An enzymatic chemiluminescence kit for detecting thymidine kinase I and a preparation method thereof

Abstract

The invention discloses an enzymatic chemiluminescent kit for detecting thymidine kinase I and a preparation method thereof. The thymidine kinase I enzymatic chemiluminescent kit comprises reagent A and reagent B, wherein reagent A is a magnetic bead suspension coated with SA-B-TK1 antibody 1; and reagent B is a carbon nanotube microparticle suspension carrying an enzyme-labeled antibody TK1 antibody 2. The preparation method of the kit comprises the following steps: S1(1) adding activated streptavidin (SA) to the magnetic bead suspension to obtain a magnetic bead suspension coated with SA; (2) coupling the magnetic bead suspension with biotinylated TK1 antibody 1 to obtain SA-B-TK1 antibody 1 labeled magnetic beads; S2(1) dispersing carboxylated carbon nanotubes in a PBS solution, adding EDC / NHS activation solution, stirring, centrifuging and washing to obtain activated carbon nanotubes; (2) adding HRP, GOD, and TK1 antibody 2 to a container containing activated carbon nanotubes to obtain an HRP-GOD-double enzyme labeled antibody complex. The kit has high detection sensitivity and excellent anti-interference performance.

Description

Technical Field

[0001] The invention relates to the field of thymidine kinase I detection reagents, and in particular to an enzymatic chemiluminescence kit for detecting thymidine kinase I and a preparation method thereof. Background Art

[0002] Thymidine kinase (TK) is one of the important enzymes in the pyrimidine nucleotide salvage synthesis pathway, including two isozymes: cytoplasmic thymidine kinase (TK1) and mitochondrial thymidine kinase (TK2). The activity of TK2 is only 5% of that of TK1, and it is mainly present in quiescent tissues. Although it also exists in proliferating cells, the content is very low. TK1 is an important marker for evaluating the proliferation of proliferating cells. It is a special enzyme in the S phase (synthesis phase) of the cell cycle and one of the key enzymes for DNA synthesis and damage repair. Its concentration is closely related to the cell proliferation state.

[0003] TK1 catalyzes the phosphorylation of deoxythymidine to generate deoxythymidine monophosphate (TMP), which then forms deoxythymidine triphosphate (TTP) to participate in DNA synthesis. Under normal conditions, the content of TK1 in adults is very low. When a large number of proliferating cells appear in the body, the cells proliferate rapidly and die, and the probability of TK1 being released into the peripheral blood increases exponentially, resulting in an increase in TK1 activity and content, which can exceed the normal level by 2 to 100 times.

[0004] Therefore, the changes in TK1 activity and content in tissues and serum can be used as indicators to monitor the proliferation level of tumor cells and quantitatively reflect the proliferation rate of tumor cells in patients. As a good cell proliferation marker, TK1 can be used for clinical physical examination screening, evaluation of surgical effects, monitoring of chemotherapy progress, prediction of recurrence risk and judgment of tumor prognosis. Many experiments have shown that TK1 can be used for health examinations, tumor screening, efficacy monitoring and prognosis evaluation, and is a sensitive and effective tumor growth marker. Methods for detecting tumor markers include enzyme-linked immunosorbent assay, fluorescent immunoassay, chemiluminescent immunoassay, electrochemical immunoassay, colloidal gold labeled immunoassay, etc.

[0005] At present, there are few detection kits for TK1. The existing detection kits for TK1 have low detection sensitivity and weak anti-interference ability. Therefore, the existing detection kits for TK1 need to be further developed. Summary of the invention

[0006] In view of the above problems, the present invention provides an enzymatic chemiluminescence kit for detecting thymidine kinase I, and a preparation method and application thereof, which has improved detection sensitivity for TK1 and strong anti-interference performance. The specific scheme is as follows:

[0007] On the one hand, the present application provides a thymidine kinase I enzymatic chemiluminescence kit, which includes reagent A and reagent B, wherein reagent A is a magnetic bead suspension coated with SA-B-TK1 antibody 1; reagent B is a carbon nanotube particle suspension carrying enzyme-labeled antibody TK1 antibody 2.

[0008] In reagent A, the magnetic beads coated with SA-B-TK1 antibody 1, the TK1 antibody 1 thereon is connected to the surface of the magnetic beads through a biotin (B)-streptavidin (SA) coupling structure; since one molecule of streptavidin has four biotin binding sites, the signal amplification effect is achieved through the cooperation of the two, thereby improving the detection sensitivity of the kit to thymidine kinase I in the sample.

[0009] In reagent B, on the carbon nanotube microparticle carrying the enzyme-labeled antibody TK1 antibody 2, there are two kinds of enzymes coated thereon, namely HRP (horseradish peroxidase) and GOD (glucose oxidase). These two enzymes act together as signal markers, cooperate with each other, achieve better signal amplification, and greatly improve the detection sensitivity and anti-interference performance of the test kit. The principle is: glucose oxidase (GOD) is an aerobic dehydrogenase. Glucose oxidase can convert glucose into gluconic acid under the condition of the presence of oxygen, producing H2O2. And HRP (horseradish peroxidase) catalyzes hydrogen donors under the effect of H2O2, and the hydrogen donors are oxidized and develop color, so that instrument detection. The hydrogen donor adopted in this application is 4-CN.

[0010] Preferably, the thymidine kinase I enzymatic chemiluminescence kit further comprises an enzyme catalytic base solution, which is a PBS solution containing 0.5 to 10 mmol / L 4-CN and 1 to 20 mmol / L glucose. The enzyme catalytic base solution is prepared by adding 4-CN and glucose to a PBS solution, wherein the concentration of 4-CN is 0.5 to 10 mmol / L and the concentration of glucose is 1 to 20 mmol / L.

[0011] Preferably, the concentration of PBS is 0.05 mol / L, and the pH of the base solution is 7.6.

[0012] Preferably, the thymidine kinase I enzymatic chemiluminescence kit further comprises a TK1 calibrator and a TK1 quality control product. Existing TK1 calibrators and TK1 quality control products can be directly used.

[0013] On the other hand, the present application also provides a method for preparing the aforementioned thymidine kinase I enzymatic chemiluminescence kit, which comprises the following steps:

[0014] S1. Preparation of reagent A:

[0015] (1) adding activated streptavidin (SA) to the aforementioned magnetic bead suspension to obtain a magnetic bead suspension coated with SA;

[0016] (2) coupling the SA-coated magnetic bead suspension with the biotinylated TK1 antibody 1 in a solution, and treating on a magnetic separator to obtain SA-B-TK1 antibody 1 labeled magnetic beads, i.e., reagent A;

[0017] S2. Preparation of reagent B:

[0018] (1) activating the carboxylated carbon nanotubes: dispersing the carboxylated carbon nanotubes in a PBS solution, adding an EDC / NHS activation solution, stirring, centrifuging and washing to obtain activated carbon nanotubes, and dispersing the activated carbon nanotubes in a PBS solution;

[0019] (2) Add HRP, GOD, and TK1 antibody 2 to the container containing activated carbon nanotubes, continue stirring at low temperature for 10 to 15 hours, and obtain an HRP-GOD-double enzyme labeled antibody complex after centrifugal washing, which is dispersed in a PBS solution to obtain reagent B.

[0020] Preferably, in step (1) of step S1 above, amino-PEG6-carboxylic acid as an extension arm is connected to the magnetic beads, and the method is: adding amino-PEG6-carboxylic acid as an extension arm to the activated magnetic bead suspension, incubating at room temperature for 1 hour, to obtain amino-PEG6-magnetic beads.

[0021] Firstly connecting amino-PEG6-carboxylic acid to the magnetic beads can ensure that the magnetic beads are not easily affected by the steric hindrance of large molecules, better expose the connection sites, and thus significantly improve the SA coating efficiency of the magnetic beads.

[0022] On the basis of the above, preferably, the following steps are further provided after step S2:

[0023] S3. Prepare standard products and quality control products; dilute human serum with a diluent to a certain multiple to prepare a basic buffer solution, use the basic buffer solution to dilute TK1 antigen into standards of different concentrations, and use the basic buffer solution to prepare TK1 antigen into high-concentration and low-concentration quality control products.

[0024] The present invention has the following beneficial effects:

[0025] In the thymidine kinase I enzymatic chemiluminescence kit, the magnetic beads coated with antibody 1 improve the coating efficiency of antibody 1 through the cooperation of streptavidin (SA) and biotin (B), thereby achieving signal amplification; on this basis, the kit adopts two markers, and coats two enzymes, HRP and GOD, on the carbon nanotube particles carrying enzyme-labeled antibody TK1 antibody 2, and further improves the signal amplification effect through the synergistic cooperation of the two. Based on the improvements in the above multiple aspects, the kit of the present application significantly improves the detection sensitivity and anti-interference performance of thymidine kinase I in the sample. DETAILED DESCRIPTION

[0026] The technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments are conventional methods in the art unless otherwise specified.

[0027] Example 1 Preparation of Thymidine Kinase I Enzymatic Chemiluminescent Kit

[0028] This embodiment provides a thymidine kinase I enzymatic chemiluminescence kit, which mainly includes: reagent A and reagent B, and may also include: calibrators, quality control products, enzyme catalytic base solution, etc.

[0029] The specific preparation method is as follows:

[0030] 1. Preparation of Reagent A

[0031] (1) Activation of magnetic beads:

[0032] 200 μL of magnetic beads (concentration of 2 mg / mL) were drawn into the EP tube, placed on a magnetic separator for 2 to 5 minutes, the supernatant was discarded, and the precipitate was retained. Then 200 μL of PBS buffer (0.1 M) was added to the tube, and after being blown evenly, the tube was placed on a magnetic separator for separation and the supernatant was discarded. This operation was repeated 2 to 4 times. Next, 100 μL of the aforementioned PBS buffer was added for resuspending, and 200 μL of 10 mg / mL EDC activator was added, and the tube was placed on an oscillator for reaction for 20 minutes, and the tube was placed on a magnetic separator for separation for 3 minutes and the supernatant was discarded.

[0033] In this example, Bioeast Mag-COOH magnetic microspheres were selected, which have carboxyl (COOH) modification on their surface and can covalently bind to proteins under EDC activation.

[0034] (2) Connection between the extension arm and the magnetic bead:

[0035] Add amino-PEG6-carboxylic acid solution (1 to 2 times the volume of the magnetic bead suspension) to the activated magnetic beads obtained in the previous step, incubate at room temperature for 1 hour to obtain amino-PEG6-magnetic beads; separate the unattached extension arms and waste liquid by a magnetic separator.

[0036] (3) Streptavidin (SA) linked to the surface of magnetic beads:

[0037] A sufficient amount of activated streptavidin (SA) was added to the aforementioned amino-PEG6-magnetic bead solution, and incubated at room temperature for 2 hours; the solution was placed on a magnetic separator for 4 minutes to remove unbound streptavidin, and finally SA-PEG6-magnetic beads were formed;

[0038] (4) Biotin-labeled TK1 antibody 1

[0039] Prepared by existing methods, such as: the molar ratio of biotin to the mouse anti-human TK1 antibody to be labeled is (10-15):1, and the following operations are performed:

[0040] Dissolve biotin in deionized water to obtain a biotin solution; mix the biotin solution with the TK1 antibody 1 solution and react at room temperature for 40 to 60 minutes; pre-clean the desalting column: take a desalting column, cut off the bottom stopper, put it in a 15mL collection tube, centrifuge at 1000 rpm for 2 minutes, and discard the filtrate. Add 2.5mL PBS solution to the desalting column, centrifuge at 1000 rpm for 2 minutes, discard the filtrate, and repeat 2-3 times. Put the desalting column into a new collection tube, add the sample, centrifuge at 1000 rpm for 2 minutes, and collect the filtrate, which is the purified labeled protein (Note: If the sample content is less than 1 mL, add 100μL of ultrapure water on the absorbed sample to increase protein recovery). Divide into 100μL / tube and place at -80℃.

[0041] (5) Preparation of SA-B-TK1 Antibody 1 Labeled Magnetic Beads

[0042] 25 μL of the above-mentioned streptavidin magnetic beads (0.06-1 μg / mL) and 50 μL of the biotinylated mouse anti-human TK1 antibody 1 solution were coupled, diluted with 0.1 mol / L PBS buffer to a total volume of 500 μL, and incubated overnight at 25°C. After the coupling was completed, a glycine solution quenching solution (1 mol / L, pH 8.0) was added and mixed at room temperature for 30 minutes; then, the mixture was placed on a magnetic separator for separation for 4-10 minutes, the supernatant was discarded, and the mixture was washed twice with 0.1% BSA in 0.01 M PBS buffer to remove substances not involved in the reaction, thereby obtaining SA-B-TK1 antibody 1 labeled magnetic beads, i.e., reagent A.

[0043] 2. Preparation of Reagent B

[0044] (1) Carboxylation of carbon nanotubes:

[0045] The carbon nanotubes were ultrasonically treated in a mixed solution of H2SO4 (98%), HNO3 (68%) and distilled water (1:3:6) at 40°C for 4-7 hours, centrifugally washed, and then dried at 50-55°C to obtain carboxylated carbon nanotubes.

[0046] (2) Activation of carbon nanotubes

[0047] Take 4 mg of carboxylated carbon nanotubes and disperse them in 1 mL of 0.01 M PBS (pH 7.4), then add 3 mL of freshly prepared 0.4 mol L -1 After vigorous stirring at room temperature for 3 to 5 hours in the EDC activation solution, the activated carbon nanotubes were obtained after centrifugation and washing, and then dispersed in 0.01 M PBS (pH 7.4).

[0048] (3) Dual enzyme and antibody labeling of carbon nanotubes

[0049] Add 200 μL HRP (1 mg / L), 200 μL GOD (1 mg / L), and 100 μL rabbit anti-human TK1 antibody 2 to the test tube containing activated carbon nanotubes, and continue stirring for 12 hours. Then, after centrifugation and washing, the HRP-GOD-double enzyme labeled antibody complex is obtained, which is then dispersed in 1 mL of PBS (0.01 M, pH 7.4) to obtain reagent B, which is stored at 4°C for use.

[0050] Example 2 Detection method of thymidine kinase I enzymatic chemiluminescence kit

[0051] 1. The kit used includes the following components:

[0052] This example uses the kit prepared in Example 1, wherein reagent A and reagent B, calibrators, quality control products, and enzyme catalytic base solution are packaged separately. The detection instrument uses a Cosma 500s chemiluminescence detector.

[0053] 2. The specific detection method includes the following steps:

[0054] 1) Add the sample to be tested to reagent A, 100 μL / well, and incubate at 37°C for 60 min;

[0055] 2) Add reagent B: add two drops or 100 μL of enzyme marker to each well;

[0056] 3) Incubation: Seal the strips with sealing film (to prevent contamination) and incubate at 37°C for 60 minutes;

[0057] 4) Washing the plate: Take out the reaction plate, wash it by magnetic separation, shake off the liquid in the plate, wash the plate 5 times, and pat it dry. Avoid generating bubbles during the washing process;

[0058] 5) Add 50 μL of enzyme catalytic base solution to each well, mix thoroughly, and catalyze the reaction for 15 to 20 minutes;

[0059] The enzyme catalytic base solution is a PBS solution (0.1 mol / L, pH 7.4) containing 1 mmol / L 4-CN and 1 mmol / L glucose.

[0060] 6) Determination: Use a semi-automatic luminometer to measure the OD value of each well;

[0061] 7) Calculation of results: Use the concentration of the calibrator as the horizontal axis and the OD value as the vertical axis. The concentration of the sample can be found on the standard curve through the OD value of the sample.

[0062] Comparative Example 1

[0063] The difference between the kit prepared in this example and Example 1 is that the magnetic beads are not connected to the extended arms in reagent A. The preparation method thereof is referred to Example 1.

[0064] Comparative Example 2

[0065] The difference between the kit prepared in this embodiment and Example 1 is that: the extended arm is connected to the surface of the magnetic bead, but streptavidin (SA) and avidin are not connected, and the extended arm of the magnetic bead is directly connected to the mouse anti-human TK1 antibody 1; the rest is the same as Example 1, and the preparation method refers to Example 1.

[0066] Comparative Example 3

[0067] The difference between the kit prepared in this example and Example 1 is that the reagent B uses only one enzyme, HRP, to label the TK1 antibody 2. The rest is the same as Example 1, and the preparation method is referred to Example 1.

[0068] Verification Example

[0069] The performance of the thymidine kinase I enzymatic chemiluminescence kit prepared in Example 1 was tested, and the kits of Comparative Examples 1 to 3 were used to perform simultaneous experiments for comparison.

[0070] 1. Precision (repeatability) evaluation

[0071] (1) Experimental methods:

[0072] High-concentration and low-concentration urine samples from cancer patients were selected as samples for testing. Each test was performed 20 times on the same plate to calculate the intra-batch precision. At the same time, one batch of each concentration level was tested every day, and each batch was tested twice. The tests were performed continuously for 6 days, a total of 12 times, and the inter-batch precision was calculated.

[0073] (2) Experimental results and analysis:

[0074] From the results in Table 1 and Table 2, it can be seen that the intra-batch and inter-batch detection precision of the kit of the present application is high, which is far superior to the technical effects of Comparative Examples 1 to 3.

[0075] Table 1 Precision test results of Example 1

[0076]

[0077]

[0078] Table 2 Precision test results of each comparative example

[0079]

[0080] 2. Interference experiment

[0081] (1) Experimental methods:

[0082] This evaluation used two common serum interfering substances, hemoglobin and triglycerides, and added a certain concentration of interfering substances to the serum test samples with high concentration TK1 and low concentration TK1, respectively, as interfering substance samples, and the volume of the added interfering substances did not exceed 5% of the total volume. The control group also added the same volume of PBS as a control sample, and then tested the two groups of samples separately, and each concentration was tested 3 times to calculate the interference rate. The kit of Example 1 and Comparative Examples 1 to 3 were tested by the above method.

[0083] The formula is as follows:

[0084] Interference rate = (measured concentration of interfering substance sample - measured concentration of control sample) / measured concentration of control sample * 100%.

[0085] (2) Experimental results and analysis

[0086] From the results in Table 3, it can be seen that the kit provided in the present application has good anti-interference ability for higher concentrations of bilirubin and triglycerides, and the interference rate is low, which is much lower than the interference rate of existing kits of this type.

[0087] At the same time, the above anti-interference experiment was carried out using comparative examples 1 to 3, and the average interference rate (the average of the interference rate of the high-concentration level sample and the low-concentration level sample of the same concentration of the interfering substance) was compared. From the results in Table 4, it can be seen that the interference rate of the kit provided in Example 1 of the present application is much lower than that of comparative examples 1 to 3. From the results, it can be seen that the use of HRP and GOD as markers and the setting of streptavidin and biotin in the present application greatly improve the anti-interference performance of the kit.

[0088] Table 3 Interference rate test results of the kit of Example 1

[0089]

[0090] Table 4 Average interference rate detection results of each comparative example

[0091]

[0092] 3. Correctness assessment

[0093] The TK1 quality control product with known concentration in the kit of Example 1 was measured three times in parallel, and the mean of the quality control product measurement results was calculated. The results are shown in Table 5 below. It can be seen that the concentration deviation of the kit is as low as 0.655%.

[0094] Table 5 Correctness evaluation results

[0095]

[0096] 4. Sensitivity (minimum detection limit):

[0097] The calibration sample was tested 10 times, and the sensitivity value was obtained by adding the concentration value corresponding to 2 times the standard deviation to the mean OD value. According to this method, the minimum detection limit of the kit was 0.001 ng / ml, while the minimum detection limit of comparative examples 2 to 3 was not less than 0.05 ng / ml.

[0098] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solution and concept of the present invention, and all these changes or substitutions should fall within the protection scope of the claims attached to the present invention.

Claims

1. A thymidine kinase I enzymatic chemiluminescence kit, characterized in that: The method comprises reagent A and reagent B, wherein reagent A is a magnetic bead suspension coated with SA-B-TK1 antibody 1; reagent B is a carbon nanotube particle suspension carrying enzyme-labeled antibody TK1 antibody 2; In reagent A, the magnetic beads coated with SA-B-TK1 antibody 1, the TK1 antibody 1 thereon is connected to the surface of the magnetic beads through a biotin (B)-streptavidin (SA) coupling structure; The magnetic beads were pre-linked with amino-PEG6-carboxylic acid as an extension arm; In reagent B, there are two types of enzymes coated on the carbon nanotube particles carrying the enzyme-labeled antibody TK1 antibody 2, namely HRP and GOD.

2. The thymidine kinase I enzymatic chemiluminescence kit according to claim 1, characterized in that: The invention also comprises an enzyme catalytic base solution, which is a PBS solution containing 0.5 to 10 mmol / L of 4-CN and 1 to 20 mmol / L of glucose.

3. The thymidine kinase I enzymatic chemiluminescence kit according to claim 1, characterized in that: Also includes TK1 Calibrator and TK1 Quality Control.

4. A method for preparing the thymidine kinase I enzymatic chemiluminescence kit as claimed in claim 1, characterized in that: The following steps are involved: S1. Preparation of reagent A: (1) adding activated streptavidin (SA) to a magnetic bead suspension to obtain a magnetic bead suspension coated with SA; (2) coupling the SA-coated magnetic bead suspension with the biotinylated TK1 antibody 1 in a solution, and treating on a magnetic separator to obtain SA-B-TK1 antibody 1 labeled magnetic beads, i.e., reagent A; S2. Preparation of reagent B: (1) activating the carboxylated carbon nanotubes: dispersing the carboxylated carbon nanotubes in a PBS solution, adding an EDC / NHS activation solution, stirring, centrifuging and washing to obtain activated carbon nanotubes, and dispersing the activated carbon nanotubes in a PBS solution; (2) adding HRP, GOD, and TK1 antibody 2 to a container containing activated carbon nanotubes, stirring continuously for 10 to 15 hours under low temperature conditions, and obtaining an HRP-GOD-dual enzyme labeled antibody complex after centrifugal washing, which is dispersed in a PBS solution to obtain reagent B; In step (1) of step S1, amino-PEG6-carboxylic acid as an extension arm is pre-connected to the magnetic beads by adding amino-PEG6-carboxylic acid as an extension arm to the activated magnetic bead suspension, incubating at room temperature for 1 hour, and obtaining amino-PEG6-magnetic beads.

5. The method for preparing the thymidine kinase I enzymatic chemiluminescence kit according to claim 4, characterized in that: The following steps are further provided after step S2: S3. Prepare standard products and quality control products; dilute human serum with a diluent to a certain multiple to prepare a basic buffer solution, use the basic buffer solution to dilute TK1 antigen into standards of different concentrations, and use the basic buffer solution to prepare TK1 antibody antigen into high-concentration and low-concentration quality control products.

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