CN111044724B - Thymidine kinase 1 magnetic particle chemiluminescence assay kit and preparation method thereof - Google Patents
Thymidine kinase 1 magnetic particle chemiluminescence assay kit and preparation method thereof Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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- Analytical Chemistry (AREA)
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- General Physics & Mathematics (AREA)
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a thymidine kinase 1 magnetic particle chemiluminescence assay kit and a preparation method thereof, wherein the kit comprises a reagent A, a reagent B and a thymidine kinase 1 (TK 1) calibrator, wherein the reagent A is a magnetic particle suspension coated with an anti-thymidine kinase 1 (TK 1) antibody, and the reagent B is an alkaline phosphatase-labeled anti-thymidine kinase 1 (TK 1) antibody enzyme conjugate reagent. Compared with the prior art, the invention provides a method for measuring the content of thymidine kinase 1 (TK 1), and the kit provided by the invention can accurately, sensitively and rapidly measure the content of thymidine kinase 1 (TK 1) in a sample according to the principle of a double-antibody sandwich method.
Description
Technical Field
The invention relates to a biological reagent, in particular to a detection kit, and especially relates to a kit for measuring human serum thymidine kinase 1 content with high sensitivity and strong specificity and a preparation method thereof.
Background
Thymidine Kinase 1 (tk1) Thymidine Kinase (TK 1) is one of the key enzymes in DNA synthesis, and catalyzes the conversion of Thymidine (TdR) to deoxythymidine monophosphate (TMP) under the participation of magnesium ions and ATP, thereby forming Thymidine triphosphate (TTP, one of the four necessary deoxynucleotides in DNA synthesis) to participate in DNA synthesis. The elevation of TK1 levels and DNA synthesis are positively correlated, with the onset of TK1 elevation in late G1 phase, highest in S phase, and declining in G2 phase in the cell cycle, due to the specific relevance of TK1 to S phase of the cell cycle, also known as "S phase key enzyme", being a key enzyme for DNA synthesis in S phase by salvage pathways.
TK1 is generally in two forms, the TK1 holoenzyme is a tetramer with a molecular weight of about 12kDa, and also in the form of a dimer. The tetramer is generally more catalytically efficient, about 22 times that of the dimer. In the presence of ATP TK1 is more prone to tetramer formation and removal of ATP reverts to dimeric form, the process is reversible. TK1 is not stable in tissue or cell extracts, but is more stable in serum and has a longer shelf life at-20 ℃.
In clinical studies, significant correlations of TK1 concentration and activity are present only in healthy people, benign tumor patients and in part malignant tumor patients (e.g. leukemia, breast cancer, gastric cancer). Typically 90-95% of malignant patients will have a higher concentration of TK1 than normal, but the corresponding TK1 activity is only 75% of this increase. Thus, the use of TK1 concentration as a tumor marker in the detection of malignant solid tumors may be more sensitive than the detection of TK1 activity, whereas the detection of TK1 protein concentration in early development of breast, prostate cancer is also more efficient than the detection of TK1 activity. At the same time, TK1 is also associated with the diagnosis and treatment of some solid tumors (e.g. lung cancer, gastric cancer). Overall, there is also a significant increase in TK1 protein levels in solid tumor patients, and the determination of TK1 concentration in solid tumor cases may even be more sensitive than the determination of TK1 activity.
Normally normal humans have TK1 levels on average around 1pM, with specific values depending on individual differences, but generally between 0-2 pM. For abnormal proliferation lesions, because the apoptosis regulation is absent in the lesion cells, the abnormal increase of TK1 level can be caused by the rapid increase of DNA synthesis, namely, the content of TK1 enzyme in serum of a patient with malignant proliferation lesions can be increased by 2-200 times compared with that of a normal person. Therefore, TK1 is also used as a nonspecific tumor marker at present and is applied to screening and post-treatment tracking monitoring of malignant proliferation lesions in physical examination and clinic.
Currently, the method for measuring TK1 is mainly enzyme-linked immunosorbent assay (ELISA). The method generally relies on manual operation, consumes labor and time, has low detection sensitivity and linear range, is mainly used for laboratory analysis, has certain limitation in clinical detection application, and can not meet clinical requirements. At present, TK1 determination reagents, especially automatic detection reagents, which are fast in speed, good in stability, high in sensitivity and strong in specificity are still lacking in the market. Therefore, the research and development production quality meets the clinical requirements, the practicability is strong, the cost performance is high, and the TK1 determination reagent which can be applied to the full-automatic luminescence analyzer becomes a hotspot in the in-vitro diagnostic reagent industry at home and abroad.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide the magnetic particle chemiluminescence detection kit for the thymidine kinase 1 and the preparation method thereof, which can realize high-flux and rapid detection of the TK1 on a full-automatic chemiluminescence analyzer, have the advantages of simplicity and convenience in operation, high sensitivity, strong specificity, accurate results and the like, effectively reduce the TK1 detection cost and are beneficial to clinical popularization and use.
The aim of the invention can be achieved by the following technical scheme: a thymidine kinase 1 magnetic particle chemiluminescence assay kit, wherein the kit comprises a reagent A, a reagent B and a thymidine kinase 1 (TK 1) calibrator, wherein the reagent A is a magnetic particle suspension coated with an anti-thymidine kinase 1 (TK 1) antibody, and the reagent B is an alkaline phosphatase-labeled anti-thymidine kinase 1 (TK 1) antibody enzyme conjugate reagent.
Further, the reagent A is prepared from 0.05-0.2mol/L buffer solution, 0.1-0.5% preservative, 1-10% surfactant and 0.01-0.08% magnetic particles coated with TK1 antibody.
Further, the magnetic particles are Fe 2 O 3 Or Fe (Fe) 3 O 4 The composite of the magnetic nano particles and the organic polymer material contains one or more active functional groups on the surface of the magnetic particles.
Further, the buffer is selected from one or more of phosphate buffer, tris buffer, 3- (N-morpholino) propane sulfonic acid buffer, 2- (N-morpholino) ethane sulfonic acid buffer, borate buffer, glycine buffer or hydroxyethyl piperazine ethane sulfonic acid buffer; the pH value of the buffer solution is 6-9;
further, the preservative is selected from one or more of sodium azide, N-methyl-isothiazolone, N '-methylene-bis [ N' - (3-hydroxymethyl-2, 5-dione-4-imidazolidinyl) ] urea, or ProClin 300;
further, the surfactant is selected from tween 20, tween 80, an ethylene oxide-propylene oxide block copolymer or polyethylene glycol octylphenyl ether;
further, the magnetic particles coated with the TK1 antibody are a compound formed by selecting magnetic particles with different particle diameters and surface active functional groups and the TK1 antibody, the surface active functional groups of the magnetic particles are selected from p-toluenesulfonyl or carboxyl, and the particle diameter of the magnetic particles is 0.1-5 mu m.
Further, the reagent B is prepared from 0.05-0.2mol/L buffer solution, 0.1-0.5% preservative, 1-10% surfactant, 1-10% protection protein and 0.1-1.0 mug/ml alkaline phosphatase labeled TK1 antibody enzyme conjugate.
Further, the buffer is selected from one or more of phosphate buffer, tris buffer, 3- (N-morpholino) propane sulfonic acid buffer, 2- (N-morpholino) ethane sulfonic acid buffer, borate buffer, glycine buffer or hydroxyethyl piperazine ethane sulfonic acid buffer; the pH value of the buffer solution is 6-9;
further, the preservative is selected from one or more of sodium azide, N-methyl-isothiazolone, N '-methylene-bis [ N' - (3-hydroxymethyl-2, 5-dione-4-imidazolidinyl) ] urea, or ProClin 300;
further, the surfactant is selected from tween 20, tween 80, sodium dodecyl sulfate or polyethylene glycol octyl phenyl ether;
further, the protective protein is selected from sodium caseinate or bovine serum albumin;
further, the alkaline phosphatase-labeled TK1 antibody enzyme conjugate is a complex formed by alkaline phosphatase and TK1 antibody.
Further, the TK1 antibodies in the reagent A and the reagent B are monoclonal antibodies with two different binding sites; the two TK1 antibodies are selected from rabbit anti-polyclonal antibodies, sheep anti-polyclonal antibodies or mouse anti-monoclonal antibodies.
Further, the volume ratio of the reagent A to the reagent B is 1:5.
The TK1 calibrator comprises a calibrator 1, a calibrator 2, a calibrator 3, a calibrator 4, a calibrator 5 and a calibrator 6. Wherein the calibrator 1 is prepared by preparing TK1 with bovine serum; calibrator 2 means that TK1 is diluted with bovine serum to a concentration of 1pmol/ml to obtain a calibrator; calibrator 3 means that TK1 is diluted with bovine serum to a concentration of 2pmol/ml to obtain a calibrator; calibrator 4 is prepared by diluting TK1 with bovine serum to a concentration of 5 pmol/ml; calibrator 5 is prepared by diluting TK1 with bovine serum to a concentration of 10 pmol/ml; calibrator 6 refers to a calibrator obtained by diluting TK1 with a bovine serum preparation to a concentration of 50 pmol/ml.
The principle of the invention is as follows: the invention uses the magnetic particles coated with TK1 antibody in the reagent A to react with TK1 in the sample and form TK1-TK1 antibody coated magnetic particle compound, and simultaneously, alkaline phosphatase marked TK1 antibody enzyme conjugate in the reagent B also reacts with TK1-TK1 antibody coated magnetic particles to form TK1 antibody coated magnetic particle-TK 1-alkaline phosphatase marked TK1 antibody compound. After incubation, the TK1 antibody complex of TK 1-alkaline phosphatase-labeled magnetic particles is retained by corresponding magnetic equipment in a full-automatic chemiluminescence analyzer, the remaining reaction liquid and the sample are washed away, a luminescent substrate is added, a generated chemiluminescent signal is measured, and the concentration of TK1 in the sample is in direct proportion to the chemiluminescent signal. Comparing the working curve of the TK1 concentration and the chemiluminescence intensity, the TK1 concentration in the sample can be obtained.
The invention also provides a preparation method of the thymidine kinase 1 magnetic particle chemiluminescence assay kit, which comprises the following steps:
(1) Preparation of reagent A:
sequentially adding buffer salt, surfactant and preservative into water, stirring until the materials are completely dissolved each time, adjusting the pH value to 6.0-9.0, then passing through a 0.22 mu m filter membrane to obtain a reagent A diluent, and then adding magnetic particles coated with TK1 antibody to obtain a reagent A;
(2) Preparation of reagent B:
sequentially adding buffer salt, surfactant, protective protein and preservative into water, stirring until the materials are completely dissolved each time, adjusting the pH value to 6.0-9.0, then passing through a 0.22 mu m filter membrane to obtain a reagent B diluent, and then adding alkaline phosphatase-labeled TK1 antibody enzyme conjugate to obtain a reagent B;
(3) The composition kit comprises: the prepared reagent A and reagent B are mixed according to the volume ratio R1: r2=1:5 split into vials to make up the kit.
Wherein, the magnetic particles coated with TK1 antibody in the step (1) are antibody-magnetic particle conjugate for binding TK1 in a sample, and are prepared by reacting the corresponding antibody and the magnetic particles, specifically:
when the active functional group on the surface of the magnetic particle is p-toluenesulfonyl, the preparation steps are as follows:
reaction of magnetic particles with antibodies: in 50mmol/L boric acid buffer solution, the TK1 antibody and the magnetic particles are mixed according to the mass ratio of 1:100 and react for 16-24 hours at 20-25 ℃ to form an antibody-magnetic particle conjugate;
blocking of antibody-magnetic particle conjugates: washing the antibody-magnetic particle conjugate with 50mmol/L Tris buffer, adding the magnetic particles into 50mmol/L Tris buffer containing 10g/L Bovine Serum Albumin (BSA), reacting at 20-25 ℃ for 16-24 hours, and sealing the antibody-magnetic particle conjugate;
iii. preservation of antibody-magnetic particle conjugate: washing the antibody-magnetic particle conjugate with 50mmol/L Tris buffer, and then adding the magnetic particles into 50mmol/L Tris buffer containing 10g/L Bovine Serum Albumin (BSA) for storage;
(II) when the active functional group on the surface of the magnetic particle is carboxyl, the preparation steps are as follows:
reaction of magnetic microparticles with antibodies: in 50 mmol/L2- (N-morpholine) ethanesulfonic acid buffer solution, TK1 antibody and magnetic particles are added according to the mass ratio of 1:100, EDC and HCl are added for reaction for 2 hours at 20-25 ℃ to form an antibody-magnetic particle conjugate;
blocking of antibody-magnetic particle conjugates: washing the antibody-magnetic particle conjugate with 50mmol/L Tris buffer, adding the magnetic particles into 50mmol/L Tris buffer containing 10g/L Bovine Serum Albumin (BSA), reacting at 20-25 ℃ for 16-24 hours, and sealing the antibody-magnetic particle conjugate;
III. preservation of antibody-magnetic particle conjugates: washing the antibody-magnetic particle conjugate with 50mmol/L Tris buffer, and then adding the magnetic particles into 50mmol/L Tris buffer containing 10g/L Bovine Serum Albumin (BSA) for storage;
the alkaline phosphatase-labeled TK1 antibody enzyme conjugate of step (2) is an antibody-alkaline phosphatase conjugate for binding TK1 in a sample, prepared by reacting the corresponding antibody with alkaline phosphatase, specifically:
(a) Activation of alkaline phosphatase: adding sodium periodate into alkaline phosphatase, uniformly mixing, and activating at 2-8 ℃ for 30min;
(b) Activation termination of alkaline phosphatase: adding ethylene glycol into the alkaline phosphatase activating solution in the step (a), uniformly mixing, and standing at 2-8 ℃ for 2 hours to terminate the activation reaction;
(c) Coupling of TK1 antibody and alkaline phosphatase: mixing the TK1 antibody and the alkaline phosphatase activated in the step (b), and standing at 2-8 ℃ for 20min for coupling after the mixture is uniformly mixed;
(d) Coupling termination of TK1 antibody and alkaline phosphatase: adding sodium borohydride into the combination solution of alkaline phosphatase and TK1 antibody in the step (c), uniformly mixing, and standing at 2-8 ℃ for 2 hours to terminate the coupling reaction;
(e) Washing of alkaline phosphatase-labeled TK1 abzyme conjugate: transferring the reaction solution of alkaline phosphatase combined with TK1 antibody to a dialysis bag, and soaking in a washing solution to remove small molecular impurities; the washing solution is 50-100 mM buffer solution, the pH value is 8.5-9.5, and the washed alkaline phosphatase marked TK1 antibody enzyme conjugate is obtained;
(f) Preservation of alkaline phosphatase-labeled TK1 abzyme conjugate: glycerol was added to the washed alkaline phosphatase-labeled TK1 antibody enzyme conjugate solution obtained in step (e) and stored at-20 ℃ for use.
Compared with the prior art, the invention has the following beneficial effects:
(1) The kit provided by the invention comprises two TK1 antibodies marked by alkaline phosphatase and TK1 antibodies coated by magnetic particles, and the concentration of TK1 in a sample can be accurately, sensitively and rapidly determined by adopting a double-antibody sandwich method.
(2) The kit adopts a more advanced chemiluminescence immunoassay method, so that the specificity and the sensitivity are greatly improved, and the kit is better used for clinical diagnosis.
(3) The kit can be operated by means of a full-automatic chemiluminescence immunoassay analyzer, all samples and reagent sample addition are completed by the operation of the analyzer, and interference of human factors on test results is reduced. Meanwhile, the test time is greatly shortened, and the clinical diagnosis result can be provided more efficiently and rapidly.
(4) When the kit is used for measuring the sample, the sample to be measured is not required to be diluted, the sample to be measured can be directly detected, errors caused by dilution operation are reduced, and the kit is also beneficial to the detection sensitivity of the low-value sample. The sample to be tested can be any one of a blank tube, an EDTA plasma tube, heparin plasma, a coagulant pigging and a separation gel pigging.
Drawings
FIG. 1 is a calibration curve of the reagent prepared in example 6 on a Tao Lumiart-II-1 chemiluminescent meter with TK1 concentration on the X-axis and luminescence (RLU) on the Y-axis.
FIG. 2 shows the correlation results of the test samples of the reagent prepared in example 6 and the Jing Huarui Tongkang reagent.
FIG. 3 is a calibration curve of the reagent prepared in example 7 on a Tao Lumiart-II-1 chemiluminescent meter with TK1 concentration on the X-axis and luminescence (RLU) on the Y-axis.
FIG. 4 is a correlation result of the reagent prepared in example 7 and a sample of Jing Huarui with kang reagent test.
Detailed Description
The invention will now be described in detail with reference to the drawings and specific examples.
The examples were obtained commercially using the starting materials. The p-toluenesulfonyl magnetic particles and carboxyl magnetic particles were purchased from Semer Feishmania technologies Inc. or Mieshiya (Shanghai) commercial Co.
Example 1
Rabbit anti-TK 1 polyclonal antibody coated p-toluenesulfonyl magnetic particles
The magnetic particles coated with the rabbit anti-TK 1 polyclonal antibody are formed by connecting p-toluenesulfonyl magnetic particles with amino groups of the rabbit anti-TK 1 polyclonal antibody, and the specific synthesis steps of the compound are as follows:
(1) Weighing 3.1g of boric acid, dissolving in 1L of deionized water, and adjusting the pH to 9.6 to prepare a buffer solution A;
(2) 7.9g of Tris-HCl is weighed and dissolved in 1L of deionized water, and the pH is adjusted to 7.6 to prepare a buffer solution B;
(3) 10g of Bovine Serum Albumin (BSA) was weighed and dissolved in 1L of the buffer solution B to prepare a buffer solution C;
(4) 1g of p-toluenesulfonyl magnetic particles and 10mg of rabbit anti-TK 1 polyclonal antibody are added into 100ml of buffer solution A, then the mixed solution is reacted for 16 to 24 hours at room temperature (20 to 25 ℃), and the container is placed on a roller machine for rolling and mixing uniformly during the reaction;
(5) Placing the reaction liquid container in the step (4) on a magnet, removing the supernatant after the magnetic beads are completely precipitated by utilizing magnetic force, adding 200ml of buffer solution B, stirring and cleaning, placing the container on the magnet, removing the supernatant after the magnetic beads are completely precipitated, and repeating the step for 4 times;
(6) Adding 200ml of buffer solution C into the magnetic particle container in the step (5), sealing the mixed solution at room temperature (20-25 ℃) for 16-24 hours, and placing the container on a roller machine for rolling and uniformly mixing during the reaction;
(7) And (3) placing the reaction liquid container in the step (6) on a magnet, removing supernatant after magnetic beads are completely precipitated by utilizing magnetic force, adding 200ml of buffer solution B, stirring and cleaning, placing the container on the magnet, removing supernatant after the magnetic beads are completely precipitated, repeating the step for 4 times, finally adding 100ml of buffer solution C to obtain magnetic particles coated with the rabbit anti-TK 1 polyclonal antibody, and storing at 4 ℃ for later use.
Example 2
Carboxyl magnetic particle coated by mouse anti-TK 1 monoclonal antibody
The magnetic particles coated with the mouse anti-TK 1 monoclonal antibody are formed by connecting carboxyl magnetic particles with amino groups of the mouse anti-TK 1 monoclonal antibody, and the specific synthesis steps of the compound are as follows:
(1) 9.7g of 2- (N-morpholinyl) ethanesulfonic acid is weighed and dissolved in 1L of deionized water, and the pH value is regulated to 5.5 to prepare a buffer solution A;
(2) 7.9g of Tris-HCl is weighed and dissolved in 1L of deionized water, and the pH is adjusted to 7.6 to prepare a buffer solution B;
(3) 10g of Bovine Serum Albumin (BSA) was weighed and dissolved in 1L of the buffer solution B to prepare a buffer solution C;
(4) Adding 1g of carboxyl magnetic particles and 10mg of mouse anti-TK 1 monoclonal antibody into 100ml of buffer solution A, adding 0.5g of EDC and HCl, reacting the mixed solution at room temperature (20-25 ℃) for 2 hours, and placing a container on a roller machine for rolling and uniformly mixing during the reaction;
(5) Placing the reaction liquid container in the step (4) on a magnet, removing the supernatant after the magnetic beads are completely precipitated by utilizing magnetic force, adding 200ml of buffer solution B, stirring and cleaning, placing the container on the magnet, removing the supernatant after the magnetic beads are completely precipitated, and repeating the step for 4 times;
(6) Adding 200ml of buffer solution C into the magnetic particle container in the step (5), sealing the mixed solution at room temperature (20-25 ℃) for 16-24 hours, and placing the container on a roller machine for rolling and uniformly mixing during the reaction;
(7) And (3) placing the reaction liquid container in the step (6) on a magnet, removing supernatant after magnetic beads are completely precipitated by utilizing magnetic force, adding 200ml of buffer solution B, stirring and cleaning, placing the container on the magnet, removing supernatant after the magnetic beads are completely precipitated, repeating the step for 4 times, finally adding 100ml of buffer solution C to obtain magnetic particles coated with the mouse anti-TK 1 monoclonal antibody, and storing at 4 ℃ for later use.
Example 3
Preparation of alkaline phosphatase-labeled goat anti-TK 1 polyclonal antibody enzyme conjugate
The sheep anti-TK 1 polyclonal antibody enzyme conjugate marked by alkaline phosphatase is formed by connecting alkaline phosphatase with TK1 antibody, and the specific synthesis steps of the compound are as follows:
(1) 2.9g of sodium bicarbonate and 1.5g of sodium carbonate are weighed and dissolved in 1L of deionized water, and the pH is adjusted to 9.5 to prepare a buffer solution A;
(2) Dissolving 100mg of alkaline phosphatase in 5ml of deionized water, uniformly mixing, adding 100mg of sodium periodate into the solution, vibrating and uniformly mixing until the sodium periodate is completely dissolved, and activating the solution at 2-8 ℃ for 30min;
(3) Adding 0.36ml of ethylene glycol into the alkaline phosphatase activating solution in the step (2), uniformly mixing, and standing at 2-8 ℃ for 2 hours to terminate the activation reaction;
(4) Adding 100mg of goat anti-TK 1 polyclonal antibody into the alkaline phosphatase reaction solution in the step (3), uniformly mixing, and standing at 2-8 ℃ for 20min for coupling reaction;
(5) Adding 10.4mg of sodium borohydride into the reaction solution of the alkaline phosphatase and the goat anti-TK 1 polyclonal antibody in the step (4), uniformly mixing, and standing at 2-8 ℃ for 2 hours to terminate the coupling reaction;
(6) Transferring the reaction solution of the sheep anti-TK 1 polyclonal antibody combined with alkaline phosphatase obtained in the step (5) to a dialysis bag, soaking the dialysis bag in a buffer solution A, and standing at 2-8 ℃ for 4 hours to remove small molecular impurities, thereby obtaining a washed alkaline phosphatase-labeled sheep anti-TK 1 polyclonal antibody enzyme conjugate;
(7) 5ml of glycerol is added to the washed alkaline phosphatase-labeled goat anti-TK 1 polyclonal antibody enzyme conjugate solution obtained in the step (6), and the mixture is stored at-20 ℃ for later use.
Example 4
The invention relates to preparation of a kit for measuring the TK1 content of a human body
Preparation of reagent 1: taking 995g of water, then sequentially adding 2.4g of sodium dihydrogen phosphate, 11.4g of disodium hydrogen phosphate, 9.0g of NaCl, 10g of Tween 20 and 0.2g of Proclin 300, stirring until the materials are completely dissolved, and adjusting the pH to 7.4; the mixture was filtered through a 0.22 μm filter to prepare 1L of reagent 1 dilution. Then, 0.2g of magnetic particles coated with a polyclonal antibody against TK1 (prepared in example 1) was added thereto, and 1L of reagent 1 was prepared by mixing the mixture.
Preparation of reagent 2: 995g of water is taken, then 2.4g of sodium dihydrogen phosphate, 11.4g of disodium hydrogen phosphate, 9.0g of NaCl, 21.6g of polyethylene glycol octyl phenyl ether, 50g of Bovine Serum Albumin (BSA) and 0.2g of Proclin 300 are sequentially added, and each time the materials are added, the materials are required to be stirred until the materials are completely dissolved, and the pH value is regulated to 7.6; the mixture was filtered through a 0.22 μm filter to prepare 1L of reagent 2 dilution. Then, 0.2mg of goat anti-TK 1 polyclonal antibody (prepared in example 3) was added thereto, and 1L of reagent 2 was prepared after mixing.
Preparation of a calibrator: taking 1L of water, then sequentially adding a buffer, sodium chloride, ethylenediamine tetraacetic acid (EDTA), sterile infant serum, thymidine kinase 1 and sodium azide, stirring until the materials are completely dissolved each time, adjusting the pH value to 7.0-8.0, preferably 7.4, and then passing through a 0.22 mu m filter membrane to obtain a calibrator solution. The calibrator was assigned using the commercially available Hua Ruitong well kit.
The composition kit comprises: the prepared reagent 1 and reagent 2 are respectively filled into bottles according to the volume ratio of 1:5 to form a reagent kit, and the packaging specification of the reagent kit is as follows: r1 (3 ml/bottle), R2 (15 ml/bottle).
Example 5
The invention relates to preparation of a kit for measuring the TK1 content of a human body
Preparation of reagent 1: 995g of water, 8g of Tris, 9.0g of NaCl, 15g of Tween 80, 10g of ethylene oxide-propylene oxide block copolymer and 0.2g of Proclin 300 are taken, and each time the materials are added, stirring is required until the materials are completely dissolved, and the pH is regulated to 7.6; the mixture was filtered through a 0.22 μm filter to prepare 1L of reagent 1 dilution. Then, 0.65g of a mouse anti-TK 1 monoclonal antibody was added to coat the carboxyl magnetic particles (prepared in example 2), and 1L of reagent 1 was prepared after mixing.
Preparation of reagent 2: 995g of water is taken, then 2.4g of sodium dihydrogen phosphate, 11.4g of disodium hydrogen phosphate, 9.0g of NaCl, 15g of polyethylene glycol octyl phenyl ether, 15g of Tween 20, 50g of Bovine Serum Albumin (BSA), 20g of sodium caseinate and 0.2g of Proclin 300 are sequentially added, and each time the materials are added, the materials are required to be stirred until being completely dissolved, and the pH value is regulated to 7.6; the mixture was filtered through a 0.22 μm filter to prepare 1L of reagent 2 dilution. Then, 0.2mg of goat anti-TK 1 polyclonal antibody (prepared in example 3) was added thereto, and 1L of reagent 2 was prepared after mixing.
Preparation of a calibrator: taking 1L of water, then sequentially adding a buffer, sodium chloride, ethylenediamine tetraacetic acid (EDTA), sterile infant serum, thymidine kinase 1 and sodium azide, stirring until the materials are completely dissolved each time, adjusting the pH value to 7.0-8.0, preferably 7.4, and then passing through a 0.22 mu m filter membrane to obtain a calibrator solution. The calibrator was assigned using the commercially available Hua Ruitong well kit.
The composition kit comprises: the prepared reagent 1 and reagent 2 are respectively filled into bottles according to the volume ratio of 1:5 to form a reagent kit, and the packaging specification of the reagent kit is as follows: r1 (3 ml/bottle), R2 (15 ml/bottle).
Example 6
Correlation analysis of TK1 assay kit of the invention (prepared in example 4)
Kit for determination of TK1 was prepared as in example 4, and 6 calibrator and 50 serum samples were compared to clinical detection results of the TK1 detection kit for darifenacin.
The experimental method comprises the following steps: the test instrument automatically draws 1 30ul of reagent, 2 150ul of reagent, 50ul of sample is added into the reaction tube, after incubation for 20min at 37 ℃, alkaline phosphatase substrate is washed and added, and then the instrument automatically detects to obtain the Relative Luminescence Unit (RLU) of the reaction liquid in the reaction tube.
And (3) making a calibration curve according to the concentrations of the RLU and the calibrator, and calculating the concentration of TK1 in the sample according to the curve and the luminous RLU obtained after sample detection.
Linear range: 0.2-50pmol/ml
Test instrument: the Lumiart-II-1 chemiluminescent analyzer.
After the reagent of this example and Hua Ruitong well reagent were calibrated separately, the reagent calibration curve of this example is shown in fig. 1, 50 clinical samples of different concentrations were tested, and the test results are shown in table 1:
TABLE 1
According to the above measurement results, the reagent has good clinical relevance with the Huaruitong TK1 measurement kit, and no obvious difference exists.
Example 7
Correlation analysis of TK1 assay kit of the invention (prepared in example 5)
Kit for determination of TK1 was prepared as in example 5, and 6 calibrator and 50 serum samples were compared to clinical detection results of the TK1 detection kit for darifenacin.
The experimental method comprises the following steps: the test instrument automatically draws 1 40ul of reagent, 2 150ul of reagent, 50ul of sample is added into the reaction tube, after incubation for 20min at 37 ℃, alkaline phosphatase substrate is washed and added, and then the instrument automatically detects to obtain the Relative Luminescence Unit (RLU) of the reaction liquid in the reaction tube.
And (3) making a calibration curve according to the concentrations of the RLU and the calibrator, and calculating the concentration of TK1 in the sample according to the curve and the luminous RLU obtained after sample detection.
Linear range: 0.2-50pmol/ml
Test instrument: the Lumiart-II-1 chemiluminescent analyzer.
After the reagent of this example and Hua Ruitong well reagent were calibrated separately, the calibration curve of the reagent of this example is shown in fig. 3, 50 clinical samples of different concentrations were tested, and the test results are shown in table 2:
TABLE 2
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It should be noted that the foregoing embodiments of the present invention are merely examples, and are not intended to limit the scope of the present invention, and all the embodiments using the content of the present invention and the attached drawings are included in the scope of the present invention.
Claims (7)
1. A thymidine kinase 1 magnetic particle chemiluminescence assay kit, wherein the kit comprises a reagent A, a reagent B and a thymidine kinase 1 (TK 1) calibrator, wherein the reagent A is a magnetic particle suspension coated with an anti-thymidine kinase 1 (TK 1) antibody, and the reagent B is an alkaline phosphatase-labeled anti-thymidine kinase 1 (TK 1) antibody enzyme conjugate reagent;
the reagent A is prepared from 0.05-0.2mol/L buffer solution, 0.1-0.5% preservative, 1-10% surfactant and 0.01-0.08% magnetic particles coated with TK1 antibody; the magnetic particles coated with the TK1 antibody are a compound formed by selecting magnetic particles with different particle diameters and surface active functional groups and the TK1 antibody, the surface active functional groups of the magnetic particles are selected from p-toluenesulfonyl or carboxyl, and the particle diameter of the magnetic particles is 0.1-5 mu m;
the reagent B is prepared from 0.05-0.2mol/L buffer solution, 0.1-0.5% preservative, 1-10% surfactant, 1-10% protection protein and 0.1-1.0 mug/ml alkaline phosphatase-marked TK1 antibody enzyme conjugate, wherein the protection protein is selected from sodium caseinate or bovine serum albumin; the alkaline phosphatase-labeled TK1 antibody enzyme conjugate is a complex formed by alkaline phosphatase and TK1 antibody.
2. The kit for chemiluminescent assay of magnetic particles of thymidine kinase 1 of claim 1 wherein the magnetic particles are Fe 2 O 3 Or Fe (Fe) 3 O 4 The composite of the magnetic nano particles and the organic polymer material contains one or more active functional groups on the surface of the magnetic particles.
3. A thymidine kinase 1 magnetic particle chemiluminescent assay kit according to claim 1 or 2 wherein, in reagent a:
the buffer solution is one or more selected from phosphate buffer solution, tris buffer solution, 3- (N-morpholino) propane sulfonic acid buffer solution, 2- (N-morpholino) ethane sulfonic acid buffer solution, borate buffer solution, glycine buffer solution or hydroxyethyl piperazine ethane sulfuric acid buffer solution; the pH value of the buffer solution is 6-9;
the preservative is selected from one or more of sodium azide, N-methyl-isothiazolone, N '-methylene-bis [ N' - (3-hydroxymethyl-2, 5-dione-4-imidazolidinyl) ] urea or ProClin 300;
the surfactant is selected from Tween 20, tween 80, ethylene oxide-propylene oxide block copolymer or polyethylene glycol octyl phenyl ether.
4. The thymidine kinase 1 magnetic particle chemiluminescent assay kit of claim 1 wherein, in reagent B:
the buffer solution is one or more selected from phosphate buffer solution, tris buffer solution, 3- (N-morpholino) propane sulfonic acid buffer solution, 2- (N-morpholino) ethane sulfonic acid buffer solution, borate buffer solution, glycine buffer solution or hydroxyethyl piperazine ethane sulfuric acid buffer solution; the pH value of the buffer solution is 6-9;
the preservative is selected from one or more of sodium azide, N-methyl-isothiazolone, N '-methylene-bis [ N' - (3-hydroxymethyl-2, 5-dione-4-imidazolidinyl) ] urea or ProClin 300;
the surfactant is selected from Tween 20, tween 80, sodium dodecyl sulfonate or polyethylene glycol octyl phenyl ether.
5. A thymidine kinase 1 magnetic particle chemiluminescent assay kit according to claim 1 wherein the TK1 antibodies in reagent a and reagent B are monoclonal antibodies having two different binding sites; the two TK1 antibodies are selected from rabbit anti-polyclonal antibodies, sheep anti-polyclonal antibodies or mouse anti-monoclonal antibodies.
6. The thymidine kinase 1 magnetic particle chemiluminescent assay kit of claim 1 wherein the volume ratio of reagent a to reagent B is 1:5.
7. A method of preparing a thymidine kinase 1 magnetic particle chemiluminescent assay kit of claim 1 comprising the steps of:
(1) Preparation of reagent A:
sequentially adding buffer salt, surfactant and preservative into water, stirring until the materials are completely dissolved each time, adjusting the pH value to 6.0-9.0, then passing through a 0.22 mu m filter membrane to obtain a reagent A diluent, and then adding magnetic particles coated with TK1 antibody to obtain a reagent A;
(2) Preparation of reagent B:
sequentially adding buffer salt, surfactant, protective protein and preservative into water, stirring until the materials are completely dissolved each time, adjusting the pH value to 6.0-9.0, then passing through a 0.22 mu m filter membrane to obtain a reagent B diluent, and then adding alkaline phosphatase-labeled TK1 antibody enzyme conjugate to obtain a reagent B;
(3) The composition kit comprises: the prepared reagent A and reagent B are mixed according to the volume ratio R1: r2=1:5 split into vials to make up the kit.
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