CN112147342A - Procalcitonin PCT-based immunoassay kit and preparation method and detection method thereof - Google Patents

Procalcitonin PCT-based immunoassay kit and preparation method and detection method thereof Download PDF

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CN112147342A
CN112147342A CN202010900416.4A CN202010900416A CN112147342A CN 112147342 A CN112147342 A CN 112147342A CN 202010900416 A CN202010900416 A CN 202010900416A CN 112147342 A CN112147342 A CN 112147342A
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pct
solution
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傅凯杰
黄飚
任志奇
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Zhejiang Boshi Biotechnology Co ltd
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Abstract

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

Description

Procalcitonin PCT-based immunoassay kit 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 procalcitonin-based time-resolved fluorescence immunoassay kit, and a preparation method and a detection method thereof.
Background
PCT is a protein whose levels in plasma are elevated when severe bacterial, fungal, parasitic infections and sepsis and multi-organ failure. PCT reflects the activity of the systemic inflammatory response. Elevated PCT levels occur in severe shock, Systemic Inflammatory Response Syndrome (SIRS), and Multiple Organ Dysfunction Syndrome (MODS), even without bacterial infection or bacterial foci. PCT concentrations in healthy individuals are very low (<0.1 ng/ml).
Clinical diagnostic significance of PCT in systemic bacterial infection/sepsis: normal human, PCT <0.05 ng/ml; SIRS, systemic inflammation, reactive syndrome, 0.05ng/ml < PCT <0.5 ng/ml; sepsis, 0.5ng/ml < PCT <2 ng/ml; severe sepsis, 2ng/ml < PCT <10 ng/ml; septic shock, PCT >10 ng/ml.
PCT assay population: chronic inflammation, autoimmune disorders; viral infections, such as acute hepatitis b; mild or localized bacterial infections; pneumonia; SIRS, combined injury, burn; severe bacterial infection, sepsis multiple organ failure.
Detection of PCT was performed by 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; there is a large Stokes shift between the excitation and emission light of the time-resolved fluorescence immune components, which is advantageousThe interference of non-specific fluorescence is eliminated, so that the specificity of measurement is enhanced; 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 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 PCT-TRFIA method is established by combining TRFIA and a magnetic particle technology and integrating all reagents on one reagent strip, the detection can be operated by full-automatic equipment, the accuracy is high, the detection is carried out by a single person, the use is convenient, and the technology is applied to chronic inflammation and autoimmune disorder; the research of viral infection can more accurately identify and diagnose chronic inflammation and autoimmune disorder; viral infections, to carry out chronic inflammation by convenient serological examination, autoimmune disorders; and evaluating the curative effect of the viral infection. Chronic inflammation, autoimmune disorders; the diagnosis and treatment of viral infection provides a very convenient and new means.
The technical scheme adopted by the invention for solving the technical problem is as follows: an immunoassay kit based on procalcitonin PCT, comprising at least one reagent rack in the kit, wherein each reagent rack comprises at least five reagent strips, and at least two bottles of lyophilized calibration products of PCT are further included 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 PCT antibody is filled in the magnetic bead holes, reaction buffer solution is filled in the reaction buffer solution holes, europium-labeled PCT antibody solution is filled in the europium-labeled freeze-dried holes, a sample to be detected is 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 included 2 vials of a lyophilized calibrator for PCT.
Further, the magnetic particle solution coated with the PCT antibody is prepared by connecting, washing and sealing activated magnetic particles with the diameter of 50-5000 nanometers with the PCT antibody; the europium-labeled PCT antibody solution is expressed by Eu3+-N2- [ P-Isocyanato-benzyl group]-diethylenetriamine tetra sodium acetate labeled PCT antibody, wherein the PCT antibody and the Eu are3+-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 lyophilized calibrator of PCT was prepared by using a mixture containing 2g/L BSA and 1g/L NaN350mmol/L of Tris-HCl pH7.8, and PCT was formulated into calibrator solutions of varying concentrations.
A preparation method of an immunoassay kit based on procalcitonin PCT comprises the following steps:
preparation of PCT calibrator solution: taking high concentration PCT 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 PCT 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; 100 μ L of the magnetic particle suspension was added with 1mL of 0.05mol/L MES buffer solution with pH 5.0 and50 μ g of PCT antibody, mixed at 25 deg.C and incubated for 2 hr, then magnetic separation is performed, the magnetic particles are retained, washed 1-3 times with 5% BSA at pH 7.2 and 0.05mol/L Tris-HCl buffer, blocked with 5% BSA at pH 7.2 and 0.05mol/L Tris-HCl buffer at 25 deg.C for 30 min, and then subjected to 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 PCT antibody solution: taking 1mg of PCT 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 a PCT antibody solution concentrated to 2 g/L; adding 0.2mg of benzyldiethylenetriamine tetraacetic acid europium sodium isothiocyanate into 500 mu L of the PCT 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. (ii) a
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 procalcitonin PCT mainly comprises the following steps:
1) respectively taking a freeze-dried calibration solution of PCT and a sample to be detected, mixing the freeze-dried calibration solution of PCT with a magnetic particle solution coating a PCT antibody and a europium-labeled PCT antibody solution diluted by a 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 fluorescence immunoassay analyzer to obtain the fluorescence values of the PCT freeze-dried calibrator solution and the sample to be tested, drawing a standard curve according to the concentration of each PCT in the PCT calibrator solution and the fluorescence value corresponding to each PCT, and substituting the fluorescence value of the sample to be tested into the corresponding PCT standard curve to obtain the content of the PCT in the sample to be tested.
The invention has the beneficial effects that: compared with the prior art, the technical scheme provided by the invention has the following advantages:
the invention provides a time-resolved fluoroimmunoassay kit for detecting PCT, which comprises a reaction buffer solution, a cleaning solution, an enhancement solution, a magnetic particle solution for coating a PCT antibody and a PCT antibody solution marked by europium, which are integrated on a reagent strip, and has the advantages of full-automatic operation, simplicity, convenience and practicability. The content of PCT 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 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 using amount of antigens is greatly reduced, the detection precision is remarkably improved, the automation is realized, the problem that the traditional microporous plate type enzyme-linked immunosorbent assay (ELISA) technology can detect samples only when the samples are accumulated to a certain amount is solved, 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 PCT antibody to form immune magnetic particles coated with the PCT antibody, the immune magnetic particles can be combined with a large amount of PCT in a short time by virtue of the overlarge specific surface area of the immune magnetic particles, the Eu-labeled PCT antibody is added for tracing, and through magnetic separation, after Eu is dissociated from a compound by using enhancement liquid, the Eu 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 PCT 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 log-log standard curve of PCT 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, 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 procalcitonin PCT comprises 5-20 reagent racks, wherein each reagent rack is provided with 5 reagent strips; the kit also included 2 vials of a lyophilized calibrator for PCT. 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 PCT antibody is filled in the magnetic bead holes, reaction buffer solution is filled in the reaction buffer solution holes, europium-labeled PCT antibody solution is filled in the europium-labeled freeze-dried holes, a sample to be detected is 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 PCT antibody is prepared by connecting, washing and sealing activated magnetic particles with the diameter of 50-5000 nanometers with the PCT antibody; the europium-labeled PCT antibody solution is expressed by Eu3+-N2-[P-Isocyanato-benzyl]-diethylenetriamine tetra sodium acetate labeled PCT antibody, wherein the PCT 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 lyophilized calibrator of PCT was prepared by using a mixture containing 2g/L BSA and 1g/L NaN350mmol/L of Tris-HCl pH7.8, and PCT was formulated into calibrator solutions of varying concentrations.
A preparation method of an immunoassay kit based on procalcitonin PCT comprises the following steps:
preparation of PCT calibrator solution: taking high concentration PCT 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 PCT 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 of 5.0 and 50 mu g of PCT antibody, uniformly mixing and incubating for 2 hours at 25 ℃, then carrying out magnetic separation, retaining the magnetic particles, washing 1-3 times by using 5% BSA with pH of 7.2 and 0.05mol/L Tris-HCl buffer solution, blocking by using 5% BSA with pH of 7.2 and 0.05mol/L Tris-HCl buffer solution at 25 ℃ for 30 minutes, and using a carrier containing 0.5% BS by mass concentrationA and NaN with the mass concentration of 0.1 percent30.05mol/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 PCT antibody solution: taking 1mg of PCT 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 a PCT antibody solution concentrated to 2 g/L; adding 0.2mg of benzyldiethylenetriamine tetraacetic acid europium sodium isothiocyanate into 500 mu L of the PCT 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. (ii) a
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 procalcitonin PCT mainly comprises the following steps:
1) after the calibration solution is calibrated, 100ul of serum to be tested is taken, mixed with 50ul of magnetic particle solution coated with the PCT antibody and europium-labeled PCT antibody solution diluted by reaction buffer solution, reacted for 12 minutes, magnetically separated, washed for 3 times by washing liquid, and then added with the enhancement solution;
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 the PCT in the sample to be detected.
The reaction principle in the detection process is shown in figure 1, a magnetic bead is coated with a PCT antibody, the PCT antibody is marked by europium, if PCT exists in a sample, a magnetic bead-PCT antibody-PCT-europium-marked PCT antibody compound is formed, after separation, 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 PCT content in the sample, a standard curve is drawn according to the fluorescence value of a freeze-dried calibration substance of the PCT, and the PCT content in the sample is calculated.
The detection results are as follows: the double logarithmic standard curve of procalcitonin PCT 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
Sample numbering Fluorescence value (cps) Concentration of
1 17951 0.10ng/mL
2 13470 0.07ng/mL
3 7723 0.04ng/mL
4 15069 0.08ng/mL
5 16030 0.09ng/mL
6 483813 2.97ng/mL
7 1052649 6.45ng/mL
8 1270551 7.78ng/mL
9 562123 3.45ng/mL
10 485444 2.98ng/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 procalcitonin PCT, characterized in that: 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 freeze-dried calibration products of PCT; 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 PCT antibody is filled in the magnetic bead holes, reaction buffer solution is filled in the reaction buffer solution holes, europium-labeled PCT antibody solution is filled in the europium-labeled freeze-dried holes, a sample to be detected is 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. A procalcitonin PCT-based immunoassay kit 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 included 2 vials of a lyophilized calibrator for PCT.
3. A procalcitonin PCT-based immunoassay kit according to claim 1, wherein: the magnetic particle solution coated with the PCT antibody is prepared by connecting, washing and sealing activated magnetic particles with the diameter of 50-5000 nanometers with the PCT antibody;
the europium-labeled PCT antibody solution is expressed by Eu3+-N2- [ P-Isocyanato-benzyl group]The-sodium diethylenetriamine tetracetate labeled PCT antibody is prepared.
4. A procalcitonin PCT-based immunoassay kit 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 lyophilized calibrator of PCT was prepared by using a mixture containing 2g/L BSA and 1g/L NaN350mmol/L of Tris-HCl pH7.8, and PCT was formulated into calibrator solutions of varying concentrations.
5. A method for the preparation of a procalcitonin PCT based immunoassay kit according to any of claims 1 to 4, comprising the following steps:
preparation of PCT calibrator solution: taking high concentration PCT 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 PCT 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 of 5.0 and 50 mu g of PCT antibody, uniformly mixing and incubating for 2 hours at 25 ℃, then performing magnetic separation, retaining the magnetic particles, washing 1-3 times with 5% BSA with pH of 7.2 and 0.05mol/L Tris-HCl buffer solution, blocking with 5% BSA with pH of 7.2 and 0.05mol/L Tris-HCl buffer solution at 25 ℃ for 30 minutes, and using 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 PCT antibody solution: taking 1mg of PCT antibody, changing buffer conditions by PD-10, and eluting with 5 containing 0.155mol/L NaCl0mmol/L of Na having a pH of 8.52CO3-NaHCO3Buffering to obtain a PCT antibody solution concentrated to 2 g/L; adding 0.2mg of benzyldiethylenetriamine tetraacetic acid europium sodium isothiocyanate into 500 mu L of the PCT 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. (ii) a
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 procalcitonin PCT based immunoassay kit according to any of claims 1 to 4, said method essentially comprising the steps of:
1) respectively taking a freeze-dried calibration solution of PCT and a sample to be detected, mixing the freeze-dried calibration solution of PCT with a magnetic particle solution coating a PCT antibody and a europium-labeled PCT antibody solution diluted by a 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 fluorescence immunoassay analyzer to obtain the fluorescence values of the PCT freeze-dried calibrator solution and the sample to be tested, drawing a standard curve according to the concentration of each PCT in the PCT calibrator solution and the fluorescence value corresponding to each PCT, and substituting the fluorescence value of the sample to be tested into the corresponding PCT standard curve to obtain the content of the PCT in the sample to be tested.
CN202010900416.4A 2020-08-31 2020-08-31 Procalcitonin PCT-based immunoassay kit and preparation method and detection method thereof Pending CN112147342A (en)

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