 0.1% BSA, 50. mu. mol/L DTPA, 0.1ml/L Tween-80 and 0.1% NaN3；
The cleaning solution is a buffer solution containing 0.48g/L of Tris, 12.49g/L of NaCl, 1.11g/L of Tween-20 and the pH value is adjusted to 7.8 by hydrochloric acid.
A detection method of a time-resolved fluoroimmunoassay kit for detecting gastrin G-17 mainly comprises the following steps:
1) respectively taking a G-17 calibrator solution and a sample to be detected, mixing the G-17 calibrator solution with a G-17 antibody coated magnetic particle solution and a europium-labeled G-17 antibody solution diluted by a reaction buffer solution, incubating, performing magnetic separation after incubation, then cleaning by using a cleaning solution, and then adding an enhancement solution;
2) respectively measuring the fluorescence value of europium in the solution added with the enhancement solution by using a time-resolved fluoroimmunoassay analyzer to obtain the fluorescence values of the G-17 calibrator solution and the sample to be detected, drawing a standard curve according to the concentration of each G-17 in the G-17 calibrator solution and the fluorescence value corresponding to each G-17, and substituting the fluorescence value of the sample to be detected into the corresponding G-17 standard curve to obtain the content of G-17 in the sample to be detected.
The invention has the beneficial effects that: compared with the prior art, the technical scheme provided by the invention has the following advantages:
(1) the invention provides a time-resolved fluoroimmunoassay kit for detecting G-17, which comprises a reaction buffer solution, a cleaning solution, an enhancement solution, a magnetic particle solution for coating a G-17 antibody and a europium-labeled G-17 antibody solution which are all integrated on a reagent strip, and has the advantages of full-automatic operation, simplicity, convenience and practicability. The content of G-17 in serum can be detected within 24min, the detection result is accurate and reliable, besides the advantages of high sensitivity, long storage time, no radioactive pollution, wide measurement range and the like of the TRFIA technology, the characteristic that the combination surface area is enlarged due to the enrichment effect of immune magnetic particles and the full diffusion of the magnetic particles in liquid can be realized, the reaction time is greatly shortened, the detection sensitivity is improved, meanwhile, the magnetic particles and an antibody are directionally connected through chemical groups, the dosage of an antigen is greatly reduced, the detection precision is remarkably improved, the automation is realized, the problem that the traditional microplate type enzyme-linked immunosorbent assay (ELISA) technology can detect the sample only when the sample is accumulated to a certain amount is solved, the instant detection of the sample is realized, and the efficiency is high;
the kit is a high-efficiency time-resolved fluorescence technology taking nano magnetic particles as carriers: the magnetic particles are connected with free amino groups of the G-17 antibody to form immune magnetic particles coated with the G-17 antibody, the immune magnetic particles can be combined with a large amount of G-17 antibody in a short time by virtue of the overlarge specific surface area of the immune magnetic particles, Eu labeled G-17 is added for tracing, Eu is dissociated from the compound by using enhancement liquid through magnetic separation and then is chelated with a chelating agent in the enhancement liquid to form a colloidal molecular group, the molecular group can emit fluorescence with strong emission wavelength under the excitation of ultraviolet light, the signal is enhanced by millions of times, and the content of the G-17 in serum can be sensitively detected.
Drawings
FIG. 1 is a schematic diagram of the detection principle of the immunoassay kit provided by the present invention.
FIG. 2 is a graph of a log-log standard curve of the G-17 standard in the immunoassay kit provided by the present invention.
FIG. 3 is a schematic structural diagram of a reagent rack integrated with 5-person reagent strips provided by the invention.
FIG. 4 is a schematic structural diagram of a reagent strip provided by the present invention.
Wherein, 1, reagent strip; 2. a reagent rack; 3. reserving a hole; 4. reserving a hole; 5. magnetic bead holes; 6. reserving a hole; 7. a reaction buffer well; 8. europium label freeze-drying holes; 9. a detection hole; 10. reserving a hole; 11-13, washing liquid holes; 14. a reinforcement liquid hole; 15. holes are reserved.
Detailed Description
The invention is further illustrated by the following specific examples. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention.
As shown in fig. 3 and 4, the time-resolved fluorescence immunoassay kit for detecting gastrin G-17 comprises 5-20 reagent racks, and each reagent rack is provided with 5 reagent strips; the kit also comprises 2 bottles of G-17 calibrator. The reagent strip comprises a magnetic bead hole, a reaction buffer liquid hole, a europium label freeze-drying hole, a detection hole, a cleaning liquid hole, a reinforcing liquid hole and a plurality of reserved holes; magnetic particle solution coated with G-17 antibody is filled in the magnetic bead holes, reaction buffer solution is filled in the reaction buffer solution holes, europium-labeled G-17 antibody solution is filled in the europium-labeled freeze-dried holes, samples to be detected are filled in the detection holes, cleaning solution is filled in the cleaning solution holes, and enhancement solution is filled in the enhancement solution holes.
The magnetic particle solution coated with the G-17 antibody is prepared by connecting activated magnetic particles with the diameter of 50-5000 nanometers with the G-17 antibody, washing and sealing; the europium-labeled G-17 antibody solution is Eu3+-N2- [ P-Isocyanato-benzyl group]A G-17 antibody marked by diethylenetriamine tetra sodium acetate, the G-17 antibody and the Eu3+-N2- [ P-Isocyanato-benzyl group]The mass ratio of the-sodium diethylenetriamine tetracetate is 5: 1.
The reactionThe buffer solution is 50mmol/L Tris-HCl with pH7.8, 8mmol/L NaCl, 0.1% BSA, 50. mu. mol/L DTPA, 0.1ml/L Tween-80 and 0.1% NaN3(ii) a The cleaning solution is a buffer solution containing 0.48g/L of Tris, 12.49g/L of NaCl and 1.11g/L of Tween-20, and the pH value is adjusted to 7.8 by hydrochloric acid; the enhancement solution is a mixed solution containing glacial acetic acid with the volume concentration of 3.6 per thousand, sodium acetate with the volume concentration of 0.5g/L, beta-naphthoyl trifluoroacetone with the volume concentration of 0.05g/L, trioctylphosphine oxide with the volume concentration of 0.03g/L and Triton X-100 with the volume concentration of 1 per thousand; the G-17 calibrator was prepared by using 2G/L BSA and 1G/L NaN350mmol/L of Tris-HCl pH7.8 reaction buffer, and preparing G-17 into calibrator solutions with different concentrations.
A preparation method of a time-resolved fluoroimmunoassay kit for detecting gastrin G-17 comprises the following steps:
preparation of G-17 calibrator solution: taking high concentration G-17 antigen solution, adding 2G/L BSA and 1G/L NaN3Preparing 50mmol/L Tris-HCl reaction buffer solution with pH of 7.8 into calibrator solutions with different concentrations, subpackaging, and storing at 2-8 ℃;
preparation of magnetic particle solution coated with G-17 antibody: activating ferroferric oxide microspheres with the diameter of 50-5000 nm by using N-hydroxysuccinimide (NHS) and 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) with the mass concentration of 0.5-2%, uniformly mixing for 4 hours at room temperature, washing for 1-3 times by using 0.05mol/L MES buffer solution with the pH value of 5.0, and suspending by using the buffer solution to prepare magnetic particle suspension with the concentration of 100 mg/mL; taking 100 mu L of the magnetic particle suspension, adding 1mL of 0.05mol/L MES buffer solution with pH 5.0 and 50 mu G of G-17 antibody, mixing uniformly at 25 ℃, incubating for 2 hours, then performing magnetic separation, retaining the magnetic particles, washing with 5% BSA with pH 7.2 and 0.05mol/L Tris-HCl buffer solution for 1-3 times, blocking with 5% BSA with pH 7.2 and 0.05mol/L Tris-HCl buffer solution at 25 ℃ for 30 minutes, and blocking with a solution containing 0.5% BSA by mass and 0.1% NaN by mass30.05mol/L Tris-HCl buffer solution with pH 7.2 is washed for 1-3 times, and the solution is washed by the solution containing BSA with mass concentration of 0.5% and NaN with mass concentration of 0.1%30.05mol/L of Tris-HCl buffer solution with pH 7.2Suspending, subpackaging and storing at 2-8 ℃;
preparation of europium-labeled G-17 antibody solution: collecting 1mg of G-17 antibody, and subjecting to PD-10 conversion buffer condition to obtain eluent containing 0.155mol/L NaCl and 50mmol/L Na with pH of 8.52CO3-NaHCO3Buffering to obtain G-17 antibody solution concentrated to 2G/L; adding 0.2mg of benzyldiethylenetriamine tetraacetic acid europium sodium isothiocyanate into 500 mu L of the G-17 antibody solution, carrying out oscillation reaction at 25 ℃ for 20 hours, transferring the reaction solution to a Sephadex G-50 column equilibrated by a Tris buffer solution with the pH of 7.8 and the concentration of 80mmol/L for chromatography, collecting protein peaks, diluting, carrying out split charging, carrying out vacuum freeze drying, and storing at 2-8 ℃;
preparation of the reinforcing liquid: glacial acetic acid with the volume concentration of 3.6 per thousand, sodium acetate with the volume concentration of 0.5g/L, beta-naphthoyl trifluoroacetone with the volume concentration of 0.05g/L, trioctylphosphine oxide with the volume concentration of 0.03g/L and Triton X-100 with the volume concentration of 1 per thousand.
The reaction buffer is 50mmol/L Tris-HCl with pH7.8, 8mmol/L NaCl, 0.1% BSA, 50. mu. mol/L DTPA, 0.1ml/L Tween-80 and 0.1% NaN3；
The cleaning solution is a buffer solution containing 0.48g/L of Tris, 12.49g/L of NaCl, 1.11g/L of Tween-20 and the pH value is adjusted to 7.8 by hydrochloric acid.
A detection method of a time-resolved fluoroimmunoassay kit for detecting gastrin G-17 mainly comprises the following steps:
1) after the calibrator solution is calibrated, 100ul of serum to be tested is taken and mixed with 50ul of magnetic particle solution coated with the G-17 antibody and europium-labeled G-17 antibody solution diluted by reaction buffer solution, the mixture reacts for 12 minutes, magnetic separation is carried out, cleaning solution is washed for 3 times, and then the enhancement solution is added;
2) and respectively measuring the fluorescence value of europium in the solution added with the enhancement solution by using a full-automatic time-resolved fluorescence immunoassay analyzer, substituting the corresponding fluorescence value of the sample to be detected into the corresponding standard curve according to the standard curve, and calculating to obtain the content of G-17 in the sample to be detected.
The reaction principle in the detection process is shown in figure 1, a G-17 antibody is coated on a magnetic bead, the G-17 antibody is labeled by europium, if G-17 exists in a sample, a magnetic bead-G-17 antibody-G-17-europium-labeled G-17 antibody compound is formed, after separation, dissociation enhancement liquid is added to dissociate europium ions, a time-resolved fluorescence instrument is adopted to detect a fluorescence value, the strength of the fluorescence value is in direct proportion to the content of G-17 in the sample, a standard curve is drawn according to the fluorescence value of a standard product of G-17, and the content of G-17 in the sample is calculated.
The detection results are as follows: the double logarithmic standard curve of the gastrin G-17 is shown in figure 2, the test result of the serum sample is shown in table 1, the detection method has better sensitivity, and the automation of fluorescence detection is realized.
TABLE 1 serum sample test results
The above embodiments are only for illustrating the invention and are not to be construed as limiting the invention, and those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention, therefore, all equivalent technical solutions also belong to the scope of the invention, and the scope of the invention is defined by the claims.
Claims (6)
1. A time-resolved fluoroimmunoassay kit for detecting gastrin G-17 is characterized in that: the kit comprises at least one reagent rack, at least five reagent strips are arranged on each reagent rack, and at least two bottles of G-17 calibration products are arranged in the kit; the reagent strip comprises a magnetic bead hole, a reaction buffer liquid hole, a europium label freeze-drying hole, a detection hole, a cleaning liquid hole, a reinforcing liquid hole and a plurality of reserved holes; magnetic particle solution coated with G-17 antibody is filled in the magnetic bead holes, reaction buffer solution is filled in the reaction buffer solution holes, europium-labeled G-17 antibody solution is filled in the europium-labeled freeze-dried holes, samples to be detected are filled in the detection holes, cleaning solution is filled in the cleaning solution holes, and enhancement solution is filled in the enhancement solution holes.
2. The time-resolved fluoroimmunoassay kit for detecting gastrin G-17 according to claim 1, wherein: the reagent box comprises 5-20 reagent racks, and each reagent rack is provided with 5 reagent strips; the kit also comprises 2 bottles of G-17 calibrator.
3. The time-resolved fluoroimmunoassay kit for detecting gastrin G-17 according to claim 1, wherein: the magnetic particle solution coated with the G-17 antibody is prepared by connecting activated magnetic particles with the diameter of 50-5000 nanometers with the G-17 antibody, washing and sealing;
the europium-labeled G-17 antibody solution is Eu3+-N2- [ P-Isocyanato-benzyl group]Preparing the antibody G-17 marked by diethylenetriamine tetra sodium acetate.
4. The time-resolved fluoroimmunoassay kit for detecting gastrin G-17 according to claim 1, wherein: the reaction buffer solution is 50mmol/L Tris-HCl with pH7.8, 8mmol/L NaCl, 0.1% BSA, 50 μmol/L DTPA, 0.1ml/L Tween-80 and 0.1% NaN3；
The cleaning solution is a buffer solution containing 0.48g/L of Tris, 12.49g/L of NaCl and 1.11g/L of Tween-20, and the pH value is adjusted to 7.8 by hydrochloric acid;
the enhancement solution is a mixed solution containing glacial acetic acid with the volume concentration of 3.6 per thousand, sodium acetate with the volume concentration of 0.5g/L, beta-naphthoyl trifluoroacetone with the volume concentration of 0.05g/L, trioctylphosphine oxide with the volume concentration of 0.03g/L and Triton X-100 with the volume concentration of 1 per thousand;
the G-17 calibrator was prepared by using 2G/L BSA and 1G/L NaN350mmol/L of reaction buffer, pH7.8Tris-HCl, G-17 was formulated into calibrator solutions of various concentrations.
5. A method of preparing the time-resolved fluoroimmunoassay kit for detecting gastrin G-17 according to any one of claims 1 to 4, comprising the steps of:
preparation of G-17 calibrator solution: taking high concentration G-17 antigen solution, adding 2G/L BSA and 1G/L NaN3Preparing 50mmol/L Tris-HCl reaction buffer solution with pH of 7.8 into calibrator solutions with different concentrations, subpackaging, and storing at 2-8 ℃;
preparation of magnetic particle solution coated with G-17 antibody: activating ferroferric oxide microspheres with the diameter of 50-5000 nm by using N-hydroxysuccinimide (NHS) and 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) with the mass concentration of 0.5-2%, uniformly mixing for 4 hours at room temperature, washing for 1-3 times by using 0.05mol/L MES buffer solution with the pH value of 5.0, and suspending by using the buffer solution to prepare magnetic particle suspension with the concentration of 100 mg/mL; taking 100 mu L of the magnetic particle suspension, adding 1mL of 0.05mol/L MES buffer solution with pH 5.0 and 50 mu G of G-17 antibody, mixing uniformly at 25 ℃, incubating for 2 hours, then performing magnetic separation, retaining the magnetic particles, washing with 5% BSA with pH 7.2 and 0.05mol/L Tris-HCl buffer solution for 1-3 times, blocking with 5% BSA with pH 7.2 and 0.05mol/L Tris-HCl buffer solution at 25 ℃ for 30 minutes, and blocking with a solution containing 0.5% BSA by mass and 0.1% NaN by mass30.05mol/L Tris-HCl buffer solution with pH 7.2 is washed for 1-3 times, and the solution is washed by the solution containing BSA with mass concentration of 0.5% and NaN with mass concentration of 0.1%3Resuspending in 0.05mol/L Tris-HCl buffer solution with pH of 7.2, subpackaging and storing at 2-8 ℃;
preparation of europium-labeled G-17 antibody solution: collecting 1mg of G-17 antibody, and subjecting to PD-10 conversion buffer condition to obtain eluent containing 0.155mol/L NaCl and 50mmol/L Na with pH of 8.52CO3-NaHCO3Buffering to obtain G-17 antibody solution concentrated to 2G/L; adding 0.2mg of benzyldiethylenetriamine tetraacetic acid europium sodium isothiocyanate into 500 mu L of the G-17 antibody solution, carrying out oscillation reaction at 25 ℃ for 20 hours, transferring the reaction solution to a Sephadex G-50 column equilibrated by a Tris buffer solution with the pH of 7.8 and the concentration of 80mmol/L for chromatography, collecting protein peaks, diluting, carrying out split charging, carrying out vacuum freeze drying, and storing at 2-8 ℃;
preparation of the reinforcing liquid: glacial acetic acid with the volume concentration of 3.6 per thousand, sodium acetate with the volume concentration of 0.5g/L, beta-naphthoyl trifluoroacetone with the volume concentration of 0.05g/L, trioctylphosphine oxide with the volume concentration of 0.03g/L and Triton X-100 with the volume concentration of 1 per thousand.
The reaction buffer is 50mmol/L Tris-HCl with pH7.8, 8mmol/L NaCl, 0.1% BSA, 50. mu. mol/L DTPA, 0.1ml/L Tween-80 and 0.1% NaN3；
The cleaning solution is a buffer solution containing 0.48g/L of Tris, 12.49g/L of NaCl, 1.11g/L of Tween-20 and the pH value is adjusted to 7.8 by hydrochloric acid.
6. The detection method of the gastrin G-17 time-resolved fluoroimmunoassay kit according to any one of claims 1 to 4, which mainly comprises the following steps:
1) respectively taking a G-17 calibrator solution and a sample to be detected, mixing the G-17 calibrator solution with a G-17 antibody coated magnetic particle solution and a europium-labeled G-17 antibody solution diluted by a reaction buffer solution, incubating, performing magnetic separation after incubation, then cleaning by using a cleaning solution, and then adding an enhancement solution;
2) respectively measuring the fluorescence value of europium in the solution added with the enhancement solution by using a time-resolved fluoroimmunoassay analyzer to obtain the fluorescence values of the G-17 calibrator solution and the sample to be detected, drawing a standard curve according to the concentration of each G-17 in the G-17 calibrator solution and the fluorescence value corresponding to each G-17, and substituting the fluorescence value of the sample to be detected into the corresponding G-17 standard curve to obtain the content of G-17 in the sample to be detected.
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