CN113030497A - Magnetic particle chemiluminescence immunoassay kit for procalcitonin and detection method thereof - Google Patents
Magnetic particle chemiluminescence immunoassay kit for procalcitonin and detection method thereof Download PDFInfo
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- CN113030497A CN113030497A CN202110396389.6A CN202110396389A CN113030497A CN 113030497 A CN113030497 A CN 113030497A CN 202110396389 A CN202110396389 A CN 202110396389A CN 113030497 A CN113030497 A CN 113030497A
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Images
Classifications
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- 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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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
The invention discloses a magnetic particle chemiluminescence immunoassay kit for procalcitonin and a detection method thereof. Belongs to the technical field of immunodetection and analysis. The kit comprises: magnetic particle reagent, PCT calibrator, PCT quality control product and PCT anti-reagent. The invention combines the chemiluminescence method with the immunomagnetic particles, provides a reaction system close to homogeneous phase, has higher detection sensitivity and specificity compared with the prior art, and achieves better performance parameters.
Description
Technical Field
The invention relates to the technical field of immunodetection and analysis, in particular to a magnetic particle chemiluminescence immunoassay kit for procalcitonin and a detection method thereof.
Background
In clinical sepsis patients, the PCT level is obviously higher than that of non-sepsis patients, and the procalcitonin, namely the PCT level of the bacterial sepsis patients is obviously higher than that of the non-bacterial sepsis patients. PCT has the highest sensitivity (85%) and highest specificity (91%) for patients with Systemic Inflammatory Response Syndrome (SIRS) in patients with sepsis compared to IL-2, IL-6, IL-8, CRP and TNF- α, and thus can be used as a biomarker for diagnosing sepsis and identifying severe bacterial infections. If sepsis is suspected, it is recommended to check PCT immediately. Current cutoff levels for PCT diagnosed sepsis are >0.5 ng/mL.
PCT increases in mass concentration in patients with SIRS, sepsis, severe sepsis, and septic shock in sequence, and there are statistical differences that positively correlate with the severity of the condition. The change trend of the PCT level can be dynamically monitored to judge the disease progress. A sustained increase in PCT suggests an increased infection or failure of treatment, and a decrease in PCT can be considered an improvement in infection and success of treatment.
A rapid decrease in PCT levels after treatment usually indicates a good prognosis, whereas a maintenance or increase in PCT meta-levels is indicative of a poor prognosis. The absolute value of initial PCT levels has limited prognostic significance, and even if initial PCT levels are very high, the prognosis is better as PCT rapidly decreases after correct treatment. Thus, the trend of PCT change is more important for prognosis judgment.
Different studies have demonstrated that proportional clinical information from PCT can further define the necessity of antibiotic treatment and optimize antibiotic courses. By monitoring PCT daily as an indication of antibiotic use, the course of antibiotic therapy can be shortened, thereby reducing unnecessary antibiotic use and reducing the rate of drug resistance and incidence of adverse reactions
Therefore, how to provide a procalcitonin detection reagent and a procalcitonin detection method is a problem to be solved urgently by the technical personnel in the field.
Disclosure of Invention
In view of this, the invention provides a magnetic particle chemiluminescence immunoassay kit for procalcitonin and a detection method thereof. Has the advantages of simple and convenient operation, rapid reaction, high sensitivity, strong specificity, suitability for on-site detection, economy, practicability and the like.
In order to achieve the purpose, the invention adopts the following technical scheme:
a magnetic particle chemiluminescence immunoassay kit for procalcitonin, comprising: magnetic particle reagent, PCT calibrator, PCT quality control product and PCT anti-reagent.
Preferably: the magnetic particle reagent is a magnetic particle solution connected with a goat anti-FITC antibody, and the preparation method comprises the following steps:
1) dispersing the carboxyl magnetic bead solution under the action of a magnetic particle buffer solution to obtain a dispersed carboxyl magnetic bead solution;
2) carrying out a connection reaction on the dispersed carboxyl magnetic bead solution and a goat anti-FITC antibody to obtain a connection product;
3) and (3) placing the connection product in a magnetic field, and washing the connection product by using a magnetic particle buffer solution to prepare a magnetic particle solution connected with the goat anti-FITC antibody.
Preferably: dispersing treatment in step 1): placing the concentrated solution of the carboxyl magnetic beads in a magnetic field for 15-20 min, removing supernatant after all the carboxyl magnetic beads are settled, adding a magnetic particle buffer solution, oscillating, placing in the magnetic field for 15-20 min, and removing supernatant;
the oscillation time is 20-30 min;
the volume ratio of the carboxyl magnetic beads to the magnetic particle buffer solution is 1 (2-5);
the dispersing treatment is carried out for 3 times;
in the step 2), the mass ratio of the dispersed carboxyl magnetic bead solution to the goat anti-FITC antibody is 100: 5-100: 1;
the concentration of the dispersed carboxyl magnetic bead solution is 10-50 mg/mL;
the temperature of the connection reaction is 2-8 ℃, and the time of the connection reaction is 16-20 h;
in the step 3), the magnetic field is placed for 13-17 min;
the number of washes was 3;
the concentration of the magnetic particle solution connected with the goat anti-FITC antibody is 10 mg/mL.
Preferably: the PCT calibrator and the PCT quality control product are both Tris buffer solutions containing PCT antigens, 0.5% BSA by mass and 0.05% prolin300 by mass; wherein, the concentration of the PCT quality control antigen is 0.8ng/mL and 33.0ng/mL respectively; the concentration of PCT calibrator antigen was 2.8ng/mL and 33.0ng/mL, respectively.
Preferably: PCT anti-agents include: FITC-labeled anti-PCT antibody 1 and AP-labeled anti-PCT antibody 2 were both present at a concentration of 0.5. + -. 0.05. mu.g/mL.
Preferably: preparation method of FITC labeled anti-PCT antibody 1:
1) preparing FITC into a FITC solution with the concentration of 1.0-5.0 mg/mL by using 0.15M sodium bicarbonate buffer solution;
2) the method comprises the following steps of (1) mixing a mouse monoclonal antibody PCT antibody 1 and a FITC solution according to a mass ratio of 1: (1-1.5) mixing, and carrying out a linking reaction to obtain a linking product of the PCT antibody and the FITC;
3) and separating and purifying the linking product by using a carbonate buffer solution with the pH value of 8-9 to obtain the FITC-labeled anti-PCT antibody.
Further: anti-PCT antibody 1 was purchased from AB1042-F, beijing zecheng biotechnology limited.
The conditions of the chain reaction are as follows: room temperature; 12 to 18 hours.
Separating and purifying by using PD10(G-25 sephadex) separation column; carbonate buffer equilibrates 5 column length volumes;
preferably: preparation method of AP-labeled anti-PCT antibody 2:
1) preparing AP into an AP solution with the concentration of 1.0-5.0 mg/mL by using TRIS buffer solution;
2) respectively activating the mouse monoclonal antibody PCT antibody 2 and the AP solution, and enabling the activated AP solution and the mouse monoclonal antibody PCT antibody to react according to the molar ratio of 1: (1-3) uniformly mixing, and carrying out coupling reaction under the action of 1M magnesium chloride solution to obtain a coupling product;
3) adding the coupling product into a separation and purification instrument, and using a Tris buffer solution with the pH value of 7.5 as a mobile phase at the flow speed of 1 ml/min; and collecting the separation product for 15-35 min to obtain the AP-labeled anti-PCT antibody.
Further: anti-PCT antibody 2 was purchased from Beijing Zernike biotechnology, Inc. AB 1042-A.
Buffer composition: 1M TRIS in percentage by mass, 0.05 percent sodium azide in percentage by volume and pH 7.0. Activation was performed with 7mg/mL EDC and NHS, respectively.
Coupling reaction conditions: 2-8 ℃ for 12-18 hours.
The invention also provides a method for detecting procalcitonin for non-diagnostic therapeutic purposes, comprising: mixing a serum sample, an FITC-labeled anti-PCT antibody 1 and an AP-labeled anti-PCT antibody 2, incubating, adding a magnetic particle reagent, incubating again, removing supernatant after the magnetic particle reagent is settled, washing twice, adding a substrate solution, oscillating and uniformly mixing, and detecting relative luminous intensity;
preferably: further comprising: calibrating and calibrating the high-value and low-value calibrators, and automatically fitting an instrument to generate a new working curve for sample measurement; and (3) taking the PCT quality control substance as a sample, performing concentration fitting by using a working curve to obtain the corresponding quality control substance concentration, and observing that the fitted quality control substance concentration is +/-30% of the standard concentration value.
Preferably: the volume ratio of the serum sample, the FITC-labeled anti-PCT antibody 1, the AP-labeled anti-PCT antibody 2 and the magnetic particle reagent is 1:1:1: 1;
incubation is accompanied with oscillation, and the oscillation time is 30-40 s;
incubating and re-incubating in water bath at 37 deg.C; the incubation time is 15-17 min; the time for re-incubation is 5-6 min;
the settling time is 3-4 min.
According to the technical scheme, compared with the prior art, the invention discloses a magnetic particle chemiluminescence immunoassay kit for procalcitonin and a detection method thereof, and the technical effects are as follows:
1. the magnetic beads are used as the solid phase, so that immunoreaction is closer to a liquid phase, the reaction is more sufficient and rapid, the combined immune complex is easier to separate, and nonspecific adsorption is reduced.
2. The used anti-reagent is a monoclonal antibody mixture, so that the affinity of immunoreaction is higher, the production batch difference phase pair of the monoclonal antibody is small, and the batch stability of the product is more easily ensured.
3. The substrate liquid for the full-automatic immune inspection system is used, so that the detection sensitivity is improved, and the linear range is wider. The minimum detection limit is 0.05ng/mL, and bacterial infection, sepsis and the like can be diagnosed.
4. Can provide a convenient, high-sensitivity, high-accuracy and stable detection method for the development of other kits.
5. The kit combines the chemiluminescence technology with the immunomagnetic particles, provides a reaction system close to homogeneous phase, has higher detection sensitivity, specificity, accuracy and precision, and achieves better performance parameters.
6. The magnetic particle reagent, the anti-reagent, the calibrator and the quality control product (which can be combined with a cleaning solution and a substrate solution for a full-automatic immunoassay system) in the kit are the optimal formula under the reaction system, and powerful guarantee is provided for the service life and the detection performance of the kit.
7. The pretreatment requirement on the sample is low, the pretreatment process is simple, and a large amount of samples can be detected quickly and at high flux; the high-specificity monoclonal antibody and the superparamagnetic, highly-dispersive magnetic microparticles with large specific surface area are adopted, the main reagent is provided in the form of working solution, and the detection method is convenient and easy to implement.
8. The magnetic particle chemiluminescence immunoassay kit has the advantages of simple structure, convenience in use, low price and convenience in carrying.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a diagram of PCT standard curve data provided by the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The embodiment of the invention discloses a magnetic particle chemiluminescence immunoassay kit for procalcitonin and a detection method thereof.
The starting materials in the examples are commercially available, for example: the carboxyl magnetic bead concentrated solution, an anti-PCT antibody 1 marked by FITC (Beijing Zecheng Biotechnology Co., Ltd AB1042-F) and an anti-PCT antibody 2 marked by AP (Beijing Zecheng Biotechnology Co., Ltd AB1042-A) are purchased from Beijing Zecheng Biotechnology Co., Ltd;
cleaning solution is purchased from WR2001 cleaning solution (number of medical instrument record voucher/number of product specification: Sutai mechanical arm No. 20160027, buffer solution containing Tris, NaCl, surfactant, etc.) of Tanzcheng Biotechnology, Inc.;
the substrate solution (for the full-automatic immunoassay system) is a buffer solution containing Lumigen APS-5 (each 1L of the buffer solution contains APS-510-14 mg) luminescent solution (the buffer solution is a tris buffer solution containing lucigenin, sodium sulfite, Sodium Dodecyl Sulfate (SDS) and Tween-20).
Operating requirements of a fully automated immunoassay system (tay zechen biotechnology limited):
1. preparation before testing
1) All reagents are required to be placed at room temperature before the test, and the instrument is started to be preheated for at least 30 minutes;
2) according to the system operation guide, the loading of reaction tubes (cups), the addition or replacement of matched reagents, the cleaning of waste liquid and waste tubes and the operation of filling liquid paths are carried out.
2. Reagent, sample Loading
1) Before loading the reagent, placing the reagent to be used on mixing equipment for fully mixing, and visually checking that the components of a reagent solution are clarified without foreign matters, precipitates and floccules and a magnetic particle reagent is a uniform suspension without obvious agglutination;
2) scanning a reagent bar code according to the system operation guide to complete the loading of the magnetic particle reagent and the anti-reagent;
3) and after being uniformly mixed, the calibrator and the quality control product are moved to a reaction cup special for the instrument and loaded at an instrument sample position, and the plasma sample obtained by centrifugation is directly loaded at the instrument sample position after being scanned.
3. Detection step
1) In order to achieve the best detection performance, the operation is carried out according to the relevant requirements of the detection kit (magnetic particle chemiluminescence method) provided by the invention and an operation manual of a full-automatic chemiluminescence determinator.
Note: the detection reaction process and related parameters are predefined in the instrument operating software.
4. Calibration of standard curve
And scanning the main curve card, reading the data of the standard curve, and automatically fitting the standard curve by the background of the instrument.
Note: the standard curve is calibrated by two calibrators (the concentrations are respectively 2.8ng/mL and 33.0ng/mL) with high and low values, and a new working curve is generated by automatic fitting of an instrument background and used for sample determination.
5. Scaling
By measuring the high and low calibrators, each calibration point on the predefined master curve will be adjusted (re-calibrated) to a new, instrument-specific measurement level, i.e. the working curve.
Example 1
A magnetic particle chemiluminescence immunoassay kit for procalcitonin, comprising: magnetic particle reagent, PCT calibrator, PCT quality control product and PCT anti-reagent.
In order to further optimize the technical scheme: the magnetic particle reagent is a magnetic particle solution connected with a goat anti-FITC antibody, and the preparation method comprises the following steps:
1) dispersing the carboxyl magnetic bead solution under the action of a magnetic particle buffer solution to obtain a dispersed carboxyl magnetic bead solution;
2) carrying out a connection reaction on the dispersed carboxyl magnetic bead solution and a goat anti-FITC antibody to obtain a connection product;
3) and (3) placing the connection product in a magnetic field, and washing the connection product by using a magnetic particle buffer solution to prepare a magnetic particle solution connected with the goat anti-FITC antibody.
In order to further optimize the technical scheme: dispersing treatment in step 1): placing the concentrated solution of carboxyl magnetic beads in a magnetic field for 15min, removing supernatant after the carboxyl magnetic beads are completely settled, adding magnetic particle buffer solution, oscillating, placing in the magnetic field for 15min, and removing supernatant;
the oscillation time is 20 min;
the volume ratio of the carboxyl magnetic beads to the magnetic particle buffer solution is 1: 2;
the dispersing treatment is carried out for 3 times;
in the step 2), the mass ratio of the dispersed carboxyl magnetic bead solution to the goat anti-FITC antibody is 100: 5;
the concentration of the dispersed carboxyl magnetic bead solution is 10 mg/mL;
the temperature of the connection reaction is 2 ℃, and the time of the connection reaction is 16 h;
in the step 3), the magnetic field is placed for 13 min;
the number of washes was 3;
the concentration of the magnetic particle solution connected with the goat anti-FITC antibody is 10 mg/mL.
In order to further optimize the technical scheme: the PCT calibrator and the PCT quality control product are both Tris buffer solutions containing PCT antigens, 0.5% BSA by mass and 0.05% prolin300 by mass; wherein, the concentration of the PCT quality control antigen is 0.8ng/mL and 33.0ng/mL respectively; the concentration of PCT calibrator antigen was 2.8ng/mL and 33.0ng/mL, respectively.
In order to further optimize the technical scheme: PCT anti-agents include: FITC-labeled anti-PCT antibody 1 and AP-labeled anti-PCT antibody 2 were both present at a concentration of 0.5. + -. 0.05. mu.g/mL.
In order to further optimize the technical scheme: preparation method of FITC labeled anti-PCT antibody 1:
1) FITC is prepared into a FITC solution with the concentration of 1.0mg/mL by using 0.15M sodium bicarbonate buffer solution;
2) the method comprises the following steps of (1) mixing a mouse monoclonal antibody PCT antibody 1 and a FITC solution according to a mass ratio of 1:1, mixing, and carrying out a linking reaction to obtain a linking product of the PCT antibody and the FITC;
3) the ligation product was isolated and purified using carbonate buffer pH 8 to obtain FITC-labeled anti-PCT antibody.
In order to further optimize the technical scheme: preparation method of AP-labeled anti-PCT antibody 2:
1) preparing AP into an AP solution with the concentration of 1.0mg/mL by using TRIS buffer solution;
2) respectively activating the mouse monoclonal antibody PCT antibody 2 and the AP solution, and enabling the activated AP solution and the mouse monoclonal antibody PCT antibody to react according to the molar ratio of 1:1, uniformly mixing, and carrying out coupling reaction under the action of a 1M magnesium chloride solution to obtain a coupling product;
3) adding the coupling product into a separation and purification instrument, and using a Tris buffer solution with the pH value of 7.5 as a mobile phase at the flow speed of 1 ml/min; and collecting the separation product for 15-35 min to obtain the AP-labeled anti-PCT antibody.
A method for detecting procalcitonin for non-diagnostic therapeutic purposes comprising: mixing a serum sample, an FITC-labeled anti-PCT antibody 1 and an AP-labeled anti-PCT antibody 2, incubating, adding a magnetic particle reagent, incubating again, removing supernatant after the magnetic particle reagent is settled, washing twice, adding a substrate solution, oscillating and uniformly mixing, and detecting relative luminous intensity;
in order to further optimize the technical scheme: calibrating and calibrating the high-value and low-value calibrators, and automatically fitting an instrument to generate a new working curve for sample measurement; and (3) taking the PCT quality control substance as a sample, performing concentration fitting by using a working curve to obtain the corresponding quality control substance concentration, and observing that the fitted quality control substance concentration is +/-30% of the standard concentration value.
In order to further optimize the technical scheme: the volume ratio of the serum sample, the FITC-labeled anti-PCT antibody 1, the AP-labeled anti-PCT antibody 2 and the magnetic particle reagent is 1:1:1: 1;
incubation is accompanied by oscillation, and the oscillation time is 30 s;
incubating and re-incubating in water bath at 37 deg.C; the incubation time is 15 min; the time for re-incubation is 5 min;
the settling time was 3 min.
Example 2
A magnetic particle chemiluminescence immunoassay kit for procalcitonin, comprising: magnetic particle reagent, PCT calibrator, PCT quality control product and PCT anti-reagent.
In order to further optimize the technical scheme: the magnetic particle reagent is a magnetic particle solution connected with a goat anti-FITC antibody, and the preparation method comprises the following steps:
1) dispersing the carboxyl magnetic bead solution under the action of a magnetic particle buffer solution to obtain a dispersed carboxyl magnetic bead solution;
2) carrying out a connection reaction on the dispersed carboxyl magnetic bead solution and a goat anti-FITC antibody to obtain a connection product;
3) and (3) placing the connection product in a magnetic field, and washing the connection product by using a magnetic particle buffer solution to prepare a magnetic particle solution connected with the goat anti-FITC antibody.
In order to further optimize the technical scheme: dispersing treatment in step 1): placing the concentrated solution of carboxyl magnetic beads in a magnetic field for 18min, removing supernatant after the carboxyl magnetic beads are completely settled, adding magnetic particle buffer solution, oscillating, placing in the magnetic field for 18min, and removing supernatant;
the oscillation time is 25 min;
the volume ratio of the carboxyl magnetic beads to the magnetic particle buffer solution is 1: 3;
the dispersing treatment is carried out for 3 times;
in the step 2), the mass ratio of the dispersed carboxyl magnetic bead solution to the goat anti-FITC antibody is 100: 3;
the concentration of the dispersed carboxyl magnetic bead solution is 30 mg/mL;
the temperature of the connection reaction is 2-8 ℃, and the time of the connection reaction is 18 h;
in the step 3), the magnetic field is placed for 15 min;
the number of washes was 3;
the concentration of the magnetic particle solution connected with the goat anti-FITC antibody is 10 mg/mL.
In order to further optimize the technical scheme: the PCT calibrator and the PCT quality control product are both Tris buffer solutions containing PCT antigens, 0.5% BSA by mass and 0.05% prolin300 by mass; wherein, the concentration of the PCT quality control antigen is 0.8ng/mL and 33.0ng/mL respectively; the concentration of PCT calibrator antigen was 2.8ng/mL and 33.0ng/mL, respectively.
In order to further optimize the technical scheme: PCT anti-agents include: FITC-labeled anti-PCT antibody 1 and AP-labeled anti-PCT antibody 2 were both present at a concentration of 0.5. + -. 0.05. mu.g/mL.
In order to further optimize the technical scheme: preparation method of FITC labeled anti-PCT antibody 1:
1) FITC is prepared into a FITC solution with the concentration of 2.0mg/mL by using 0.15M sodium bicarbonate buffer solution;
2) the method comprises the following steps of (1) mixing a mouse monoclonal antibody PCT antibody 1 and a FITC solution according to a mass ratio of 1: 1.2, mixing, and carrying out a linking reaction to obtain a linking product of the PCT antibody and the FITC;
3) the ligation product was isolated and purified using carbonate buffer pH 8 to obtain FITC-labeled anti-PCT antibody.
In order to further optimize the technical scheme: preparation method of AP-labeled anti-PCT antibody 2:
1) preparing AP into an AP solution with the concentration of 3.0mg/mL by using TRIS buffer solution;
2) respectively activating the mouse monoclonal antibody PCT antibody 2 and the AP solution, and enabling the activated AP solution and the mouse monoclonal antibody PCT antibody to react according to the molar ratio of 1:2, uniformly mixing, and carrying out coupling reaction under the action of a 1M magnesium chloride solution to obtain a coupling product;
3) adding the coupling product into a separation and purification instrument, and using a Tris buffer solution with the pH value of 7.5 as a mobile phase at the flow speed of 1 ml/min; and collecting the separation product for 15-35 min to obtain the AP-labeled anti-PCT antibody.
A method for detecting procalcitonin for non-diagnostic therapeutic purposes comprising: mixing a serum sample, an FITC-labeled anti-PCT antibody 1 and an AP-labeled anti-PCT antibody 2, incubating, adding a magnetic particle reagent, incubating again, removing supernatant after the magnetic particle reagent is settled, washing twice, adding a substrate solution, oscillating and uniformly mixing, and detecting relative luminous intensity;
in order to further optimize the technical scheme: calibrating and calibrating the high-value and low-value calibrators, and automatically fitting an instrument to generate a new working curve for sample measurement; and (3) taking the PCT quality control substance as a sample, performing concentration fitting by using a working curve to obtain the corresponding quality control substance concentration, and observing that the fitted quality control substance concentration is +/-30% of the standard concentration value.
In order to further optimize the technical scheme: the volume ratio of the serum sample, the FITC-labeled anti-PCT antibody 1, the AP-labeled anti-PCT antibody 2 and the magnetic particle reagent is 1:1:1: 1;
incubation is accompanied by oscillation, and the oscillation time is 35 s;
incubating and re-incubating in water bath at 37 deg.C; the incubation time is 16 min; the time for re-incubation is 6 min;
the settling time was 4 min.
Example 3
A magnetic particle chemiluminescence immunoassay kit for procalcitonin, comprising: magnetic particle reagent, PCT calibrator, PCT quality control product and PCT anti-reagent.
In order to further optimize the technical scheme: the magnetic particle reagent is a magnetic particle solution connected with a goat anti-FITC antibody, and the preparation method comprises the following steps:
1) dispersing the carboxyl magnetic bead solution under the action of a magnetic particle buffer solution to obtain a dispersed carboxyl magnetic bead solution;
2) carrying out a connection reaction on the dispersed carboxyl magnetic bead solution and a goat anti-FITC antibody to obtain a connection product;
3) and (3) placing the connection product in a magnetic field, and washing the connection product by using a magnetic particle buffer solution to prepare a magnetic particle solution connected with the goat anti-FITC antibody.
In order to further optimize the technical scheme: dispersing treatment in step 1): placing the concentrated solution of carboxyl magnetic beads in a magnetic field for 20min, removing supernatant after the carboxyl magnetic beads are completely settled, adding magnetic particle buffer solution, oscillating, placing in the magnetic field for 20min, and removing supernatant;
the oscillation time is 30 min;
the volume ratio of the carboxyl magnetic beads to the magnetic particle buffer solution is 1: 5;
the dispersing treatment is carried out for 3 times;
in the step 2), the mass ratio of the dispersed carboxyl magnetic bead solution to the goat anti-FITC antibody is 100: 1;
the concentration of the dispersed carboxyl magnetic bead solution is 50 mg/mL;
the temperature of the connection reaction is 2-8 ℃, and the time of the connection reaction is 20 h;
in the step 3), the magnetic field is placed for 17 min;
the number of washes was 3;
the concentration of the magnetic particle solution connected with the goat anti-FITC antibody is 10 mg/mL.
In order to further optimize the technical scheme: the PCT calibrator and the PCT quality control product are both Tris buffer solutions containing PCT antigens, 0.5% BSA by mass and 0.05% prolin300 by mass; wherein, the concentration of the PCT quality control antigen is 0.8ng/mL and 33.0ng/mL respectively; the concentration of PCT calibrator antigen was 2.8ng/mL and 33.0ng/mL, respectively.
In order to further optimize the technical scheme: PCT anti-agents include: FITC-labeled anti-PCT antibody 1 and AP-labeled anti-PCT antibody 2 were both present at a concentration of 0.5. + -. 0.05. mu.g/mL.
In order to further optimize the technical scheme: preparation method of FITC labeled anti-PCT antibody 1:
1) FITC is prepared into a FITC solution with the concentration of 5.0mg/mL by using 0.15M sodium bicarbonate buffer solution;
2) the method comprises the following steps of (1) mixing a mouse monoclonal antibody PCT antibody 1 and a FITC solution according to a mass ratio of 1: 1.5, mixing, and carrying out a linking reaction to obtain a linking product of the PCT antibody and the FITC;
3) and separating and purifying the linking product by using a carbonate buffer solution with the pH value of 8-9 to obtain the FITC-labeled anti-PCT antibody.
In order to further optimize the technical scheme: preparation method of AP-labeled anti-PCT antibody 2:
1) preparing AP into an AP solution with the concentration of 5.0mg/mL by using TRIS buffer solution;
2) respectively activating the mouse monoclonal antibody PCT antibody 2 and the AP solution, and enabling the activated AP solution and the mouse monoclonal antibody PCT antibody to react according to the molar ratio of 1:3, uniformly mixing, and carrying out coupling reaction under the action of a 1M magnesium chloride solution to obtain a coupling product;
3) adding the coupling product into a separation and purification instrument, and using a Tris buffer solution with the pH value of 7.5 as a mobile phase at the flow speed of 1 ml/min; and collecting the separation product for 15-35 min to obtain the AP-labeled anti-PCT antibody.
A method for detecting procalcitonin for non-diagnostic therapeutic purposes comprising: mixing a serum sample, an FITC-labeled anti-PCT antibody 1 and an AP-labeled anti-PCT antibody 2, incubating, adding a magnetic particle reagent, incubating again, removing supernatant after the magnetic particle reagent is settled, washing twice, adding a substrate solution, oscillating and uniformly mixing, and detecting relative luminous intensity;
in order to further optimize the technical scheme: calibrating and calibrating the high-value and low-value calibrators, and automatically fitting an instrument to generate a new working curve for sample measurement; and (3) taking the PCT quality control substance as a sample, performing concentration fitting by using a working curve to obtain the corresponding quality control substance concentration, and observing that the fitted quality control substance concentration is +/-30% of the standard concentration value.
In order to further optimize the technical scheme: the volume ratio of the serum sample, the FITC-labeled anti-PCT antibody 1, the AP-labeled anti-PCT antibody 2 and the magnetic particle reagent is 1:1:1: 1;
incubation is accompanied with oscillation, and the oscillation time is 30-40 s;
incubating and re-incubating in water bath at 37 deg.C; the incubation time is 17 min; the time for re-incubation is 6 min;
the settling time was 3 min.
The performance indexes of the detection kit provided by the invention are as follows:
1. linearity
The linear range (0.2-110) ng/mL, and the correlation coefficient r is more than or equal to 0.9900.
The operation method comprises the following steps:
diluting the serum or serum substitute close to the upper limit of the linear range into five concentrations according to a certain proportion by using a sample diluent, respectively carrying out 3 times of repeated measurement on 6 concentration samples before and after dilution by using a chemiluminescence determinator, calculating an average value, carrying out linear fitting by using a least square method according to the average value and the dilution proportion, and calculating a linear correlation coefficient r (obtained by carrying out linear fitting on an X/Y axis in a column of 1-1/512 and an RLU average value), wherein the result is not less than 0.9900.
Data case:
2. accuracy of
The addition recovery rate is 85-115%.
The operation method comprises the following steps:
a sample A at a concentration of 110.0 ng/mL. + -. 20% is added to a sample B of serum or other corresponding substrate at a concentration of 0.8 ng/mL. + -. 20%, preferably in a volume not exceeding 10% of the total volume (A + B), and the recovery R is calculated according to equation (1).
In the formula:
r: recovery rate
V: volume of sample a fluid;
volume of V0 sample B liquid;
c: detecting the concentration of the sample solution B after adding the solution A;
c0: the concentration of sample B;
cs: concentration of sample a liquid.
Data case (V0.1 mL, V0 0.9mL, C0 0.8ng/mL, Cs 110.0ng/mL, where V and V0 are in a 1:9 relationship as described above, and C0 and Cs are the concentrations of the high and low concentration points in the calibrator, respectively):
3. minimum limit of detection
The lowest detection limit is less than or equal to 0.05 ng/mL.
The operation method comprises the following steps:
the measurement was repeated 20 times using a zero concentration calibrator or sample diluent as the sample to be measured. Obtaining RLU values (relative luminescence values) of 20 measurement results, calculating the average value (M) and the Standard Deviation (SD) of the RLU values, obtaining M +2SD, performing two-point regression fitting according to the concentration-RLU value result between the zero-concentration calibrator and the adjacent calibrator to obtain a linear equation, substituting the RLU value of the M +2SD into the equation, and calculating a corresponding concentration value, namely the lowest detection limit.
4. Repeatability of
Coefficient of variation CV is less than or equal to 8 percent. The operation method comprises the following steps:
taking 1(0.8 +/-0.16) ng/mL and 2(33.0 +/-6.6) ng/mL of the quality control product as samples to be tested, and respectively carrying out 10 repeated determinations on the Procalcitonin (PCT) content in the samples to be tested by using a chemiluminescence determinator. The Coefficient of Variation (CV) is calculated according to equation (2).
In the formula:
Xi: each time measuring concentration value
Data case:
5. difference between batches
Coefficient of variation CV is less than or equal to 15 percent.
The operation method comprises the following steps:
taking the quality control product 1 or the quality control product 2 in the same batch of kits as a sample to be detected, using 3 different batches of kits, and using a chemiluminescence determinator to repeatedly determine the content of abnormal prothrombin in the sample to be detected for 10 times respectively, wherein the total number of the determination is 30.
The inter-batch Coefficient of Variation (CV) was calculated according to equation (3).
In the formula:
Xi: each time measuring concentration value
6. Specificity of
a) The concentration of the calcium-resistant element is not lower than 1200ng/mL, and the determination result on the kit is not higher than the lowest detection limit level;
b) the calcitonin with the concentration not lower than 1000ng/mL, and the determination result on the kit should not be higher than the minimum detection limit level.
The operation method comprises the following steps:
2 parts of sample with zero PCT content are respectively added with anticalcin and calcitonin to ensure that the concentration of the anticalcin in the sample is not lower than 1200ng/mL and the concentration of the calcitonin is not lower than 1000ng/mL, and a PCT reagent (kit) is used for detecting the two parts of sample to determine the content of the PCT in the sample.
7. Calibrator measurement accuracy
The relative deviation should be within + -10%.
The operation method comprises the following steps:
and (3) repeatedly detecting the same bottle of calibrator respectively, and calculating the relative measurement deviation B of the two calibrators with different concentrations according to a formula (4).
In the formula:
b: relative deviation of
M: mean value of measured concentration
T: calibration concentration
8. Homogeneity of calibrator
Uniformity among bottles, CV is less than or equal to 10 percent.
The operation method comprises the following steps:
detecting 5 different bottles of calibrators in the same batch for 1 time respectively to obtain 5 test results XiCalculating the average value
And standard deviation (S)1) (ii) a The same 1 bottle of the batch of the calibrator was tested 5 times each, and the standard deviation (S) of the test results was calculated2) (ii) a The vial-to-vial imprecision CV (%) was calculated according to the following formula (5).
When S1 is less than S2, let CV bottle be 0
In the formula:
s: standard deviation;
Xi: the concentration values were determined each time.
Data case (tcam concentration refers to target concentration of sample):
9. quality control measurement value
The assay results for the same set of quality controls should be within the ranges specified in the kits provided in the examples.
The operation method comprises the following steps:
using the kit provided in the same batch of examples, the quality control material was subjected to repeated measurement 3 times using a chemiluminescence analyzer, and the average value was calculated to be within. + -. 30% of the value of the target concentration of the quality control material.
10. Quality control product measurement accuracy
The relative deviation should be within + -10%.
The operation method comprises the following steps:
and (3) repeatedly detecting the quality control products in the same bottle respectively, and calculating the relative measurement deviation B of the quality control products with two different concentrations according to the formula (6).
In the formula:
b: relative deviation of
M: mean value of measured concentration
T: calibration concentration
11. Homogeneity of quality control product
Coefficient of variation CV is less than or equal to 8 percent.
The operation method comprises the following steps:
detecting 10 bottles of quality control material with the same concentration in the same batch for 1 time, and calculating the average value of the measured values
And standard deviation (S)1) (ii) a Repeating the measurement of the concentration control substance in the same batch for 10 times, and calculating average value of the measured values
And standard deviation (S)2) (ii) a The vial-to-vial imprecision CV (%) was calculated according to the following formula (7).
When S1 is less than S2, let CV bottle be 0
In the formula:
S1: standard deviation of the measured concentration of 10 bottles each measured 1 time;
S2: standard deviation of measured concentration was determined 10 times for 1 vial.
Data case (tcam concentration refers to target concentration of sample):
the embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. A procalcitonin magnetic particle chemiluminescence immunoassay kit is characterized by comprising: magnetic particle reagent, PCT calibrator, PCT quality control product and PCT anti-reagent.
2. The kit for the magnetic particle chemiluminescence immunoassay of procalcitonin according to claim 1, wherein the magnetic particle reagent is a magnetic particle solution connected with goat anti-FITC antibody, and the preparation method comprises:
1) dispersing the carboxyl magnetic bead solution under the action of a magnetic particle buffer solution to obtain a dispersed carboxyl magnetic bead solution;
2) carrying out a connection reaction on the dispersed carboxyl magnetic bead solution and a goat anti-FITC antibody to obtain a connection product;
3) and (3) placing the connection product in a magnetic field, and washing the connection product by using a magnetic particle buffer solution to prepare a magnetic particle solution connected with the goat anti-FITC antibody.
3. The kit for the magnetic particle chemiluminescence immunoassay of procalcitonin according to claim 2, wherein: the dispersion treatment in step 1): placing the concentrated solution of the carboxyl magnetic beads in a magnetic field for 15-20 min, removing supernatant after all the carboxyl magnetic beads are settled, adding a magnetic particle buffer solution, oscillating, placing in the magnetic field for 15-20 min, and removing supernatant;
the oscillation time is 20-30 min;
the volume ratio of the carboxyl magnetic beads to the magnetic particle buffer solution is 1 (2-5);
the dispersing treatment is carried out for 3 times;
in the step 2), the mass ratio of the dispersed carboxyl magnetic bead solution to the goat anti-FITC antibody is (100: 5-100): 1;
the concentration of the dispersed carboxyl magnetic bead solution is 10-50 mg/mL;
the temperature of the connection reaction is 2-8 ℃, and the time of the connection reaction is 16-20 h;
in the step 3), the magnetic field is placed for 13-17 min;
the number of the washing times is 3;
the concentration of the magnetic particle solution connected with the goat anti-FITC antibody is 10 mg/mL.
4. The kit for the magnetic particle chemiluminescence immunoassay of procalcitonin according to claim 1, wherein: the PCT calibrator and the PCT quality control product are both Tris buffer solutions containing PCT antigens, 0.5% BSA (bovine serum albumin) and 0.05% prolin300 by mass; wherein, the concentration of the PCT quality control antigen is 0.8ng/mL and 33.0ng/mL respectively; the concentration of the PCT calibrator antigen was 2.8ng/mL and 33.0ng/mL, respectively.
5. The kit for a magnetic particle chemiluminescence immunoassay of procalcitonin according to claim 1, wherein said PCT anti-reagent comprises: FITC-labeled anti-PCT antibody 1 and AP-labeled anti-PCT antibody 2 were both present at a concentration of 0.5. + -. 0.05. mu.g/mL.
6. The kit for the magnetic particle chemiluminescence immunoassay of procalcitonin according to claim 5, wherein the FITC-labeled anti-PCT antibody 1 is prepared by the following steps:
1) preparing FITC into a FITC solution with the concentration of 1.0-5.0 mg/mL by using 0.15M sodium bicarbonate buffer solution;
2) the method comprises the following steps of (1) mixing a mouse monoclonal antibody PCT antibody 1 and a FITC solution according to a mass ratio of 1: (1-1.5) mixing, and carrying out a linking reaction to obtain a linking product of the PCT antibody and the FITC;
3) and separating and purifying the linked product by using a carbonate buffer solution with the pH value of 8-9 to obtain the FITC-labeled anti-PCT antibody.
7. The kit for the magnetic particle chemiluminescence immunoassay of procalcitonin according to claim 5, wherein the preparation method of AP-labeled anti-PCT antibody 2 comprises the following steps:
1) preparing AP into an AP solution with the concentration of 1.0-5.0 mg/mL by using TRIS buffer solution;
2) respectively activating the mouse monoclonal antibody PCT antibody 2 and the AP solution, and enabling the activated AP solution and the mouse monoclonal antibody PCT antibody to react according to the molar ratio of 1: (1-3) uniformly mixing, and carrying out coupling reaction under the action of 1M magnesium chloride solution to obtain a coupling product;
3) adding the coupling product into a separation and purification instrument, and using a Tris buffer solution with the pH value of 7.5 as a mobile phase at the flow speed of 1 ml/min; and collecting the separation product for 15-35 min to obtain the AP-labeled anti-PCT antibody.
8. A method for detecting procalcitonin for non-diagnostic therapeutic purposes comprising: mixing a serum sample, an FITC-labeled anti-PCT antibody 1 and an AP-labeled anti-PCT antibody 2, incubating, adding a magnetic particle reagent, incubating again, removing supernatant after the magnetic particle reagent is settled, washing twice, adding a substrate solution, oscillating and mixing uniformly, and detecting relative luminous intensity.
9. The method of claim 8, further comprising the step of: calibrating and calibrating the high-value and low-value calibrators, and automatically fitting an instrument to generate a new working curve for sample measurement; and (3) taking the PCT quality control substance as a sample, performing concentration fitting by using a working curve to obtain the corresponding quality control substance concentration, and observing that the fitted quality control substance concentration is +/-30% of the standard concentration value.
10. The method for detecting procalcitonin for non-diagnostic therapeutic purposes according to claim 9, wherein the volume ratio of the serum sample, FITC-labeled anti-PCT antibody 1, AP-labeled anti-PCT antibody 2 and magnetic microparticle reagent is 1:1:1: 1;
the incubation is accompanied by oscillation, and the oscillation time is 30-40 s;
the incubation and the secondary incubation are carried out in water bath, and the temperature is 37 ℃; the incubation time is 15-17 min;
the re-incubation time is 5-6 min;
and the settling time is 3-4 min.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108205059A (en) * | 2017-11-28 | 2018-06-26 | 泰州泽成生物技术有限公司 | A kind of kit and its test method for measuring calcitonin content |
| CN111175492A (en) * | 2020-02-27 | 2020-05-19 | 江苏泽成生物技术有限公司 | Kit for detecting procalcitonin content in serum and use method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108205059A (en) * | 2017-11-28 | 2018-06-26 | 泰州泽成生物技术有限公司 | A kind of kit and its test method for measuring calcitonin content |
| CN111175492A (en) * | 2020-02-27 | 2020-05-19 | 江苏泽成生物技术有限公司 | Kit for detecting procalcitonin content in serum and use method thereof |
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