CN112147328A - Immunoassay kit based on pepsinogen PG I, and preparation method and detection method thereof - Google Patents

Immunoassay kit based on pepsinogen PG I, and preparation method and detection method thereof Download PDF

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CN112147328A
CN112147328A CN202010898602.9A CN202010898602A CN112147328A CN 112147328 A CN112147328 A CN 112147328A CN 202010898602 A CN202010898602 A CN 202010898602A CN 112147328 A CN112147328 A CN 112147328A
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solution
antibody
tris
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任志奇
黄飚
万文成
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Zhejiang Boshi Biotechnology Co ltd
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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
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • 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"
    • 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
    • G01N33/534—Production of labelled immunochemicals with radioactive label
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
    • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
    • G01N2333/96472—Aspartic endopeptidases (3.4.23)
    • G01N2333/96475—Aspartic endopeptidases (3.4.23) with definite EC number
    • G01N2333/96477—Pepsin (3.4.23.1; 3.4.23.2; 3.4.23.3)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/06—Gastro-intestinal diseases
    • G01N2800/062—Gastritis or peptic ulcer disease

Abstract

The invention discloses an immunoassay kit based on pepsinogen PG I, a preparation method and a detection method thereof, belonging to the fields of immunoassay analysis technology and nanometer biotechnology. The kit can provide a reaction system close to homogeneous phase, detect the content of PG I, shorten the detection time, improve the efficiency and provide convenience for clinical use.

Description

Immunoassay kit based on pepsinogen PG I, and preparation method and detection method thereof
Technical Field
The invention belongs to the fields of immunoassay and nano-biotechnology, and particularly relates to a time-resolved fluorescence immunoassay kit based on pepsinogen I, and a preparation method and a detection method thereof.
Background
Pepsinogen (PG) is a precursor of pepsin secreted from the stomach and involved in digestion, and generally about 1% of PG can enter blood circulation through the gastric mucosa, and can be divided into two different subgroups with characteristics of biochemical and immunological activities of pgi and pgi, and the cell sources and the distribution in tissues are different. Serum PG concentration mainly reflects the level of secretion by gastric gland cells. PG I is an indication for detecting the function of the cells of the oxyntic gland, PG I is increased when gastric acid secretion is increased, and PG I is decreased when the gastric acid secretion is not increased; the PG I has larger relevance with the pathological changes of gastric fundus mucosa, and the increase of the PG I is related to atrophy of gastric fundus glandular tubes, metaplasia of gastric epithelium or metaplasia and abnormal proliferation of pyloric gland; progressive reduction of the PG I/I ratio is associated with progression of atrophy of the gastric mucosa. The combined determination of PG I, PG I and PG I/I ratio can play a role in serological biopsy of gastric fundus gland mucosa. The levels of PG I and PG I are measured before and after gastroscopy, and regular tracking is carried out, so that the state and the function of the gastric mucosa can be effectively monitored.
Time-resolved fluoroimmunoassay (TRFIA) is a novel 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 detection is repeated 1000 times, and the average fluorescence count value is taken as a result, so that the detection accuracy is improved. TRFIA isOne of the main development directions of domestic and foreign immunoassay.
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 the PG I-TRFIA method, can be operated by full-automatic equipment, has high accuracy, can be detected by a single person, is convenient to use, is applied to the research of stomach lesions, can more accurately identify and diagnose the gastric lesions such as superficial gastritis, gastric mucosal erosion ulcer, atrophic gastritis, chronic gastrectasia, chronic duodenitis, duodenal ulcer and the like, and can evaluate the curative effect of the stomach lesions by convenient serological examination. Provides a very convenient and fast new means for the diagnosis and treatment of stomach lesions.
The technical scheme adopted by the invention for solving the technical problem is as follows: an immunoassay kit based on pepsinogen PG I, which comprises at least one reagent rack, at least five reagent strips on each reagent rack, and at least two bottles of freeze-dried calibrator PG I; 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 PG I antibody is filled in the magnetic bead hole, reaction buffer solution is filled in the reaction buffer solution hole, europium-labeled PG I antibody solution is filled in the europium-labeled freeze-dried hole, a sample to be detected is filled in the detection hole, cleaning solution is filled in the cleaning solution hole, and enhancement solution is filled in the enhancement solution hole.
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 freeze-dried calibrator PG I.
Further, the magnetic particle solution coated with the PG I antibody is prepared by connecting, washing and sealing activated magnetic particles with the diameter of 50-5000 nanometers with the PG I antibody; the europium-labeled PG I antibody solution is prepared by using Eu3+-N2- [ P-Isocyanato-benzyl group]The-sodium diethylenetriamine tetracetate marked PG I antibody is prepared,the PG I antibody 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 freeze-dried calibrator for PG I is prepared by using 2g/L BSA and 1g/L NaN350mmol/L of Tris-HCl pH7.8 reaction buffer, PG I was prepared into calibrator solutions of different concentrations.
A preparation method of an immunoassay kit based on pepsinogen PG I comprises the following steps:
preparation of PG I calibrator solution: taking high concentration PG I antigen solution containing 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 PG I 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 PG I 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 mixture 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 PG I antibody solution: taking 1mg of PG I antibody, buffering by PD-10 conversion, eluting with 50mmol/L Na containing 0.155mol/L NaCl and having pH of 8.52CO3-NaHCO3Buffering to obtain PG I antibody solution concentrated to 2 g/L; adding 0.2mg of benzyldiethylenetriamine tetraacetic acid europium sodium isothiocyanate into 500 mu L of the PG I antibody solution, carrying out oscillation reaction for 20 hours at 25 ℃, transferring the reaction solution to a Sephadex G-50 column which is balanced 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 an immunoassay kit based on pepsinogen PG I mainly comprises the following steps:
1) respectively taking a freeze-dried calibrator solution of PG I and a sample to be detected, mixing the freeze-dried calibrator solution of PG I with a magnetic particle solution coated with PG I antibody and a europium-labeled PG I antibody solution diluted by reaction buffer solution, incubating, performing magnetic separation after incubation, then cleaning with 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 freeze-dried PG I calibrator solution and the sample to be detected, drawing a standard curve according to the concentration of each PG I in the PG I calibrator solution and the fluorescence value corresponding to each PG I, and substituting the fluorescence value of the sample to be detected into the corresponding PG I standard curve to obtain the content of PG I 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 PG I, which comprises a reaction buffer solution, a cleaning solution, an enhancement solution, a magnetic particle solution for coating PG I antibody and a PG I antibody solution marked by europium, which are all integrated on a reagent strip, and has the advantages of full-automatic operation, simplicity, convenience and practicability. The content of PG I 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 detection method can also greatly shorten the reaction time and improve the detection sensitivity by the enrichment effect of immune magnetic particles and the characteristic that the magnetic particles are fully diffused in liquid to enlarge the combined surface area, meanwhile, the magnetic particles are directionally connected with antibodies through chemical groups, the use amount of antigens is greatly reduced, the detection precision is remarkably improved, the automation is realized, the defect that the traditional micropore plate type enzyme-linked immunosorbent assay (ELISA) technology can detect samples only when the samples are accumulated to a certain amount is overcome, the instant detection of the samples 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 PG I antibody to form immune magnetic particles coated with the PG I antibody, the immune magnetic particles can be combined with a large amount of PG I in a short time by virtue of the overlarge specific surface area of the immune magnetic particles, the Eu-labeled PG I antibody is added for tracing, and after Eu is dissociated from a compound by using enhancement liquid through magnetic separation, the Eu is chelated with a chelating agent in the enhancement liquid to form a colloidal molecular group, and the colloidal molecular group can emit strong fluorescence with emission wavelength under the excitation of ultraviolet light, so that the signal is enhanced by millions of times, and the content of the PG I 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 of PG I standard in the immunoassay kit of 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. a diluent aperture; 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, cleaning solution 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 immunoassay kit based on pepsinogen PG I comprises 5-20 reagent racks, and each reagent rack is provided with 5 reagent strips; the kit also comprises 2 bottles of freeze-dried calibrator PG I. 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 PG I antibody is filled in the magnetic bead hole, reaction buffer solution is filled in the reaction buffer solution hole, europium-labeled PG I antibody solution is filled in the europium-labeled freeze-dried hole, a sample to be detected is filled in the detection hole, cleaning solution is filled in the cleaning solution hole, and enhancement solution is filled in the enhancement solution hole.
The magnetic particle solution coated with the PG I antibody is prepared by connecting activated magnetic particles with the diameter of 50-5000 nanometers with the PG I antibody, washing and sealing; the europium-labeled PG I antibody solution is prepared by using Eu3+-N2- [ P-Isocyanato-benzyl group]A sodium diethylenetriamine tetracetate marked PG I antibody is prepared, and the PG I antibody and the Eu are3+-N2- [ P-Isocyanato-benzyl group]The mass ratio of the-sodium diethylenetriamine tetracetate is 5: 1.
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(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 freeze-dried calibrator for PG I is prepared by using 2g/L BSA and 1g/L NaN350mmol/L of Tris-HCl pH7.8 reaction buffer, PG I was prepared into calibrator solutions of different concentrations.
A preparation method of an immunoassay kit based on pepsinogen PG I comprises the following steps:
preparation of PG I calibrator solution: taking high concentration PG I antigen solution containing 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 PG I 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 PG I 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 mixture 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%30.05 mol-Resuspending the L in a Tris-HCl buffer solution with the pH of 7.2, subpackaging and storing at 2-8 ℃;
preparation of europium-labeled PG I antibody solution: taking 1mg of PG I antibody, buffering by PD-10 conversion, eluting with 50mmol/L Na containing 0.155mol/L NaCl and having pH of 8.52CO3-NaHCO3Buffering to obtain PG I antibody solution concentrated to 2 g/L; adding 0.2mg of benzyldiethylenetriamine tetraacetic acid europium sodium isothiocyanate into 500 mu L of the PG I antibody solution, carrying out oscillation reaction for 20 hours at 25 ℃, transferring the reaction solution to a Sephadex G-50 column which is balanced 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 an immunoassay kit based on pepsinogen PG I 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 PG I antibody and the europium-labeled PG I antibody solution diluted by the reaction buffer solution, the mixture reacts for 12 minutes, magnetic separation is carried out, the 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, and substituting the corresponding fluorescence value of the sample to be detected into the corresponding standard curve according to the standard curve to calculate the content of PG I in the sample to be detected.
The reaction principle in the detection process is shown in figure 1, the PG I antibody is coated on a magnetic bead, the europium labels the PG I antibody, if PG I exists in a sample, a magnetic bead-PG I antibody-PG I-europium labels PG I antibody compound is formed, after separation, enhancement liquid is added to dissociate europium ions, a time-resolved fluorescence instrument is adopted to detect the fluorescence value, the strength of the fluorescence value is in direct proportion to the content of PG I in the sample, a standard curve is drawn according to the fluorescence value of a freeze-dried calibrator of PG I, and the content of PG I in the sample is calculated.
The detection results are as follows: the double logarithmic standard curve of the pepsinogen I is shown in a figure 2, the test result of a serum sample is shown in a table 1, and the detection method has good sensitivity and realizes the automation of fluorescence detection.
TABLE 1 serum sample test results
Sample numbering Fluorescence value (cps) Concentration of
1 590138 186.5ng/mL
2 539072 169.4ng/mL
3 465863 145.0ng/mL
4 445497 138.2ng/mL
5 441602 136.9ng/mL
6 218726 64.6ng/mL
7 163189 47.2ng/mL
8 1061533 348.3ng/mL
9 1280220 425.1ng/mL
10 1445546 483.7ng/mL
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. An immunoassay kit based on pepsinogen PG I 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 lyophilized calibrator PG I 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 PG I antibody is filled in the magnetic bead hole, reaction buffer solution is filled in the reaction buffer solution hole, europium-labeled PG I antibody solution is filled in the europium-labeled freeze-dried hole, a sample to be detected is filled in the detection hole, cleaning solution is filled in the cleaning solution hole, and enhancement solution is filled in the enhancement solution hole.
2. The pepsinogen PG I-based immunoassay kit as claimed in 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 freeze-dried calibrator PG I.
3. The pepsinogen PG I-based immunoassay kit as claimed in claim 1, wherein: the magnetic particle solution coated with the PG I antibody is prepared by connecting activated magnetic particles with the diameter of 50-5000 nanometers with the PG I antibody, washing and sealing;
the europium-labeled PG I antibody solution is prepared by using Eu3+-N2- [ P-Isocyanato-benzyl group]Preparing the-sodium diethylenetriamine tetracetate marked PG I antibody.
4. The pepsinogen PG I-based immunoassay kit as claimed in 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 freeze-dried calibrator for PG I is prepared by using 2g/L BSA and 1g/L NaN350mmol/L of Tris-HCl pH7.8 reaction buffer, PG I was prepared into calibrator solutions of different concentrations.
5. A process for the preparation of an immunoassay kit according to any of claims 1 to 4 based on pepsinogen PG I, comprising the following steps:
preparation of PG I calibrator solution: taking high concentration PG I antigen solution containing 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 PG I 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 PG I 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 mixture 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 PG I antibody solution: taking 1mg of PG I antibody, buffering by PD-10 conversion, eluting with 50mmol/L Na containing 0.155mol/L NaCl and having pH of 8.52CO3-NaHCO3A buffer solution is added to the reaction kettle,obtaining PG I antibody solution concentrated to 2 g/L; adding 0.2mg of benzyldiethylenetriamine tetraacetic acid europium sodium isothiocyanate into 500 mu L of the PG I antibody solution, carrying out oscillation reaction for 20 hours at 25 ℃, transferring the reaction solution to a Sephadex G-50 column which is balanced 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. A method for detecting the pepsinogen PG I-based immunoassay kit as claimed in any one of claims 1 to 4, characterized in that said method essentially comprises the following steps:
1) respectively taking a freeze-dried calibrator solution of PG I and a sample to be detected, mixing the freeze-dried calibrator solution of PG I with a magnetic particle solution coated with PG I antibody and a europium-labeled PG I antibody solution diluted by reaction buffer solution, incubating, performing magnetic separation after incubation, then cleaning with 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 freeze-dried PG I calibrator solution and the sample to be detected, drawing a standard curve according to the concentration of each PG I in the PG I calibrator solution and the fluorescence value corresponding to each PG I, and substituting the fluorescence value of the sample to be detected into the corresponding PG I standard curve to obtain the content of PG I in the sample to be detected.
CN202010898602.9A 2020-08-31 2020-08-31 Immunoassay kit based on pepsinogen PG I, and preparation method and detection method thereof Pending CN112147328A (en)

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