Electrochemiluminescence detection kit for procalcitonin

Abstract

The invention discloses a procalcitonin electrochemical luminescence detection kit. The kit comprises a magnetic microsphere reagent coated by a procalcitonin capture antibody and a procalcitonin detection antibody reagent labeled by an electrically neutral ruthenium compound, the structural general formula of the electrically neutral ruthenium compound is # imgabs0 # n = 0-9; the magnetic microsphere reagent coated with the procalcitonin capture antibody comprises magnetic microspheres coated with the procalcitonin capture antibody and a diluent; the electrically neutral ruthenium compound labeled procalcitonin detection antibody reagent comprises an electrically neutral ruthenium compound labeled procalcitonin detection antibody and a diluent; the diluent is prepared from piperazine-1, 4-diethyl sulfonic acid or 3-[N-N-bis (2-ethoxyl) amino]-2-hydroxy propanesulfonic acid. When the procalcitonin electrochemical luminescence detection kit is used for detection, a reaction system does not contain streptavidin-coated magnetic beads or biotinylated antibodies, so that interference of biotin possibly existing in a sample on a test result is effectively avoided, and wide application can be realized.

Description

Technical Field

[0001] The invention belongs to the field of medical detection, and in particular relates to a procalcitonin electrochemiluminescence detection kit. Background Art

[0002] Procalcitonin (PCT) is the prohormone of calcitonin, composed of 116 amino acids and a molecular weight of approximately 12.8kD. It is a glycoprotein without hormonal activity and an endogenous non-steroidal anti-inflammatory substance produced by the thyroid gland in the absence of infection. When a systemic infection occurs, different types of cells in many organs secrete procalcitonin after being stimulated by a pro-inflammatory response, especially when infected with bacteria. PCT is mainly produced by the induction of bacterial toxins and infection-related inflammatory factors, while viral infection cannot stimulate the production of PCT. Therefore, the blood PCT concentration does not increase significantly during viral infection. PCT is an important marker that can specifically distinguish between bacterial infection and inflammatory reactions caused by other reasons. It is also an ideal indicator for auxiliary diagnosis of severe bacterial infections and diseases such as sepsis and septicemia. It has high sensitivity and specificity for bacterial infections, sepsis and septicemia.

[0003] So far, the chemiluminescence methods used to detect PCT in human serum or plasma mainly include: enzymatic magnetic particle chemiluminescence method, acridinium ester magnetic particle chemiluminescence method and traditional terpyridine ruthenium electrochemiluminescence method. Most of the above methodologies use (streptavidin) avidin / biotin coupling, and PCT detection is easily interfered by biotin in the sample. Summary of the invention

[0004] In order to solve the technical problem in the above-mentioned prior art that PCT detection is easily interfered by biotin in the sample, the purpose of the present invention is to provide a procalcitonin electrochemiluminescence detection kit to achieve high sensitivity, good repeatability, wide linear range, strong anti-interference ability, and effectively shorten the detection time.

[0005] The present invention provides a procalcitonin electrochemiluminescence detection kit, which comprises a procalcitonin capture antibody-coated magnetic microsphere reagent and a procalcitonin detection antibody reagent labeled with an electrically neutral ruthenium compound;

[0006] The general structural formula of the electrically neutral ruthenium compound (NRC) is as follows:

[0007]

[0008] Wherein, n=0~9;

[0009] The procalcitonin capture antibody-coated magnetic microsphere reagent comprises procalcitonin capture antibody-coated magnetic microspheres and a diluent;

[0010] The electrically neutral ruthenium compound labeled procalcitonin detection antibody reagent comprises an electrically neutral ruthenium compound labeled procalcitonin detection antibody and a diluent;

[0011] The diluent includes piperazine-1,4-diethanesulfonic acid or 3-[NN-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid.

[0012] The present invention uses procalcitonin capture antibody to coat magnetic microspheres, uses electrically neutral ruthenium compounds to label procalcitonin detection antibodies, and the two reagent components undergo immune binding reaction with procalcitonin in the object to be detected, and finally a double antibody sandwich complex structure of magnetic microspheres·antibody-antigen-antibody*ruthenium compound (such as Figure 1 As shown). On the one hand, the electrically neutral ruthenium compound used in the present invention is used to label the procalcitonin detection antibody, which can reduce nonspecific interference in immunoassay and generate a strong luminescent signal; on the other hand, the diluent in the two reagent components (including piperazine-1,4-diethanesulfonic acid or 3-[NN-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid) can not only maintain a stable pH value, but also provide the necessary ion strength and ion composition, through which a stable reaction environment can be provided. The above two aspects work synergistically, so that the procalcitonin electrochemiluminescence detection kit of the present invention can significantly improve the sensitivity, repeatability, linear range and anti-interference ability of the detection, and can effectively shorten the detection time. The reaction system of the procalcitonin electrochemiluminescence detection kit of the present invention when used for detection does not contain streptavidin-coated magnetic beads or biotinylated antibodies, which effectively avoids the interference of biotin that may exist in the sample on the test results, and can be widely used.

[0013] Procalcitonin Capture Antibody is a procalcitonin antibody that is used as a capture antibody for coating magnetic microspheres.

[0014] Procalcitonin Detection Antibody is a procalcitonin antibody used as a detection antibody for labeling with electrically neutral ruthenium compounds.

[0015] Preferably, n=2-5.

[0016] Preferably, the kit further comprises a procalcitonin calibrator 1 and a procalcitonin calibrator 2, wherein the procalcitonin antigen concentration range in the procalcitonin calibrator 1 is 0.05 to 0.25 ng / mL, and the procalcitonin antigen concentration range in the procalcitonin calibrator 2 is 40 to 60 ng / mL.

[0017] Further preferably, the kit further comprises a procalcitonin calibrator 1 and a procalcitonin calibrator 2, wherein the procalcitonin antigen concentration range in the procalcitonin calibrator 1 is 0.08 to 0.12 ng / mL, and the procalcitonin antigen concentration range in the procalcitonin calibrator 2 is 45 to 55 ng / mL.

[0018] Preferably, in the procalcitonin capture antibody-coated magnetic microsphere reagent, the particle size of the magnetic microsphere is 1 to 5 μm, and the concentration range of the procalcitonin capture antibody-coated magnetic microsphere is 0.2 to 0.5 mg / mL.

[0019] Further preferably, in the procalcitonin capture antibody-coated magnetic microsphere reagent, the particle size of the magnetic microsphere is 2-3 μm; and the concentration range of the procalcitonin capture antibody-coated magnetic microsphere is 0.2-0.3 mg / mL.

[0020] Preferably, in the diluent, the concentration of the piperazine-1,4-diethanesulfonic acid is 10 to 200 mmol / L, or the concentration of the 3-[NN-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid is 10 to 200 mmol / L.

[0021] More preferably, in the diluent, the concentration of the piperazine-1,4-diethanesulfonic acid is 50 to 100 mmol / L, or the concentration of the 3-[NN-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid is 50 to 100 mmol / L.

[0022] Preferably, the pH value of the diluent is 6-8.

[0023] More preferably, the pH value of the diluent is 7.0.

[0024] Preferably, the diluent further comprises Tween-20 and bovine serum albumin (BSA).

[0025] Preferably, in the diluent, the concentration of Tween-20 is 0.02-0.1% g / mL, and the concentration of bovine serum albumin (BSA) is 0.5-2% g / mL.

[0026] Preferably, the preparation method of the procalcitonin capture antibody-coated magnetic microsphere reagent is:

[0027] S1. Mix the magnetic microspheres with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution;

[0028] S2. Remove the supernatant by magnetic separation, add procalcitonin capture antibody and mix well;

[0029] S3. Add sealing agent and mix well;

[0030] S4. After the supernatant is removed by magnetic separation, a diluent is added to resuspend the magnetic microspheres to obtain a magnetic microsphere reagent coated with procalcitonin capture antibody.

[0031] Preferably, in the electrically neutral ruthenium compound labeled procalcitonin detection antibody reagent, the concentration range of the electrically neutral ruthenium compound labeled procalcitonin detection antibody is 0.5 to 3 μg / mL.

[0032] Further preferably, in the electrically neutral ruthenium compound labeled procalcitonin detection antibody reagent, the concentration range of the electrically neutral ruthenium compound labeled procalcitonin detection antibody is 1 to 2 μg / mL.

[0033] Preferably, in the procalcitonin detection antibody labeled with the electrically neutral ruthenium compound, the molar binding ratio of the electrically neutral ruthenium compound to the procalcitonin detection antibody is (3:1) to (9:1).

[0034] In the procalcitonin detection antibody labeled with an electrically neutral ruthenium compound described in this scheme, the molar binding ratio of the electrically neutral ruthenium compound to the procalcitonin detection antibody is (3:1) to (9:1), which can not only enhance the intensity of the luminescent signal, but also ensure that the procalcitonin detection antibody labeled with an electrically neutral ruthenium compound can fully undergo an immune binding reaction with the procalcitonin in the object to be detected.

[0035] Further preferably, in the procalcitonin detection antibody reagent labeled with an electrically neutral ruthenium compound, the molar binding ratio of the electrically neutral ruthenium compound to the procalcitonin detection antibody is (5:1) to (8:1).

[0036] Preferably, the preparation method of the procalcitonin detection antibody reagent labeled with an electrically neutral ruthenium compound is:

[0037] S1. Mixing 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution, N-hydroxysulfosuccinimide solution, and electrically neutral ruthenium compound solution;

[0038] S2. Add procalcitonin detection antibody and incubate;

[0039] S3. Remove the unbound electrically neutral ruthenium compound, add a diluent, and obtain the procalcitonin detection antibody reagent labeled with the electrically neutral ruthenium compound.

[0040] Preferably, the reaction volume ratio of the magnetic microsphere reagent coated with the procalcitonin capture antibody to the procalcitonin detection antibody reagent labeled with an electrically neutral ruthenium compound is (50-100): (50-100).

[0041] Further preferably, the reaction volume ratio of the magnetic microsphere reagent coated with the procalcitonin capture antibody to the procalcitonin detection antibody reagent labeled with an electrically neutral ruthenium compound is (70-90): (70-90).

[0042] Preferably, the incubation time for detection using the kit is 5 to 15 minutes. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Schematic diagram of the double antibody sandwich complex structure formed in the electrochemiluminescent immunoreaction of the present invention.

[0044] Figure 2 This is a calibration curve diagram of the procalcitonin calibrator of Example 1 on the YnY3030 fully automatic electrochemiluminescence immunoassay analyzer in the 5-minute incubation time detection procedure (the assigned concentration of PCT calibrator 1 is 0.10 ng / mL, and the assigned concentration of PCT calibrator 2 is 54 ng / mL).

[0045] Figure 3 This is a calibration curve diagram of the procalcitonin calibrator of Example 2 on the YnY3030 fully automatic electrochemiluminescence immunoassay analyzer in the 5-minute incubation time detection procedure (the assigned concentration of PCT calibrator 1 is 0.10 ng / mL, and the assigned concentration of PCT calibrator 2 is 54 ng / mL).

[0046] Figure 4 This is a calibration curve diagram of the procalcitonin calibrator of Example 3 on the YnY3030 fully automatic electrochemiluminescence immunoassay analyzer in the 5-minute incubation time detection procedure (the assigned concentration of PCT calibrator 1 is 0.10 ng / mL, and the assigned concentration of PCT calibrator 2 is 54 ng / mL).

[0047] Figure 5 This is a calibration curve diagram of the procalcitonin calibrator of Example 4 on the YnY3030 fully automatic electrochemiluminescence immunoassay analyzer in the 5-minute incubation time detection procedure (the assigned concentration of PCT calibrator 1 is 0.10 ng / mL, and the assigned concentration of PCT calibrator 2 is 54 ng / mL).

[0048] Figure 6 This is a calibration curve diagram of the procalcitonin calibrator of comparative example 1 on the YnY3030 fully automatic electrochemiluminescence immunoassay analyzer in the 5-minute incubation time detection procedure (the assigned concentration of PCT calibrator 1 is 0.10 ng / mL, and the assigned concentration of PCT calibrator 2 is 54 ng / mL).

[0049] Figure 7This is the calibration curve of the procalcitonin calibrator of comparative example 2 on the YnY3030 fully automatic electrochemiluminescence immunoassay analyzer in the 5-minute incubation time detection procedure (the assigned concentration of PCT calibrator 1 is 0.10 ng / mL, and the assigned concentration of PCT calibrator 2 is 54 ng / mL). DETAILED DESCRIPTION

[0050] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.In addition, should be understood that after reading content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope of the appended claims limitation of the application.

[0051] PCT monoclonal antibody (capture antibody) and PCT monoclonal antibody (detection antibody) were purchased from Hangzhou Huakui Jinpei Biotechnology Co., Ltd.

[0052] The preparation methods of the freshly prepared 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) solution and N-hydroxysulfosuccinimide solution used in the following examples and comparative examples are as follows:

[0053] 10 mg / mL and 40 mmol / L 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solutions were prepared using 2-morpholineethanesulfonic acid (MES) buffer (pH=6.0), respectively.

[0054] A 100 mmol / L N-hydroxysulfosuccinimide (Sulfo-NHS) solution was prepared using 2-morpholineethanesulfonic acid (MES) buffer (pH=6.0).

[0055] Example 1

[0056] The preparation method of the electrochemiluminescence detection kit for procalcitonin of this embodiment is as follows:

[0057] The preparation of the PCT monoclonal antibody (capture antibody) coated magnetic microsphere reagent is as follows:

[0058] S1. Mix 1 mL of magnetic microspheres (20 mg / mL) with a particle size of 2.7 μm with 80 μL of freshly prepared 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) solution (10 mg / mL) and rotate at room temperature for 30 minutes;

[0059] S2. Remove the supernatant by magnetic separation, resuspend the magnetic microspheres with 1 mL of 25 mM 2-morpholineethanesulfonic acid buffer (MES buffer, pH 6.0), add 0.4 mg PCT monoclonal antibody (capture antibody), and rotate and mix at room temperature for 4 hours;

[0060] S3. Add an equal volume of 1% (mass / volume, g / mL) bovine serum albumin (BSA) solution and rotate and mix at room temperature for 30 minutes;

[0061] S4. After removing the supernatant by magnetic separation, add 80 mL of diluent containing 50 mmol / L piperazine-1,4-diethanesulfonic acid, 0.05% Tween-20 (w / v, g / mL), and 1% (w / v, g / mL) BSA (pH adjusted to 7.0) to resuspend the magnetic beads;

[0062] S5. The concentration of the magnetic microspheres coated with PCT monoclonal antibody (capture antibody) after dilution is 0.25 mg / mL.

[0063] The preparation of the PCT monoclonal antibody (detection antibody) reagent labeled with an electrically neutral ruthenium compound is as follows:

[0064] S1.5mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution (40mmol / L), 5mL N-hydroxysulfosuccinimide solution (100mmol / L), 5mL electrically neutral ruthenium compound solution (10mmol / L, solvent is purified water), mix at room temperature for 45 minutes to obtain an electrically neutral ruthenium compound mixed solution, the specific structural formula of the electrically neutral ruthenium compound is as follows:

[0065]

[0066] S2. Add 5 μmol of PCT monoclonal antibody (detection antibody) to the above-mentioned electrically neutral ruthenium compound mixed solution and incubate at room temperature for 4 hours;

[0067] S3. removing unbound electrically neutral ruthenium compounds through a desalting column;

[0068] S4. The labeled antibody was quantified by bicinchoninic acid (BCA) protein assay. According to the extinction coefficient of the labeled molecule, the absorbance measurement of the electrically neutral ruthenium compound at 455 nm was related to the labeled amount. It was determined that the actual binding molar ratio of the electrically neutral ruthenium compound to the PCT monoclonal antibody (detection antibody) in the PCT monoclonal antibody (detection antibody) labeled with the electrically neutral ruthenium compound was 6:1.

[0069] S5. Dilute the PCT monoclonal antibody (detection antibody) labeled with an electrically neutral ruthenium compound to 1 μg / mL using a diluent containing 50 mmol / L piperazine-1,4-diethanesulfonic acid, 0.05% Tween-20 (w / v, g / mL), and 1% (w / v, g / mL) BSA (pH adjusted to 7.0).

[0070] The procalcitonin calibrator was prepared as follows:

[0071] Preparation of PCT calibrator 1: weigh 0.1 mL of PCT antigen stock solution (0.5 μg / mL), dilute to 500 mL with antigen diluent, the concentration of PCT calibrator 1 is 0.1 ng / mL, and lyophilize after aliquoting;

[0072] Preparation of PCT calibrator 2: Weigh 50 mL of PCT antigen stock solution (0.5 μg / mL), dilute to 500 mL with antigen diluent, the concentration of PCT calibrator 2 is 50 ng / mL, and lyophilize after aliquoting.

[0073] Example 2

[0074] In the preparation method of the electrochemiluminescence detection kit for procalcitonin of this embodiment, the specific structural formula of the electrically neutral ruthenium compound used is as follows:

[0075] Other than that, the same as in Example 1.

[0076] Example 3

[0077] In the preparation method of the electrochemiluminescence detection kit for procalcitonin of this embodiment, except that piperazine-1,4-diethanesulfonic acid is replaced by 3-[NN-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid, the rest is the same as in Example 1.

[0078] Example 4

[0079] In the preparation method of the electrochemiluminescence detection kit for procalcitonin of this embodiment, except that the preparation method of the PCT monoclonal antibody (detection antibody) reagent labeled with an electrically neutral ruthenium compound is as follows, the rest is the same as in Example 1;

[0080] Preparation method of PCT monoclonal antibody (detection antibody) reagent labeled with electrically neutral ruthenium compound:

[0081] S1.5mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution (40mmol / L), 5mL N-hydroxysulfosuccinimide solution (100mmol / L), 5mL electrically neutral ruthenium compound solution (7mmol / L, solvent is purified water), mix at room temperature for 45 minutes to obtain an electrically neutral ruthenium compound mixed solution, the specific structural formula of the electrically neutral ruthenium compound is as follows:

[0082]

[0083] S2. Add 5 μmol of PCT monoclonal antibody (detection antibody) to the above-mentioned electrically neutral ruthenium compound mixed solution and incubate at room temperature for 4 hours;

[0084] S3. removing unbound electrically neutral ruthenium compounds through a desalting column;

[0085] S4. The labeled antibody was quantified by bicinchoninic acid (BCA) protein assay. According to the extinction coefficient of the labeled molecule, the absorbance measurement of the electrically neutral ruthenium compound at 455 nm was related to the labeled amount. It was determined that the actual binding molar ratio of the electrically neutral ruthenium compound to the PCT monoclonal antibody (detection antibody) in the PCT monoclonal antibody (detection antibody) labeled with the electrically neutral ruthenium compound was 4:1.

[0086] S5. Dilute the PCT monoclonal antibody (detection antibody) labeled with an electrically neutral ruthenium compound to 1 μg / mL using a diluent containing 50 mmol / L piperazine-1,4-diethanesulfonic acid, 0.05% Tween-20 (w / v, g / mL), and 1% (w / v, g / mL) BSA (pH adjusted to 7.0).

[0087] Comparative Example 1

[0088] In the preparation method of the procalcitonin electrochemiluminescence detection kit of this comparative example, the specific structural formula of the electrically neutral ruthenium compound used is as follows:

[0089] Other than that, the same as in Example 1.

[0090] Comparative Example 2

[0091] In the preparation method of the electrochemiluminescence detection kit for procalcitonin of this comparative example, except that piperazine-1,4-diethanesulfonic acid is replaced by N-(2-hydroxyethyl)piperazine-N-3-propanesulfonic acid, the rest is the same as in Example 1.

[0092] Performance Testing

[0093] 5-minute incubation time test procedure - calibration of procalcitonin calibrator

[0094] S1. Pipette 85 μL of PCT monoclonal antibody (detection antibody) reagent labeled with neutral ruthenium compound and 30 μL of reconstituted calibrant (the solvent is purified water) into the test tube, mix for 3 to 5 seconds, pipette 85 μL of PCT monoclonal antibody (capture antibody) coated magnetic microsphere reagent into the test tube, mix for 3 to 5 seconds, and incubate at 37°C for 5 minutes;

[0095] S2. The reaction mixture after incubation is sucked into the measuring pool, and the magnet under the measuring pool fixes the magnetic beads in the reaction mixture on the electrode surface;

[0096] S3. Absorb the cleaning solution to clean other substances not fixed to the electrode surface, start the voltage, and the ruthenium compound undergoes an electrochemical reaction to form light emission;

[0097] S4. The intensity of the light signal detected by the photomultiplier tube is positively correlated with the amount of PCT captured on the surface of the magnetic beads, so PCT can be quantitatively analyzed;

[0098] S5. Establish a calibration curve using the two calibrator concentrations and corresponding luminescent signal values;

[0099] S6. Procalcitonin calibrator was calibrated using the YnY 3030 fully automatic electrochemiluminescence immunoassay analyzer from Shenzhen Ansai Diagnostics Technology Co., Ltd.

[0100] The detection method of the procalcitonin electrochemiluminescence detection kit comprises the following steps:

[0101] S1. Pipette 85 μL of PCT monoclonal antibody (detection antibody) reagent labeled with neutral ruthenium compound and 30 μL of sample into the test tube, mix for 3 to 5 seconds, pipette 85 μL of PCT monoclonal antibody (capture antibody) coated magnetic microsphere reagent into the test tube, mix for 3 to 5 seconds, and incubate at 37°C for 5 minutes;

[0102] S2. The reaction mixture after incubation is sucked into the measuring pool, and the magnet under the measuring pool fixes the magnetic beads in the reaction mixture on the electrode surface;

[0103] S3. Absorb the cleaning solution to clean other substances not fixed to the electrode surface, start the voltage, and the electrically neutral ruthenium compound undergoes an electrochemical reaction to form light emission;

[0104] S4. The intensity of the light signal detected by the photomultiplier tube is positively correlated with the amount of PCT captured on the surface of the magnetic beads, so PCT can be quantitatively analyzed;

[0105] S5. The PCT content in the sample is determined by the calibration curve.

[0106] Detection limit evaluation

[0107] The calibration curve was established by 5-minute incubation time detection procedure-procalcitonin calibrator calibration (such as Figures 2 to 7 As shown in the figure, take 5 low-value samples with a concentration close to the detection limit (0.01ng / mL), test them with the kit, and test each sample 5 times. The following requirements should be met: the number of test results with a value lower than the given blank limit (0.005ng / mL) should be less than or equal to 3.

[0108] Table 1 Detection limit evaluation results (unit: ng / mL)

[0109]

[0110]

[0111] As can be seen from Table 1, the detection limit assessment of the kits prepared using Examples 1, 2, 3 and 4 using the 5-minute incubation detection program of the YnY 3030 fully automatic electrochemiluminescence immunoassay analyzer of Shenzhen Ansai Diagnostics Technology Co., Ltd. all meets the requirements; the detection limit assessment of the kits prepared using Comparative Examples 1 and 2 does not meet the requirements.

[0112] Repeatability assessment

[0113] The calibration curve was established by 5-minute incubation time detection procedure-procalcitonin calibrator calibration (such as Figures 2 to 7 As shown in the figure, the samples with concentrations between (0.5±0.1) ng / mL and (10±2.0) ng / mL were measured 10 times, and the mean (M) and standard deviation (SD) of the 10 measurement results were calculated to obtain the coefficient of variation (CV), which should satisfy CV≤8%.

[0114] CV=SD / M×100%

[0115] Table 2 Repeatability evaluation results (unit: ng / mL)

[0116]

[0117]

[0118] As can be seen from Table 2, the repeatability evaluation of the kits prepared using Example 1, Example 2, Example 3 and Example 4 in the 5-minute incubation detection program of the YnY 3030 fully automatic electrochemiluminescence immunoassay analyzer of Shenzhen Ansai Diagnostics Technology Co., Ltd. all met the requirements; the repeatability evaluation of the kits prepared in Comparative Examples 1 and 2 did not meet the requirements.

[0119] Linear range assessment

[0120] The calibration curve was established by 5-minute incubation time detection procedure-procalcitonin calibrator calibration (such as Figures 2 to 7 As shown in the figure, the high-value linear sample close to the upper limit of the linear interval (100ng / mL) is diluted proportionally to 5 concentrations, among which the low-value linear sample should be close to the lower limit of the linear interval (0.01ng / mL). The sample of each concentration is measured 3 times, and the average value is calculated. The average value of the measured concentration and the theoretical concentration are linearly fitted using the least squares method, and the linear correlation coefficient r is calculated, which should meet r≥0.99.

[0121] Table 3 Linear range evaluation results (unit: ng / mL)

[0122]

[0123]

[0124] As can be seen from Table 3, the 5-minute incubation detection program of the YnY 3030 fully automatic electrochemiluminescence immunoassay analyzer of Shenzhen Ansai Diagnostics Technology Co., Ltd., and the linear range evaluation of the kits prepared using Example 1, Example 2, Example 3, Example 4, Comparative Example 1 and Comparative Example 2 all meet the requirements.

[0125] Biotin Interference Assessment

[0126] The calibration curve was established by 5-minute incubation time detection procedure-procalcitonin calibrator calibration (such as Figures 2 to 7 As shown), add biotin to a sample with a PCT concentration of (0.5±0.1) ng / mL to make its concentration reach 1000000 ng / mL. The addition ratio does not exceed (1:9). Repeat the test for this sample 3 times. The interference deviation should be within the range of ±10%.

[0127] Table 4 Biotin interference evaluation results (unit: ng / mL)

[0128]

[0129] As can be seen from Table 4, the 5-minute incubation detection program of the YnY 3030 fully automatic electrochemiluminescence immunoassay analyzer of Shenzhen Ansai Diagnostics Technology Co., Ltd., and the biotin interference assessment of the kits prepared using Example 1, Example 2, Example 3, Example 4, Comparative Example 1 and Comparative Example 2 all meet the requirements.

[0130] Comparison with imported test kits:

[0131] Performance comparison with imported test kits

[0132] Table 5 Performance comparison of Examples 1 to 4 and imported kits

[0133] Comparison indicators Embodiments 1 to 4 Imported test kit Detection limit 0.01ng / mL 0.02ng / mL Linear range 0.01~100 ng / mL 0.02~100 ng / mL Incubation time 5 minutes 18 minutes Maximum concentration of biotin interference 1000000ng / mL 1200ng / mL

[0134] As shown in Table 5, the kits prepared using Examples 1 to 4 are superior to the imported reagents in terms of detection limit, linear range, incubation time, and biotin interference, among which the detection limit, incubation time, and biotin interference are significantly improved.

[0135] The above contents are examples of specific embodiments of the present invention. Reagents, equipment, operating methods, etc. that are not described in detail should be understood as being implemented by using conventional reagents, equipment, operating methods, etc. available in the art.

[0136] The above is a description of the embodiments of the present invention. Through the above description of the disclosed embodiments, professionals and technicians in the field can implement or use the present invention. Various modifications to these embodiments will be obvious to professionals and technicians in the field. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but will conform to the widest range consistent with the principles and novel features disclosed herein.

Claims

1. A procalcitonin electrochemiluminescence detection kit, characterized in that: The kit comprises a magnetic microsphere reagent coated with a procalcitonin capture antibody and a procalcitonin detection antibody reagent labeled with an electrically neutral ruthenium compound; The general structural formula of the electrically neutral ruthenium compound is as follows: Wherein, n=0~9; The procalcitonin capture antibody-coated magnetic microsphere reagent comprises procalcitonin capture antibody-coated magnetic microspheres and a diluent; The electrically neutral ruthenium compound labeled procalcitonin detection antibody reagent comprises an electrically neutral ruthenium compound labeled procalcitonin detection antibody and a diluent; The diluent includes piperazine-1,4-diethanesulfonic acid or 3-[NN-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid.

2. The electrochemiluminescence detection kit for procalcitonin according to claim 1, characterized in that: The kit also includes a procalcitonin calibrator 1 and a procalcitonin calibrator 2, wherein the procalcitonin antigen concentration range of the procalcitonin calibrator 1 is 0.05-0.25 ng / mL, and the procalcitonin antigen concentration range of the procalcitonin calibrator 2 is 40-60 ng / mL.

3. The electrochemiluminescence detection kit for procalcitonin according to claim 1, characterized in that: In the procalcitonin capture antibody-coated magnetic microsphere reagent, the particle size of the magnetic microsphere is 1-5 μm, and the concentration range of the procalcitonin capture antibody-coated magnetic microsphere is 0.2-0.5 mg / mL.

4. The electrochemiluminescence detection kit for procalcitonin according to claim 1, characterized in that: In the diluent, the concentration of the piperazine-1,4-diethanesulfonic acid is 10 to 200 mmol / L, or the concentration of the 3-[NN-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid is 10 to 200 mmol / L.

5. The electrochemiluminescence detection kit for procalcitonin according to claim 1, characterized in that: The preparation method of the procalcitonin capture antibody-coated magnetic microsphere reagent is as follows: S1. Mix the magnetic microspheres with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution; S2. Remove the supernatant by magnetic separation, add procalcitonin capture antibody and mix well; S3. Add sealing agent and mix well; S4. After removing the supernatant by magnetic separation, add diluent and resuspend the magnetic microspheres to obtain the magnetic microsphere reagent coated with procalcitonin capture antibody.

6. The electrochemiluminescence detection kit for procalcitonin according to claim 1, characterized in that: In the electrically neutral ruthenium compound labeled procalcitonin detection antibody reagent, the concentration range of the electrically neutral ruthenium compound labeled procalcitonin detection antibody is 0.5 to 3 μg / mL.

7. The electrochemiluminescence detection kit for procalcitonin according to claim 1, characterized in that: In the procalcitonin detection antibody labeled with the electrically neutral ruthenium compound, the molar binding ratio of the electrically neutral ruthenium compound to the procalcitonin detection antibody is (3:1) to (9:1).

8. The electrochemiluminescence detection kit for procalcitonin according to claim 1, characterized in that: The preparation method of the electrically neutral ruthenium compound labeled procalcitonin detection antibody reagent is as follows: S1. Mixing 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution, N-hydroxysulfosuccinimide solution, and electrically neutral ruthenium compound solution; S2. Add procalcitonin detection antibody and incubate; S3. Remove the unbound electrically neutral ruthenium compound, add diluent, and obtain the procalcitonin detection antibody reagent labeled with the electrically neutral ruthenium compound.

9. The electrochemiluminescence detection kit for procalcitonin according to claim 1, characterized in that: The reaction volume ratio of the magnetic microsphere reagent coated with the procalcitonin capture antibody to the procalcitonin detection antibody reagent labeled with an electrically neutral ruthenium compound is (50-100): (50-100).

10. The electrochemiluminescence detection kit for procalcitonin according to any one of claims 1 to 9, characterized in that: The incubation time for detection using the kit is 5 to 15 minutes.

Images