Kit for detecting thymidine kinase 1 as well as preparation method and application of kit

Abstract

The invention relates to a kit for detecting thymidine kinase 1 as well as a preparation method and application of the kit, and relates to the technical field of immunodetection. The kit comprises a reagent M and a reagent N, the reagent M comprises a magnetic bead working solution and a magnetic microsphere coated with a TK1 mouse monoclonal antibody; and the reagent N comprises a labeled antibody diluent and an acridinium ester labeled TK1 mouse monoclonal antibody. The magnetic bead working solution and the labeled antibody diluent are adopted, the stability of the magnetic microspheres coated with the TK1 mouse monoclonal antibody and the stability of the acridinium ester labeled TK1 mouse monoclonal antibody are improved, the result can be obtained within 8 minutes, the repeatability is within 2.8%, and the sensitivity reaches 0.89 pmol / mL; the kit provided by the invention is more stable during transportation and storage, can be kept for a long time at 2-8 DEG C, and can be stable for 2 years.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassays, and in particular to a kit for detecting thymidine kinase 1, a preparation method thereof and an application thereof. Background Art

[0002] Current research indicates that the abnormal or uncontrolled proliferation of malignant tumor cells is caused by mutations in genes associated with proliferation in normal cells, leading to overexpression of related oncoproteins, loss of inhibitory proteins, or expression of mutant protein products, accompanied by uncontrolled regulation of a series of enzymes and proteins associated with cell growth. The proliferation of malignant tumor cells in vivo does not follow the diploid division pattern of normal cells. Based on this characteristic of tumor cells with endless malignant division, researchers have established the theoretical basis for tumor cell proliferation markers. By culturing cells in vitro, researchers observed that thymidine (Thd) is involved in DNA synthesis.

[0003] Thymidine kinase (TK) is an enzyme that catalyzes the phosphorylation of thymidine to form thymidine monophosphate. It is known as an enzyme in the pyrimidine salvage pathway and plays a key role in assessing the proliferation of proliferating cells. Human cells contain two TK isoenzymes: cytoplasmic thymidine kinase (TK1), located in the cytoplasm, and mitochondrial thymidine kinase (TK2), located in the mitochondria. TK1 is a cell cycle S-phase-dependent enzyme, and its level in vivo is directly positively correlated with cell proliferation and DNA synthesis rates. During the cell cycle, TK1 levels begin to increase in the late G1 phase, reach their highest level in the S phase, and begin to decline in the G2 phase. Due to its special correlation with the S phase of the cell cycle, TK1 is also known as the "S-phase key enzyme" and is associated with cell proliferation. For abnormal proliferative lesions, since the diseased cells lack apoptosis regulation, the dramatic increase in DNA synthesis will lead to an abnormal increase in TK1 levels. Abnormally proliferative cells contain high levels of TK1 in the S phase or G2 phase. Cell necrosis causes the release of cell contents into the blood, resulting in high TK1 levels in the serum. Therefore, TK1 detection can be used for physical examinations and clinical screening of malignant proliferative lesions and follow-up monitoring after treatment.

[0004] Currently, methods for detecting Tk1 levels in serum include enzyme immunoassay (ELISA) with chemiluminescence and double-antibody sandwich assays. However, these methods all have certain drawbacks. Therefore, the present invention provides a kit for detecting thymidine kinase 1, its preparation method, and its use. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a kit for detecting thymidine kinase 1, and its preparation method and application. The technical solution of the present invention to solve the above technical problem is as follows:

[0006] In a first aspect, a kit for detecting thymidine kinase 1 comprises reagent M and reagent N; the reagent M comprises magnetic bead working solution and magnetic microspheres coated with TK1 mouse monoclonal antibody; the reagent N comprises labeled antibody diluent and acridinium ester-labeled TK1 mouse monoclonal antibody.

[0007] Here’s how it works:

[0008] A sample, magnetic microspheres coated with TK1 mouse monoclonal antibody, and acridinium ester-labeled TK1 mouse monoclonal antibody are added to a reaction tube. After incubation, the TK1 protein in the sample binds to the TK1 mouse monoclonal antibody coated on the magnetic beads and simultaneously binds to the acridinium ester-labeled TK1 mouse monoclonal antibody, forming an antibody-antigen-antibody sandwich complex. After the reaction is complete, the magnetic beads are attracted by a magnetic field and unbound substances are washed away. A chemiluminescent substrate (pre-excitation solution / chemiluminescent substrate solution A, excitation solution / chemiluminescent substrate solution B) is added to the reaction tube to generate chemiluminescence. The number of photons produced by the reaction is measured by a photomultiplier tube, and the number of photons produced is proportional to the concentration of the analyte in the sample. The amount of analyte in the sample is determined by a calibration curve.

[0009] The present invention has the following beneficial effects: the present invention adopts a highly specific anti-TK1 antibody, which can simultaneously detect the TK1 value in human serum, plasma or urine samples with a single sampling; the present invention adopts a magnetic bead working solution and a labeled antibody diluent to improve the stability of the magnetic microspheres coated with the TK1 mouse monoclonal antibody and the acridinium ester-labeled TK1 mouse monoclonal antibody, and the results are available in 8 minutes, with a repeatability within 2.8% and a sensitivity of 0.89 pmol / mL; the kit of the present invention is more stable during transportation and storage, and can be kept stable for 2 years at 2-8°C for a long time. It can be kept stable for 13 days after an aging test at 37°C. The calibrators and quality control products matched with the reagents do not need to be lyophilized and are ready for use in liquid form. They can be kept stable for 2 years at 2-8°C for a long time.

[0010] On the basis of the above technical solution, the present invention can also be improved as follows.

[0011] Furthermore, the mass ratio of TK1 mouse monoclonal antibody to magnetic microspheres in the magnetic microspheres coated with TK1 mouse monoclonal antibody is 3-5:50-100.

[0012] Furthermore, the mass ratio of acridinium ester to TK1 mouse monoclonal antibody in the acridinium ester-labeled TK1 mouse monoclonal antibody is 5-9:30-50.

[0013] Furthermore, each 1L of the magnetic bead working solution includes the following components: HEPES 11.91g, sodium chloride 9.00g, BSA 25.00g, trehalose 100.00g, glucose 80.00g, Tween-20 1mL, Proclin300 1mL, polyethylene glycol-20000 80.00g, alkyl polyglycoside glycerol ether 7.2g, pH=7.4.

[0014] Furthermore, each 1 L of the labeled antibody dilution solution includes the following components: 2.42 g of tris(hydroxymethyl)aminomethane, 1.41 mL of hydrochloric acid, 9.00 g of sodium chloride, 10.00 g of BSA, 0.5 mL of Proclin 300, 4.5 g of dextran, 7.4 g of isodecyl citrate monoester, and 0.01 g of amaranth.

[0015] Furthermore, the kit also includes a calibrator, a quality control product and a chemiluminescent substrate; the chemiluminescent substrate is a pre-excitation solution / chemiluminescent substrate A solution and an excitation solution / chemiluminescent substrate B solution.

[0016] In a second aspect, a method for preparing a kit for detecting thymidine kinase 1 comprises the following steps:

[0017] Magnetic bead coating: activating magnetic microspheres in a magnetic bead coating buffer using an activator to obtain activated magnetic beads; adding TK1 mouse monoclonal antibodies to the activated magnetic beads for coupling reaction to obtain magnetic microspheres coated with the TK1 mouse monoclonal antibody; and storing the magnetic microspheres coated with the TK1 mouse monoclonal antibody in a magnetic bead working solution to obtain reagent M;

[0018] Acridinium ester labeling: TK1 mouse monoclonal antibody is labeled with acridine in a luminophore labeling buffer to obtain an antibody labeling mixture; the antibody labeling mixture is ultrafiltered to obtain acridinium ester-labeled TK1 mouse monoclonal antibody, and the acridinium ester-labeled TK1 mouse monoclonal antibody is stored in a labeled antibody diluent to obtain reagent N.

[0019] Furthermore, each 1 L of the magnetic bead coating buffer comprises the following components: MES 9.762 g, Triton-100 0.1 mL, alkyl polyglycoside sulfosuccinate disodium salt 5.00 g;

[0020] Each 1L of the luminophore labeling buffer comprises the following components: 13.435g of disodium hydrogen phosphate dodecahydrate, 0.8265g of sodium dihydrogen phosphate dihydrate, 2.3g of α-sulfo fatty acid methyl ester (mono) sodium salt, and 0.6g of fatty alcohol sulfate (ester);

[0021] The activator is EDC; the mass ratio of the activator to the magnetic beads is 10:0.5-2;

[0022] The activation parameters are: room temperature, 20 min to 40 min;

[0023] The coupling reaction parameters are: room temperature, 3 h to 8 h.

[0024] The dosage ratio of the magnetic microspheres coated with TK1 mouse monoclonal antibody to the magnetic bead working solution only needs to meet the requirements.

[0025] Furthermore, the parameters of the labeling reaction are: 37°C, 20 min to 40 min;

[0026] The ultrafiltration parameters are: 14000xg, 5-15 min.

[0027] The dosage ratio of the acridinium ester-labeled TK1 mouse monoclonal antibody to the labeled antibody diluent only needs to meet the requirements.

[0028] In a third aspect, a kit for detecting thymidine kinase 1 is used to detect the tumor marker thymidine kinase 1. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the TK1 calibration curve diagram of the present invention;

[0030] Figure 2 This is the linear regression correlation diagram of TK1 of the present invention. DETAILED DESCRIPTION

[0031] The principles and features of the present invention are described below. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in this field or the product instructions are used. Where the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be purchased through regular channels.

[0032] Example

[0033] 1. Materials and reagents.

[0034] (1) Antigen and Antibody:

[0035] TK1 monoclonal magnetic microsphere-coated antibody and anti-TK1 labeled antibody were purchased from Beijing Baixinyi Biotechnology Co., Ltd. at a concentration of 10 mg / mL; TK1 antigen was purchased from Beijing Aibosheng Biotechnology Co., Ltd. at a concentration of 5 mg / mL.

[0036] (2) Reagents:

[0037] Acridinium ester (AE) was purchased from Helison (Xiamen) Biotechnology Co., Ltd.; carboxyl magnetic beads were purchased from Nanjing Ruibeixi Biotechnology Co., Ltd. at a concentration of 10 mg / mL; pre-excitation solution / chemiluminescent substrate solution A and excitation solution / chemiluminescent substrate solution B were purchased from Guangzhou Hongrunkang Technology Development Co., Ltd.; disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate, HEPES (4-hydroxyethylpiperazineethanesulfonic acid), glucose, sodium chloride, sucrose, 3-morpholinopropanesulfonic acid (MOPS), N-tris(hydroxymethyl)methylglycine (Tricine), sodium caseinate, hydrazine yellow, fruit green, lactose, Tween-20, Triton-100, glycerol, tris(hydroxymethyl)aminomethane hydrochloride (Tris), 2-(N-morpholino)ethanesulfonic acid (MES) Conventional chemical reagents such as ethylene glycol, hydrochloric acid, sodium periodate, and sodium borohydride were purchased from Sinopharm Chemical Reagent Co., Ltd.; 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), bovine serum albumin (BSA), Proclin-300, and polyethylene glycol 6000 were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; amaranth (standard solution) was purchased from Tianjin Guangfu Fine Chemical Research Institute; sorbitan polyoxyethylene ether tetraoleate, methyl glucoside sesquistearate (CAS: 68936-95-8), diethylene glycol monolaurate (CAS: 141-20-8), and polyoxypropylene stearate (CAS: 25190-52-7) were purchased from Shanghai Kao Co., Ltd.

[0038] Polypropylene fumarate was prepared from the compound in the article “Wu Yongchao, Zheng Qixin, Guo Xiaodong, et al. Synthesis, cross-linking, biomechanics and in vitro degradation of polypropylene fumarate [J]. Orthopedic Biomaterials and Clinical Research, 2003.”; isodecyl citrate monoester and lauryl citrate monoester were prepared from the compound in the article “Huang Di et al. Study on the solution properties of a new surfactant citric acid monoester and its application in gel oil. Modern Food Science and Technology 34.5”.

[0039] (2018):10.》 The compound in the article; the surfactant Coltide HSi adopts the compound in the article "Li Linping. Application of a new surfactant in laundry detergent. China Detergent Industry 8(2014):3."

[0040] (3) Instrument: SMART 500S fully automatic chemiluminescence immunoassay analyzer produced by Chongqing Cosmay Biotechnology Co., Ltd. (Yu Medical Device Registration No. 20192220077).

[0041] 2. Experimental method.

[0042] 2.1 Reagent preparation:

[0043] (1) Magnetic bead coating buffer:

[0044] Add 9.762 g of MES, 0.1 mL of Triton-100, and 5.00 g of disodium alkyl polyglycoside sulfosuccinate, and dilute to 1 L with purified water.

[0045] (2) Magnetic bead working solution:

[0046] HEPES (4-hydroxyethylpiperazineethanesulfonic acid) 11.91 g, sodium chloride 9.00 g, BSA 25.00 g, trehalose 100.00 g, glucose 80.00 g, Tween-20 1 mL, Proclin 300 1 mL, polyethylene glycol 20000 80.00 g, alkyl polyglycoside glycerol 7.2 g, adjust the pH to 7.4, and dilute to 1 L with purified water.

[0047] (3) Luminescent labeling buffer:

[0048] 13.435 g of disodium hydrogen phosphate dodecahydrate, 0.8265 g of sodium dihydrogen phosphate dihydrate, 2.3 g of α-sulfo fatty acid methyl ester (mono) sodium salt, 0.6 g of fatty alcohol sulfate (ester), and dilute to 1 L with purified water.

[0049] (4) Luminescent blocking buffer:

[0050] 12.1 g of tris(hydroxymethyl)aminomethane, 9.0 g of sodium chloride, 10.0 g of lysine, 100 g of BSA, 1.5 g of lauryl citrate, 5.0 g of sorbitan polyoxyethylene ether tetraoleate, and dilute to 1 L with purified water.

[0051] (5) Acridinium ester labeled antibody dilution solution:

[0052] 2.42 g of tris(hydroxymethyl)aminomethane, 1.41 mL of hydrochloric acid, 9.00 g of sodium chloride, 10.00 g of BSA, 0.5 mL of Proclin 300, 4.5 g of dextran, 7.4 g of isodecyl citrate, and 0.01 g of amaranth were diluted to 1 L with purified water.

[0053] (6) Calibrator diluent:

[0054] 8.19 g of 3-morpholinepropanesulfonic acid (MOPS), 9.00 g of sodium chloride, 26.00 g of sorbitol, 5.00 g of sodium caseinate, 5.0 g of glycine, 40 mL of β-mercaptoethanol, 1.45 g of magnesium chloride, 0.58 g of zinc chloride, 200 mL of glycerol, 5.00 g of hydrazine yellow, 5.00 g of fruit green, 1 mL of Proclin 300, and dilute to 1 L with purified water.

[0055] (7) Quality control diluent:

[0056] 8.19 g of 3-morpholinepropanesulfonic acid (MOPS), 9.00 g of sodium chloride, 26.00 g of sorbitol, 5.00 g of sodium caseinate, 5.0 g of glycine, 40 mL of β-mercaptoethanol, 1.45 g of magnesium chloride, 0.58 g of zinc chloride, 200 mL of glycerol, 5.00 g of hydrazine yellow, 5.00 g of fruit green, 1 mL of Proclin 300, and dilute to 1 L with purified water.

[0057] (8) Working cleaning fluid:

[0058] 9.76 g of N-tris(hydroxymethyl)methylglycine (Tricine), 0.70 mL of hydrochloric acid, 9.00 g of sodium chloride, 5.0 g of monolauryl phosphate MAP, 2.5 mL of Triton 100, 40 g of surfactant Coltide HSi, 1 mL of Proclin 300, and dilute to 1 L with purified water.

[0059] 2.2 Magnetic bead coating:

[0060] The method for preparing magnetic microspheres coated with TK1 mouse monoclonal antibody comprises the following steps:

[0061] (1) Magnetic bead washing: Place 10 mL of magnetic beads on a magnetic rack. After all the magnetic beads are adsorbed, discard the supernatant. Add 20 mL of magnetic bead coating buffer and mix at 30 rpm / min at room temperature for 10 min. Place the magnetic beads on a magnetic rack. After all the magnetic beads are adsorbed, discard the supernatant. Repeat this washing process twice and discard the supernatant.

[0062] (2) Activation of magnetic beads: Weigh 10.00 mg of EDC and dissolve it in 1 mL of magnetic bead coating buffer. Once fully dissolved, add it to the magnetic beads. Simultaneously add 49.00 mL of magnetic bead coating buffer and mix at 30 rpm / min at room temperature for 30 min. Then, repeat step 1.2.2.1 to wash the magnetic beads and discard the supernatant.

[0063] (3) Magnetic Bead Coating: Add 5 mg of TK1 magnetic microsphere coating antibody to the magnetic beads, then add 50 mL of magnetic bead coating buffer and mix at 30 rpm / min at room temperature for 5 h. Then, repeat step 1.2.2.1 to wash the magnetic beads and discard the supernatant.

[0064] (4) Magnetic bead storage: Add 500 mL of magnetic bead working solution to the magnetic beads at 30 rpm / min, mix at room temperature for 30 min, and store at 2-8°C.

[0065] 2.3 Preparation of luminescent marker (TK1 luminescent marker working solution as an example):

[0066] (1) Antibody labeling:

[0067] ① Take 1uL of acridinium ester mother solution (5mM) and add 9uL of DMF (N,N-dimethylformamide) to prepare the acridinium ester working solution (acridinium ester mother solution: anhydrous dimethylformamide = 1:9).

[0068] ② Add 50 μg of anti-TK1 labeled antibody to a centrifuge tube, add luminescent labeling buffer to 300 μL (the volume here increases with the amount of antibody), add 30 μL of acridine working solution, and shake in a 37°C incubator for 30 minutes;

[0069] ③ Add 100uL of luminescent blocking solution and shake in a 37°C incubator for 30 minutes;

[0070] (2) Ultrafiltration of antibody after labeling:

[0071] ① Rinse: Pipette 500 μL of luminescent labeling buffer into the sample pool, close the lid, and centrifuge at 10,000 x g for 10 minutes. Aspirate the liquid in the filtrate receiver. Repeat the rinse cycle.

[0072] ②Ultrafiltration: Add labeled antibodies to the sample pool, cover the lid, and centrifuge at 14,000 x g for 10 minutes to concentrate the labeled antibodies in the sample pool;

[0073] ③ Washing: Add 200 μL of acridinium ester labeled antibody diluent to the sample pool, centrifuge at 14000xg for 20 minutes, and concentrate the labeled antibody in the sample pool after washing;

[0074] ④ Collection: Add 100 μL of acridinium ester labeled antibody diluent to the sample pool, pipette and reconstitute (pipe and reconstitute more than 30 times to avoid bubbles), transfer to a 1.5 ml centrifuge tube, add acridinium ester labeled antibody diluent to 1 ml, and store at -20°C. (Luminescent marker storage solution, concentration is 0.05 mg / ml)

[0075] (3) Preparation of luminescent marker working solution: Take 5 mL of luminescent marker stock solution stored at -20°C in the dark, add 495 mL of acridinium ester labeled antibody diluent, mix at 30 rpm / min, 2-8°C in the dark for 30 min, and then store at 2-8°C in the dark.

[0076] 2.4 Preparation of calibrators:

[0077] Add 0.5 μL of TK1 antigen to 999 μL of Calibrator Diluent and mix thoroughly at room temperature to prepare a 2.5 μg / mL stock solution. Then, serially dilute the stock solution using Calibrator Diluent to six concentration points: 1500 ng / mL, 750 ng / mL, 250 ng / mL, 50 ng / mL, 25 ng / mL, and 0 ng / mL. Store at 2-8°C.

[0078] 2.5 Preparation of quality control products:

[0079] Add 0.5 μL of TK1 antigen to 999 μL of Calibrator Diluent and mix at room temperature to prepare a 2.5 μg / mL stock solution. Then, dilute the stock solution to 200 ng / mL for Control Level 1 and 600 ng / mL for Control Level 2 using Control Diluent. Store at 2-8°C.

[0080] 2.6 Detection method:

[0081] (1) Testing procedures:

[0082] When loading the reagent bottle onto the machine for the first time, it should be gently turned over 30 times before opening, and the reagent bottle should be observed to ensure that the magnetic beads are completely suspended. If the magnetic beads are still attached to the bottom of the reagent bottle, continue to turn the reagent bottle until they are completely suspended. If they still cannot be suspended, the bottle of reagent cannot be used.

[0083] The system requires a sample volume of 10 μL per measurement. Before applying for a test, you should prepare all the materials required for the test and carefully read the chemiluminescence immunoassay user manual to obtain information on system operating procedures, sample management, usage precautions, maintenance, and other information. The main steps for applying for a test are as follows:

[0084] ① Enter the sample application interface and enter the sample number, sample rack number, location number, sample type, remarks and other information according to the test requirements;

[0085] ② Select the test item, select the sample type in the item options, enter the number of repetitions and other information;

[0086] ③ After preparing the test sample and placing it correctly, click the start button to start the test. The chemiluminescence immunoassay analyzer will perform the following operations in sequence:

[0087] a. The sample processing system transfers the sample rack to the sample suction position;

[0088] b. The cuvette loading and scheduling system loads the cuvette from the loading area to the sample loading position;

[0089] c. The sample injection system completes the sample injection and cleaning of the sample needle;

[0090] The reagent processing system provides the reagents required for the test, mixes each reagent, and sends it to the reagent aspiration position for aspiration; the sample injection system completes the reagent dispensing and cleaning of the reagent needle;

[0091] d. Mix the reaction mixture with the counter-liquid mixing system and incubate in the reaction plate;

[0092] e. After the incubation is completed, the magnetic separation system uses a cleaning solution to clean and separate the reaction mixture;

[0093] f. The substrate system injects the preheated substrate solution into the reaction cup after magnetic separation, mixes, incubates, and waits for light detection;

[0094] g. The optical reaction system sends each reaction cup to the signal collection position to collect signals for calculating the luminescence value;

[0095] h. Calculate the amount of analyte in the sample.

[0096] (2) Calibration:

[0097] Use the matching reagent kit and calibrator to perform calibration tests; before starting the calibration of the reagent kit, the master curve in the 2D barcode of the reagent kit should be scanned and imported into the system.

[0098] The chemiluminescence immunoassay analyzer uses the results of calibrator tests to adjust the master curve and generate the calibration curve for the current system. The instrument operating software includes a calibration check function that automatically verifies the validity of the calibration curve. If calibration fails, consider the instrument settings, status, location of the calibrators (place low-value calibrators first, then high-value calibrators), and expiration dates.

[0099] A valid calibration curve is required before all tests. Recalibration is required in the following situations:

[0100] ① Use a new batch of test kit;

[0101] ② The same batch of test kits has been used on the analyzer for more than 30 days;

[0102] ③After instrument maintenance;

[0103] ④The quality control value exceeds the specified range;

[0104] (3) Quality control

[0105] To ensure the reliability of test results, it is recommended that both high and low level quality control products be tested once every 24 hours. In addition, quality control testing is also recommended after each calibration test, reagent batch change, maintenance and troubleshooting.

[0106] The test values of quality control samples should be within the specified range. If they are outside the specified range, the user should check the detection system, such as the location, expiration date, storage method, calibration process, instrument performance and status, and recalibrate if necessary. If the results are still outside the range after retesting, please contact customer service.

[0107] (4) Calculation:

[0108] Using stored calibration data, the system software automatically determines the sample test results using a weighted four-parameter logarithmic curve (4PLC, Y-weighted) mathematical method. Results are given in ng / mL.

[0109] 3. Experimental results.

[0110] 3.1 Construction of calibration curve:

[0111] The construction of the calibration curve is shown in Table 1 and Figure 1 .

[0112] Table 1 Calibration results of reagent TK1 of the present invention

[0113]

[0114] 3.2 Minimum detection limit:

[0115] Using PBS buffer as a blank sample, the detection and comparison reagents prepared in this invention were tested 20 times simultaneously. The test and comparison values from the test strips prepared in this invention, plus twice their standard deviation, were substituted into the fitted curve for regression. The results showed that the minimum detection limit of the TK1 test kit prepared in this invention was 0.89 pmol / mL, demonstrating the high sensitivity of the test strip preparation method prepared in this invention. The results are shown in Table 2.

[0116] Table 2 Results of minimum detection limits of reagents

[0117]

[0118] 3.3 Repeatability:

[0119] The test kit of the present invention was tested repeatedly for 10 times at two levels of quality control, and the CV was calculated. The results showed that the CV of the quality control level 1 of the reagent TK1 of the present invention was 2.847%, and the CV of the quality control level 2 was 2.49%. It can be seen that the preparation method of the test strip prepared by the present invention has good repeatability. The results are shown in Tables 3 and Figure 2 .

[0120] Table 3 TK1 repeatability results

[0121]

[0122] 3.4 Linearity:

[0123] A high-value sample containing 2 pmol / mL of TK1 was diluted with PBS buffer, and 5 samples (xi) with different gradients were diluted 2-fold to test the test strips prepared by the present invention. The sample of each dilution concentration was tested 3 times, and the mean value (yi) of the measurement results was calculated. The linear regression equation and linear regression coefficient were calculated with the dilution concentration (xi) as the independent variable and the mean value (yi) of the measurement result as the dependent variable. The results showed that the linear correlation coefficient (r) of the reagent of the present invention for TK1 was not less than 0.9990 in the range of [1.5 to 20] pmol / mL. It can be seen that the preparation method of the test strips prepared by the present invention has good linearity. The results are shown in Table 4.

[0124] Table 4 Linearity test results of the inventive reagent TK1

[0125]

[0126]

[0127] 3.5 Accuracy:

[0128] A high-concentration reference substance (Solution A) was added to a low-concentration reference substance (Solution B), with the volume ratio between the high-concentration reference substance (Solution A) and the low-concentration reference substance (Solution B) being no greater than 1:9. Each sample was measured three times and the average value was obtained. The results calculated according to formula (1) indicate that the TK1 assay prepared in the present invention has good accuracy. The results are shown in Table 5.

[0129]

[0130] Where: R is the recovery rate; V is the volume of solution A added; V0 is the volume of serum sample B; c is the detection concentration of serum sample after adding solution A; c0 is the detection concentration of serum sample B; c s is the concentration of solution A.

[0131] Table 5 Accuracy test results of the inventive reagent TK1

[0132]

[0133]

[0134] 3.6 Stability:

[0135] 3.6.1 Long-term stability at 2-8°C:

[0136] (1) Three batches of the high-sensitivity chemiluminescent immunoassay kit for the combined tumor marker TK1 of the present invention were stored in a finished product warehouse at 2-8°C in a dry environment. The temperature of the finished product warehouse was monitored using a thermometer and required to be between 2-8°C to ensure compliance with the product storage conditions during the stability study.

[0137] (2) Three batches of the reagent of the present invention stored in a finished product warehouse at 2-8°C were randomly sampled for testing at month 0, month 6, month 12, month 18, month 24, and month 25. The test items were: appearance, detection limit, accuracy, linearity, repeatability, inter-batch variation, accuracy of calibrator value assignment, uniformity of calibrator, validity of target value of quality control product, and uniformity of quality control product.

[0138] (3) Judgment of test content and results

[0139] ①Appearance

[0140] a. All components of the test kit should be complete and intact, with no leakage of liquid; the Chinese packaging label should be clear and not worn;

[0141] b. The magnetic bead reagent component is a liquid containing brown solid particles. The magnetic beads are not caking, and the liquid is free of flocs and foreign matter.

[0142] c. Other reagent components are clear and uniform liquids without precipitation or flocs.

[0143] ② Accuracy: The recovery rate should be within the range of 85% to 115%;

[0144] ③ Detection limit: no more than 1.25 pmol / mL;

[0145] ④ Linearity: The detection range of the kit is TK1[1.25-20] pmol / mL, and the correlation coefficient R of the linear interval should be no less than 0.9900;

[0146] ⑤ Repeatability: coefficient of variation CV is not greater than 5%;

[0147] ⑥ Inter-batch difference: inter-batch coefficient of variation CV is not greater than 8%;

[0148] ⑦ Accuracy of calibrator value assignment: The relative deviation of the results of the calibration sample detected by the chemiluminescence immunoassay analyzer calibrated with the calibrator is ±8%;

[0149] ⑧ Homogeneity of calibration material: homogeneity ≤ 8%

[0150] ⑨ Target value validity of quality control products: When quality control products are tested using a chemiluminescence immunoassay analyzer calibrated with the calibrator, the results should be within the target value range;

[0151] ⑩ Homogeneity of quality control products: Homogeneity ≤ 8%

[0152] (4) Three batches of the kits of the present invention were stored at 2-8°C for 25 months, and all performance characteristics met the requirements. This indicates that the kits are stable when stored at 2-8°C for 25 months. Therefore, the kits of the present invention are stored at 2-8°C and have a shelf life of 24 months. The results are detailed in Tables 6-8.

[0153] Table 6 Real-time stability test results (first batch)

[0154]

[0155]

[0156] Table 7 Real-time stability test results (second batch)

[0157]

[0158]

[0159] Table 8 Real-time stability test results (third batch)

[0160]

[0161] 3.6.2 37℃ aging stability:

[0162] (1) The main raw material of the detection kit of the present invention is the coated antibody. Since temperature has a great influence on the activity of the antibody, the higher the temperature, the more serious the attenuation of the antibody activity. The accelerated stability test design adopts the destruction test at 37°C. The stability study is carried out by examining the appearance, detection limit, accuracy, linearity, repeatability, batch difference, calibration value assignment accuracy, calibration uniformity, target value validity of the quality control product, and quality control product uniformity performance indicators, which are required to be no less than the product design requirements.

[0163] (2) Three batches of test kits, 4 boxes each, totaling 12 boxes, were placed in a 37°C incubator according to the commercial packaging for accelerated testing. The full performance (appearance, detection limit, accuracy, linearity, repeatability, inter-batch variation, accuracy of calibrator value assignment, uniformity of calibrator, validity of target values of quality control products, and uniformity of quality control products) of the test kits were tested on day 0, day 5, day 10, and day 13, respectively.

[0164] (3) Judgment of test content and results: consistent with long-term stability at 2-8°C;

[0165] (4) After accelerating the test kit at 37°C for 13 days, all performances of the three batches of the test kit of the present invention met the requirements, indicating that the performance of the test kit is stable within 13 days of accelerating the test kit at 37°C. The results are shown in Tables 9 to 11.

[0166] Table 9 Accelerated stability test results (first batch)

[0167]

[0168]

[0169] Table 10 Accelerated stability test results (second batch)

[0170]

[0171] Table 11 Accelerated stability test results (third batch)

[0172]

[0173] In summary, the kit of the present invention adopts the magnetic microparticle chemiluminescence-double antibody sandwich method. Compared with other methodologies such as enzyme-linked immunosorbent assay and fluorescent immunochromatography, it is simple to operate and can be tested on the machine directly after adding the sample. In addition, through the independently developed acridinium ester working solution, magnetic bead working solution and working cleaning solution, after adding the sample, the incubation time of the magnetic beads and acridinium ester with the sample is greatly shortened. It only takes 3 minutes to reach the optimal reaction time, achieving the purpose of real-time detection, fully meeting the diagnosis and treatment needs of time-sensitive diseases, and providing patients with accurate test results.

[0174] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A kit for detecting thymidine kinase 1, characterized in that: The kit comprises reagent M and reagent N; the reagent M comprises magnetic bead working solution and magnetic microspheres coated with TK1 mouse monoclonal antibody; the reagent N comprises labeled antibody diluent and acridinium ester-labeled TK1 mouse monoclonal antibody.

2. A kit for detecting thymidine kinase 1 according to claim 1, characterized in that: The mass ratio of the TK1 mouse monoclonal antibody to the magnetic microspheres in the magnetic microspheres coated with the TK1 mouse monoclonal antibody is 3-5:50-100.

3. A kit for detecting thymidine kinase 1 according to claim 1, characterized in that: The mass ratio of acridinium ester to TK1 mouse monoclonal antibody in the acridinium ester-labeled TK1 mouse monoclonal antibody is 5-9:30-50.

4. A kit for detecting thymidine kinase 1 according to claim 1, characterized in that: Each 1L of the magnetic bead working solution includes the following components: HEPES 11.91g, sodium chloride 9.00g, BSA 25.00g, trehalose 100.00g, glucose 80.00g, Tween-20 1mL, Proclin 300 1mL, polyethylene glycol-20000 80.00g, alkyl polyglycoside glycerol ether 7.2g, pH=7.

4.

5. A kit for detecting thymidine kinase 1 according to claim 1, characterized in that: Each 1 L of the labeled antibody dilution solution includes the following components: 2.42 g of tris(hydroxymethyl)aminomethane, 1.41 mL of hydrochloric acid, 9.00 g of sodium chloride, 10.00 g of BSA, 0.5 mL of Proclin 300, 4.5 g of dextran, 7.4 g of isodecyl citrate monoester, and 0.01 g of amaranth.

6. A kit for detecting thymidine kinase 1 according to claim 1, characterized in that: The kit further comprises a calibrator, a quality control product and a chemiluminescent substrate; the chemiluminescent substrate is a pre-excitation solution / chemiluminescent substrate A solution and an excitation solution / chemiluminescent substrate B solution.

7. A method for preparing a kit for detecting thymidine kinase 1 according to any one of claims 1 to 6, characterized in that: The steps include: Magnetic bead coating: activating magnetic microspheres in a magnetic bead coating buffer using an activator to obtain activated magnetic beads; adding TK1 mouse monoclonal antibodies to the activated magnetic beads for coupling reaction to obtain magnetic microspheres coated with the TK1 mouse monoclonal antibody; and storing the magnetic microspheres coated with the TK1 mouse monoclonal antibody in a magnetic bead working solution to obtain reagent M; Acridinium ester labeling: TK1 mouse monoclonal antibody is labeled with acridine in a luminophore labeling buffer to obtain an antibody labeling mixture; the antibody labeling mixture is ultrafiltered to obtain acridinium ester-labeled TK1 mouse monoclonal antibody, and the acridinium ester-labeled TK1 mouse monoclonal antibody is stored in a labeled antibody diluent to obtain reagent N.

8. The method for preparing a kit for detecting thymidine kinase 1 according to claim 7, characterized in that: The activator is EDC; the mass ratio of the activator to the magnetic beads is 10:0.5-2; The activation parameters are: room temperature, 20 min to 40 min; The coupling reaction parameters are: room temperature, 3 h to 8 h.

9. The method for preparing a kit for detecting thymidine kinase 1 according to claim 7, characterized in that: The labeling reaction parameters are: 37° C., 20 min to 40 min; The ultrafiltration parameters are: 14000xg, 5-15 min.

10. Use of a kit for detecting thymidine kinase 1, characterized in that: The kit for detecting thymidine kinase 1 according to any one of claims 1 to 6 is used to prepare a product for detecting the tumor marker thymidine kinase 1.

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