CN112730839A - Kit for measuring content of cytokeratin 19 fragments by magnetic particle chemiluminescence method - Google Patents

Kit for measuring content of cytokeratin 19 fragments by magnetic particle chemiluminescence method Download PDF

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CN112730839A
CN112730839A CN202110072767.5A CN202110072767A CN112730839A CN 112730839 A CN112730839 A CN 112730839A CN 202110072767 A CN202110072767 A CN 202110072767A CN 112730839 A CN112730839 A CN 112730839A
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cytokeratin
solution
antibody
fragment
alkaline phosphatase
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CN112730839B (en
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柳建敏
郭俊美
汪云峰
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Ningbo Haiyi Biotechnology Co ltd
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    • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
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Abstract

The invention provides a kit for measuring the content of a cytokeratin 19 fragment by a magnetic particle chemiluminescence method, which comprises a magnetic particle reagent marked by a cytokeratin 19 fragment antibody and an alkaline phosphatase reagent marked by a cytokeratin 19 fragment antibody; in the magnetic particle reagent marked by the cytokeratin 19 fragment antibody, the concentration of the magnetic particles marked by the cytokeratin 19 fragment antibody is 0.05-1 mg/ml; the concentration of the cytokeratin 19 fragment antibody-labeled alkaline phosphatase in the cytokeratin 19 fragment antibody-labeled alkaline phosphatase reagent is 0.05-2. mu.g/ml. According to the invention, the mode of directly marking the magnetic particles by the antibody is adopted, so that the interference of endogenous biotin to the test result is effectively avoided, and the analysis sensitivity and precision of the kit are improved.

Description

Kit for measuring content of cytokeratin 19 fragments by magnetic particle chemiluminescence method
Technical Field
The invention belongs to the technical field of in-vitro detection, and relates to a kit for determining the content of cytokeratin 19 fragments by a magnetic particle chemiluminescence method.
Background
Cytokeratin 19 fragment (CYFRA21-1) is a tumor marker mainly used for auxiliary diagnosis of lung cancer. Especially has higher diagnosis value for non-small cell lung cancer, and the positive detection rate for various non-small cell lung cancers can reach 70 to 85 percent. When the concentration of CYFRA21-1 in the serum of a patient exceeds 30ng/mL and the imaging test shows that unclear annular shadows exist in the lung, the possibility of diagnosing primary bronchopulmonary carcinoma is high.
The concentration level of CYFRA21-1 in serum is high and low, and is generally in positive correlation with the treatment effect and clinical stage of tumor, the successful treatment mark is that the concentration of CYFRA21-1 in serum is rapidly reduced, otherwise, the disease relapse or the focus is not completely cleared, so the CYFRA21-1 can be used as one of effective indexes for evaluating the operation treatment effect and monitoring the prognostic relapse of the lung cancer patient.
At present, the detection method of the non-small cell lung cancer related antigen CYFRA21-1 mainly takes an electrochemiluminescence method and a chemiluminescence method immunoassay method as main methods, 2 methods have the advantages of high sensitivity, high specificity, high stability and the like, but over 95 percent of market share is occupied by foreign brands, and the cost of the kit is high. Therefore, the development of the domestic CYFRA21-1 chemiluminescence immunoassay kit with independent intellectual property rights, stable evaluation and use performance and excellent performance is an effective way for breaking the long-term monopoly situation of foreign brands, and can greatly reduce the cost of clinical test CYFRA 21-and reduce the medical expense.
At present, a manufacturer of an electrochemical luminescence method on the market is mainly Roche, a biotin-streptavidin system is mainly used, two strains of antibodies respectively mark biotin and terpyridyl ruthenium, and magnetic particles are coated by streptavidin; the direct light-emitting manufacturer mainly adopts Yapei, adopts a reaction mode that an antibody directly coats magnetic beads, and the other antibody marks acridine ester; the reagent Mairui made in China adopts a mode that an antibody directly marks magnetic particles, and the other antibody marks alkaline phosphatase; the domestic reagent WantakayRuri adopts a direct luminescence method, and two antibodies respectively coat magnetic beads and acridinium ester. Other manufacturers mostly adopt plate-type luminescence, and the labeling substance mostly adopts a mode of labeling horseradish peroxidase (HRP) by using an antibody. Tubular luminescence is more simple and convenient than plate-type luminescence, and the reaction time is shorter, and is automatic higher, can do single test, and is obvious to the cost saving advantage. Therefore, the tube-type luminescence has the advantages of short reaction time, high sensitivity, good precision, wider linear range and the like, and is more applied to the development of luminescent reagents. In the manufacturers using magnetic particles in the market at present, the biotin system cannot eliminate the interference caused by human endogenous biotin, so that the abnormal condition of the test result often occurs; the anti-FITC antibody is mainly derived from goat antibody or mouse antibody, the titer is low after labeling, and the sensitivity of the reagent is greatly influenced. The mode of directly wrapping magnetic beads by using the antibody is adopted, and due to the use of different methods, the state of the antibody magnetic particle connector cannot be well controlled, and the conditions that magnetic particle agglomeration is difficult to disperse and the test result is influenced are frequently generated. Therefore, the problems of large batch difference, deviation of stability, further improvement of specificity and the like often occur. And the use rate of the antibody is generally low, and the production cost is high.
Disclosure of Invention
Aiming at the defects existing in CYFRA21-1 detection in the prior art, the invention provides the kit for determining the content of the cytokeratin 19 fragment by the magnetic particle chemiluminescence method, and the method of directly marking the magnetic particles by using the antibody effectively avoids the interference of endogenous biotin to the test result and improves the analysis sensitivity and precision of the kit.
The invention provides a kit for measuring the content of a cytokeratin 19 fragment by a magnetic particle chemiluminescence method, which comprises a magnetic particle reagent marked by a cytokeratin 19 fragment antibody and an alkaline phosphatase reagent marked by a cytokeratin 19 fragment antibody; in the magnetic particle reagent marked by the cytokeratin 19 fragment antibody, the concentration of the magnetic particles marked by the cytokeratin 19 fragment antibody is 0.05-1 mg/ml; the concentration of the cytokeratin 19 fragment antibody-labeled alkaline phosphatase in the cytokeratin 19 fragment antibody-labeled alkaline phosphatase reagent is 0.05-2. mu.g/ml.
The cytokeratin 19 fragment antibody is preferably a cytokeratin 19 fragment monoclonal antibody.
Preferably, the preparation method of the magnetic particle reagent marked by the cytokeratin 19 fragment antibody comprises the following steps: adding the cytokeratin 19 fragment antibody after the magnetic particles are resuspended, adding a reaction catalyst, carrying out suspension reaction for 4-24h at 24-42 ℃, then adding a sealant, carrying out suspension reaction for 6-24 h at 24-42 ℃, carrying out sealing, washing, and adding a magnetic particle preservation solution for resuspension to obtain the magnetic particle reagent marked by the cytokeratin 19 fragment antibody.
The magnetic particle is colloidal composite material formed by combining magnetic nano particles with organic molecules or inorganic molecules, which can be uniformly dispersed in a certain base solution and has high stability, and the surface of the magnetic particle contains one or more active functional groups such as tosyl, amino, carboxyl, hydroxyl, ethylene oxide and the like.
Preferably, the magnetic particles used in the present invention are Fe2O3Or Fe3O4The magnetic particle surface contains tosyl, and the particle diameter of the magnetic particle is 0.1-3 μm.
In the preparation method of the magnetic particle reagent marked by the cytokeratin 19 fragment antibody, the temperature range of the reaction between the magnetic particles and the antibody is wider, the magnetic particles can be marked at higher temperature, and the combination efficiency of the antibody and the magnetic particles can be effectively improved at high temperature. The use of the reaction catalyst can have an effect of promoting the accelerated reaction of the antibody with the groups on the magnetic fine particles. In the labeling process, the non-binding sites on the magnetic particles can be filled by using a sealant, so that the effect of sealing is achieved, and in the reaction, the object to be detected can not directly react with the magnetic particles to generate non-specific adsorption.
The preparation method of the magnetic particle reagent marked by the cytokeratin 19 fragment antibody specifically comprises the following steps: the magnetic particles are re-suspended by buffer solution with pH of 5.0-9.5 and 0.05-0.3mol/L, the buffer solution can be one or more of Hepes, MES, boric acid and phosphate buffer solution, and the concentration of the magnetic particles after re-suspension is 5-10 mg/ml; adding the cytokeratin 19 fragment antibody so that the mass ratio of the cytokeratin 19 fragment antibody to the magnetic particles is (0.1-2): 100, uniformly mixing, adding a reaction catalyst which can be 0.5-4mol/L ammonium sulfate solution, wherein the addition volume of the reaction catalyst is 0.5-2 times of the sum of the magnetic particles and the cytokeratin 19 fragment antibody, and suspending and reacting for 4-24h at 24-42 ℃; then adding a sealant, adding 0.05-0.25ml of sealant into every 10mg of magnetic particles, and carrying out suspension reaction for 6-24 hours at the temperature of 24-42 ℃; then washing with buffer solution, wherein the buffer solution can be Tris buffer solution and boric acid solution, one or more of 0.05-2% Tween20, SDS and Tween80 is added, the volume of the buffer solution is 0.5-2 times of the total volume, and washing is carried out for 2-5 times; after cleaning, adding a magnetic particle preservation solution for resuspension to obtain a magnetic particle reagent marked by the cytokeratin 19 fragment antibody for later use, wherein the concentration of the magnetic particle marked by the cytokeratin 19 fragment antibody is 5-10 mg/ml; then, the magnetic particle reagent marked by the cytokeratin 19 fragment antibody is diluted by the magnetic particle preservation solution, so that the concentration of the magnetic particles marked by the cytokeratin 19 fragment antibody is 0.05-1 mg/ml.
Preferably, in the method for preparing the magnetic microparticle reagent labeled with the cytokeratin 19 fragment antibody, the blocking agent used is 0.5-3 wt% bovine serum albumin solution, 0.5-3 wt% casein solution, or BlockmasterTM CE210、BlockmasterTM CE510、BlockmasterTM DB1130、BlockmasterTMOne or more of PA 1080.
More preferably, in the method for preparing the magnetic microparticle reagent labeled with the antibody against the cytokeratin 19 fragment, the blocking agent used is a casein solution of 0.5 to 3 wt% or a BlockmasterTM CE210、BlockmasterTMCE510 was measured at a volume ratio of 1: (0.8-1.5): (0.8-1.5) to form a mixed solution.
The molecular weight of PEG in CE210 is 2000, the molecular weight of PEG in CE510 is 5000, and because the sites on the surface of the magnetic particles are different in size, different molecular weight blocking agents are mixed for use, so that all the exposed sites on the surface of the magnetic particles can be blocked more effectively. The casein is mixed with the CE210 and the CE510 for use, so that the dispersion stability of the magnetic particles can be improved, and the sensitivity, the precision and the accuracy of the kit can be improved.
Preferably, the magnetic particle preservation solution has a pH of 6 to 9 and comprises: 0.01-0.2mol/L Tris buffer solution, 0.1-0.5mol of sodium chloride, 0.1-10 wt% of one or more of bovine serum albumin, casein and gamma globulin (cattle), 1.0-5 wt% of Triton X-405, 0.01-5 wt% of surfactant S9, 0.5-5 wt% of one or two of surfactant RPE1740 and surfactant RPE2520, 0.5-10 wt% of one or more of PEG2000, PEG4000, PEG6000, PEG8000, PEG10000 and PEG20000, 0.5-10 wt% of dextran, 0.5-10 wt% of gelatin, 0.1-5.0 wt% of hexadecyl trimethyl ammonium bromide and/or lauryl trimethyl ammonium bromide, and 0.05-5.0 wt% of Proclin series preservative and/or sodium azide. The Proclin series preservative comprises one or more of Proclin 150, Proclin 200, Proclin300 and Proclin 5000.
Triton X-405, surfactant S9, surfactant RPE1740 and surfactant RPE2520 in the magnetic particle preservation solution can effectively reduce the aggregation phenomenon among magnetic particles due to charge effect, and the surfactant has the function of renaturation protein, can reduce the non-specific binding of antibodies and improve the specific recognition capability. One or more of PEG 2000-20000, dextran and gelatin (derived from one or more of pig or fish skin) in the magnetic particle preservation solution can significantly improve the specificity and precision of the kit, and the luminescent signal and precision are greatly improved by adding the substances into the magnetic particles with the same working concentration. The hexadecyl trimethyl ammonium bromide or lauryl trimethyl ammonium bromide in the magnetic particle preservation solution can effectively reduce bubbles generated in a cleaning link by magnetic particles, the bubbles can be quickly eliminated within 5-10 seconds, the conditions that the cleaning link is incomplete, the magnetic particles are lost and the reaction tube wall is polluted due to the bubbles can be effectively avoided, and the effect of improving the reliability and the precision of the result is remarkable.
Preferably, the method for preparing the cytokeratin 19 fragment antibody-labeled alkaline phosphatase reagent comprises the following steps:
s1, activated antibody: passing cytokeratin 19 fragment antibody through desalting column, eluting with 0.01-0.05mol/L Hepes buffer solution with pH of 7.0-7.5, and concentrating to 2-4 mg/ml; adding Dithiothreitol (DTT) or tris (2-carbonylethyl) phosphate (TCEP), and reacting at 20-30 ℃ for 10-30 min; adding 0.5-2mol/L glycine solution with pH of 7.0-7.4, continuing to react for 5-10min, passing through desalting column, eluting with 0.01-0.05mol/L Hepes buffer solution with pH of 8.0-9.0, and concentrating to 2-4mg/ml to obtain activated cytokeratin 19 fragment antibody solution;
s2, activated alkaline phosphatase: passing alkaline phosphatase through desalting column, eluting with 0.01-0.05mol/L Hepes buffer solution with pH of 7.0-7.5, and concentrating to 2-4 mg/ml; dissolving LC-SMCC in an organic solvent to obtain 5-8mg/ml LC-SMCC solution, adding the LC-SMCC solution into eluent, adding 2.5-7.5 mu L of LC-SMCC solution into the eluent according to the mg of alkaline phosphatase, and reacting for 10-30min at 20-30 ℃; adding glycine solution with pH of 7.0-7.4 and 0.5-2mol/L, adding 2.5-7.5 mu L of glycine solution per milligram of alkaline phosphatase, continuing to react for 10-20min, passing through a desalting column, eluting with Hepes buffer solution with pH of 8.0-9.0 and 0.01-0.05mol/L, and concentrating to 2-4mg/ml to obtain activated alkaline phosphatase solution;
s3, mixing the cytokeratin 19 fragment antibody and alkaline phosphatase in a mass ratio of 1: (0.5-2) mixing the activated cytokeratin 19 fragment antibody solution and the activated alkaline phosphatase solution, adding 0.1-0.5mol/L magnesium chloride solution, adding 1-5 μ L magnesium chloride solution per ml reaction volume, and reacting at 2-8 ℃ for 8-20 h;
s4, purifying the connection substance, for example, passing the connection substance through a superdex200 molecular sieve column, collecting according to the peak condition of the protein, and diluting with a buffer solution with pH7.0-7.5 and 0.01-0.05mol/L Hepes for later use after purification.
The desalting column used in step S1 and step S2 is exemplified by PD-10 desalting column, which is passed through a desalting column to remove salt, and the buffer is replaced.
The DTT or TCEP added in step S1 is in the form of a solution, the DTT solution or TCEP solution is dissolved in a Hepes buffer solution of pH8.0-9.00.01-0.05mol/L to a concentration of 10-50mmol/L, and a volume of 0.5-2.5. mu.l per mg of antibody mass is added. The glycine solution is added in the same volume as the DTT solution or TCEP solution in steps S1 and S2.
The alkaline phosphatase used in the present invention is preferably one or more of the following alkaline phosphatase, heat-sensitive alkaline phosphatase, calf intestinal alkaline phosphatase.
Preferably, the kit further comprises an antibody buffer solution, wherein the pH of the antibody buffer solution is 7.0-9.0, and the antibody buffer solution comprises one or more of bovine serum albumin, casein, trehalose and a blocking agent.
Preferably, the kit further comprises a cytokeratin 19 fragment series calibrator, wherein the cytokeratin 19 fragment series calibrator is obtained by diluting cytokeratin 19 fragments to a plurality of series concentrations by a calibrator buffer.
Preferably, the calibrator buffer comprises 1-10g/L HEPES, 20-60g/L bovine serum albumin, 5-10g/L sodium chloride, 0.05-0.2ml of 0.5-2mol/L magnesium chloride solution, 0.05-0.2ml of 0.1-0.5mol/L zinc chloride solution, 1-4g/L preservative and water, and the pH of the adjusting buffer is 7.0-8.0.
Preferably, the kit also comprises a cytokeratin 19 fragment quality control product, wherein the cytokeratin 19 fragment quality control product is obtained by diluting the cytokeratin 19 fragment with a quality control product diluent to obtain a plurality of concentrations. The quality control product diluent comprises: 1-10g/L HEPES, 20-60g/L bovine serum albumin, 5-10g/L sodium chloride, 0.05-0.2ml of 0.5-2mol/L magnesium chloride solution, 0.05-0.2ml of 0.1-0.5mol/L zinc chloride solution, 1-4g/L preservative and water, and the pH value of the buffer solution is adjusted to 7.0-8.0.
The detection method for detecting the content of the cytokeratin 19 fragment by using the kit provided by the invention comprises the following steps:
mixing a sample to be detected, a magnetic particle reagent marked by a cytokeratin 19 fragment antibody, an alkaline phosphatase reagent marked by a cytokeratin 19 fragment antibody and an antibody buffer solution, wherein the volume ratio of the sample to be detected, the magnetic particle reagent marked by the cytokeratin 19 fragment antibody, the alkaline phosphatase reagent marked by the cytokeratin 19 fragment antibody to the antibody buffer solution is 1: (2-5): (2-5): (2-5), reacting at 35-42 ℃ for 5-15min to form a sandwich compound magnetic suspension;
placing the sandwich compound magnetic suspension in a magnetic field, settling for 2-4min in the magnetic field, removing supernatant, and repeatedly cleaning the magnetic antigen-antibody compound with cleaning solution;
adding chemical fermentation substrate liquid into the washed magnetic antigen-antibody complex, suspending for 2-5s, and detecting by using a luminescence detector.
Compared with the prior art, the invention has the following beneficial effects:
(1) according to the cytokeratin 19 fragment magnetic particle chemiluminescence detection kit, the mode that antibodies directly wrap magnetic particles is adopted, compared with the mode that other manufacturers adopt biotin systems, the interference of endogenous biotin to detection results can be effectively avoided, the direct wrapping mode is higher than that of labeled biotin and the like, the titer of a connector is higher, and the sensitivity is obviously improved;
(2) according to the invention, the antibody is adopted to mark the tosyl magnetic particles, and a catalyst, a sealant and a magnetic particle preservation solution which are suitable for the system are obtained by groping, so that the problems of large antibody usage amount, low linker activity, high cost and the like of the carboxyl magnetic beads adopted in the market for marking the antibody are effectively solved, the utilization rate of the antibody is effectively improved, the analysis sensitivity of the kit is improved, and the reagent cost is reduced;
(3) the invention adopts 0.5-3 wt% casein solution and BlockmasterTM CE210、BlockmasterTMCE510 was measured at a volume ratio of 1: (0.8-1.5): (0.8-1.5) as a sealant, and adopting different molecular weight sealants to mix, the mixed solution can more effectively seal all exposed sites on the surface of the magnetic particles, improve the dispersion stability of the magnetic particles, and is beneficial to improving the sensitivity, precision and accuracy of the kit;
(4) the invention adopts special magnetic particle preserving fluid, which can make the marked magnetic particles uniformly distributed without obvious agglomeration, and make the difference between batches of the reagent smaller, the detection result more stable and the specificity stronger;
(5) according to the technology for labeling alkaline phosphatase by using the antibody, the DTT or TCEP is adopted, the disulfide bonds between heavy chains of the antibody can be directionally opened, the targeted labeling is realized, the labeled antibody keeps better specificity, the linear range and the sensitivity are better improved, and the difference between labeled batches can be effectively reduced by the directional labeling.
Drawings
FIG. 1 is a microscopic view of a magnetic microparticle reagent in example 1 of the present invention;
FIG. 2 is a microscopic view of a magnetic microparticle reagent of comparative example 1 of the present invention;
FIG. 3 is a microscopic image of the magnetic microparticle reagent of comparative example 2 of the present invention.
Detailed Description
The technical solution of the present invention will be further described and explained with reference to the following embodiments and the accompanying drawings. The raw materials used in the examples of the present invention are those commonly used in the art, and the methods used in the examples are those conventional in the art, unless otherwise specified.
Magnetic particles: MS160/TOSYl magnetic beads manufactured by JSR Life Science corporation
CYFRA21-1 antibody: medix corporation 1604
BlockmasterTMThe CE 210: CE210, manufactured by JSR Life Science corporation
BlockmasterTMCE 510: CE510 manufactured by JSR Life Science corporation
And (3) glucan: purchased from Sigma; 31394
Gelatin: from Sigma G7041
PD-10 desalting column: GE Cat No. 17-0851-01
Alkaline phosphatase (alkaline phosphatase): purchased from BBI Solutions, cat #: ALPI 2G;
LC-SMCC：Thermo 22362
superdex200 molecular sieve column: GE 28-9893-35.
Example 1
The kit for determining the content of the cytokeratin 19 fragment comprises a magnetic particle reagent marked by a cytokeratin 19 fragment antibody, an alkaline phosphatase reagent marked by a cytokeratin 19 fragment antibody, an antibody buffer solution, a series of cytokeratin 19 fragment calibrators and a quality control product.
The preparation process of the magnetic particle reagent marked by the cytokeratin 19 fragment antibody comprises the following steps:
resuspending the magnetic particles with 0.05mol/L, pH 8.0.0 boric acid buffer solution, wherein the concentration of the resuspended magnetic particles is 10 mg/ml; adding the cytokeratin 19 fragment antibody so that the mass ratio of the cytokeratin 19 fragment antibody to the magnetic particles is 0.5: 100, uniformly mixing, adding 1mol/L ammonium sulfate solution, wherein the adding volume of the ammonium sulfate solution is 2 times of the sum of the magnetic particles and the cytokeratin 19 fragment antibody, and then suspending and reacting for 12 hours at 37 ℃; then adding 0.2ml of sealant into every 10mg of magnetic particles, and suspending and reacting for 12 hours at 37 ℃; washing with 0.2% Tween20 in pH7.4 boric acid buffer solution, adding 1 time of total reaction volume, and washing for 3 times; after cleaning, adding a magnetic particle preservation solution for resuspension to obtain a magnetic particle reagent marked by 10mg/ml cytokeratin 19 fragment antibody for later use; then, the magnetic particle reagent marked by the cytokeratin 19 fragment antibody is diluted by the magnetic particle preservation solution to the concentration of 0.5 mg/ml.
Wherein the blocking agent is 0.2 wt% casein water solution, BlockmasterTM CE210、BlockmasterTMCE510 was measured at a volume ratio of 1: 1: 1, and (2) forming a mixed solution;
the pH of the magnetic particle preservation solution is 8.0, and the magnetic particle preservation solution comprises 0.2mol/L sodium chloride, 0.2 wt% of bovine serum albumin, 1.0 wt% of Triton X-405, 0.05 wt% of surfactant S9, 0.8 wt% of surfactant RPE1740, 1.0 wt% of PEG2000, 0.5 wt% of PEG10000, 1 wt% of dextran, 1 wt% of gelatin, 0.5 wt% of hexadecyl trimethyl ammonium bromide and 0.05mol/L Tris buffer solution of 0.1 wt% of Proclin 300.
The preparation method of the cytokeratin 19 fragment antibody labeled alkaline phosphatase reagent comprises the following steps:
s1, activated antibody: passing the cytokeratin 19 fragment antibody through a PD-10 desalting column, eluting with a Hepes buffer solution with the pH value of 7.30.01mol/L, and concentrating to 3 mg/ml; adding 2.0 μ L of 20mmol/L dithiothreitol solution (dissolved in 0.01mol/L Hepes buffer solution with pH 8.5) per mg of antibody, and reacting at 25 deg.C for 20 min; adding 2.0 microliter of pH7.3 and 1mol/L glycine solution according to the mass of each milligram of antibody, continuously reacting for 10min at 25 ℃, passing through a PD-10 desalting column, eluting by using a Hepes buffer solution with the pH of 8.50.01mol/L and concentrating to 3mg/ml to obtain an activated cytokeratin 19 fragment antibody solution;
s2, activated alkaline phosphatase: passing alkaline phosphatase through PD-10 desalting column, eluting with Hepes buffer solution (pH7.30.01mol/L), and concentrating to 3 mg/ml; dissolving LC-SMCC in DMF to obtain 6mg/ml LC-SMCC solution, adding LC-SMCC solution into eluent, adding 2.5 μ L LC-SMCC solution per mg alkaline phosphatase, and reacting at 25 deg.C for 20 min; adding glycine solution with pH of 7.3 and 1mol/L, adding 2.5 μ L glycine solution per mg of alkaline phosphatase, reacting at 25 deg.C for 10min, passing through PD-10 desalting column, eluting with Hepes buffer solution with pH of 8.50.01mol/L, and concentrating to 3mg/ml to obtain activated alkaline phosphatase solution;
s3, mixing the cytokeratin 19 fragment antibody and alkaline phosphatase in a mass ratio of 1: 1, mixing an activated cytokeratin 19 fragment antibody solution and an activated alkaline phosphatase solution, adding 0.2mol/L magnesium chloride solution, adding 2 mu L magnesium chloride solution into each milliliter of reaction solution, and reacting for 18 hours at 4 ℃;
s4, passing the link through a superdex200 molecular sieve column, collecting according to the peak condition of protein, and adding a buffer solution to dilute to 0.5 mu g/ml, wherein the buffer solution is pH7.4 and 0.02mol/L Hepes.
The antibody buffer solution has a pH of 8.0 and comprises 5g/L BSA, 1g/L casein, 20g/L trehalose, 5g/L gelatin, and 1ml/L ProClin300 in 2.38g/L Hepes buffer solution.
The cytokeratin 19 fragment series calibrator is prepared by diluting cytokeratin 19 fragment with calibrator buffer to 0, 0.5, 5.0, 50, 500, 1000 ng/ml. The cytokeratin 19 fragment quality control product is prepared by diluting cytokeratin 19 fragment with quality control buffer solution to concentration of 10, 100 ng/ml. The pH value of the buffer solution of the calibrator and the quality control material is 8.0, and the buffer solution of the calibrator and the quality control material comprises 30g/L bovine serum albumin, 6g/L sodium chloride, 0.1ml of 1mol/L magnesium chloride solution, 0.1ml of 0.1mol/L zinc chloride solution and 2g/L ProClin300 of 1-10g/L HEPES buffer solution.
Example 2
Example 2 differs from example 1 in that the blocking agent of example 2 is a 0.2 wt% aqueous casein solution.
Example 3
Example 3 differs from example 1 in that the blocking agent of example 3 is a BlockmasterTM CE210。
Example 4
Example 4 differs from example 1 in that the blocking agent of example 4 is a BlockmasterTM CE510。
Comparative example 1
Comparative example 1 is different from example 1 in that the magnetic particle preservation solution of comparative example 1 has a pH of 8.0 and includes 0.2mol/L sodium chloride, 0.2 wt% bovine serum albumin, 1.0 wt% Triton X-405, 0.05 wt% surfactant S9, 0.8 wt% surfactant RPE1740, 0.5 wt% cetyltrimethylammonium bromide, 0.1 wt% Proclin300 in 0.05mol/L Tris buffer.
Comparative example 2
Comparative example 2 is different from example 1 in that the magnetic particle preservation solution of comparative example 2 has a pH of 8.0 and comprises 0.2mol/L sodium chloride, 0.2 wt% bovine serum albumin, 1.0 wt% Triton X-405, 0.05 wt% surfactant S9, 0.8 wt% surfactant RPE1740, 1.0 wt% PEG2000, 0.5 wt% PEG10000, 0.5 wt% cetyltrimethylammonium bromide, 0.1 wt% Proclin300 in 0.05mol/L Tris buffer.
Comparative example 3
Comparative example 3 is different from example 1 in that the magnetic particle preservation solution of comparative example 3 has a pH of 8.0 and includes 0.2mol/L sodium chloride, 0.2 wt% bovine serum albumin, 1.0 wt% Triton X-405, 0.05 wt% surfactant S9, 0.8 wt% surfactant RPE1740, 1 wt% dextran, 1 wt% gelatin, 0.5 wt% cetyltrimethylammonium bromide, 0.1 wt% Proclin300 in 0.05mol/L Tris buffer.
Comparative example 4
Comparative example 4 is different from example 1 in that the magnetic particle preservation solution of comparative example 4 has a pH of 8.0 and comprises 0.2mol/L sodium chloride, 0.2 wt% bovine serum albumin, 1.0 wt% Triton X-405, 0.05 wt% surfactant S9, 0.8 wt% surfactant RPE1740, 1.0 wt% PEG2000, 0.5 wt% PEG10000, 1 wt% gelatin, 0.5 wt% cetyltrimethylammonium bromide, 0.05mol/L Tris buffer of Proclin 300.
Comparative example 5
Comparative example 5 differs from example 1 in that the magnetic particle preservation solution of comparative example 5 has a pH of 8.0 and comprises 0.2mol/L sodium chloride, 0.2 wt% bovine serum albumin, 1.0 wt% Triton X-405, 1.0 wt% PEG2000, 0.5 wt% PEG10000, 1 wt% dextran, 1 wt% gelatin, 0.5 wt% cetyltrimethylammonium bromide, 0.1 wt% Proclin300 in 0.05mol/L Tris buffer.
Determination of kit performance index
1.1 detection method
Mixing 15 mu L of a sample to be detected, 50 mu L of a magnetic particle reagent marked by a cytokeratin 19 fragment antibody, 50 mu L of an alkaline phosphatase reagent marked by a cytokeratin 19 fragment antibody and 50 mu L of an antibody buffer solution, and reacting at 37 ℃ for 10min to form a sandwich compound magnetic suspension;
placing the sandwich compound magnetic suspension in a magnetic field, settling for 2min in the magnetic field, removing supernatant, and repeatedly washing the magnetic antigen-antibody compound for 3 times by using a cleaning solution;
and adding 100 mu L of chemical chromogenic substrate liquid into the washed magnetic antigen-antibody complex, suspending for 3s, and detecting by using a luminescence detector. And (4) according to the relative luminous intensity RLU of the sample to be detected, calculating the concentration of the cytokeratin 19 fragment in the sample from the standard curve.
1.2 sensitivity
The assay was performed using a zero concentration calibrator as a sample, and the assay was repeated 20 times using the kit of example 1 to obtain luminescence (RLU) values for 20 measurements, as shown in Table 1. Calculating the average value (M) and the Standard Deviation (SD) of the light-emitting values to obtain the light-emitting values corresponding to M +2SD, performing two-point regression fitting according to the concentration-RLU value result between the zero-concentration calibrator and the adjacent calibrator to obtain a linear equation, substituting the light-emitting values of M +2SD into the equation, and calculating the corresponding concentration value, namely the lowest detection limit. The sensitivity of the kit is calculated to be 0.012ng/ml, and the linear range of the kit is 0-1000 ng/ml.
TABLE 1
Figure BDA0002906470160000121
1.3 magnetic particle dispersibility
The magnetic microparticle reagents of example 1 and comparative examples 1 to 2 were observed under a microscope, and the results of the observation are shown in fig. 1 to 3, fig. 1 is a microscope image of the magnetic microparticle reagent of example 1, fig. 2 is a microscope image of the magnetic microparticle reagent of comparative example 1, and fig. 3 is a microscope image of the magnetic microparticle reagent of comparative example 2, and it can be seen from the images that the magnetic microparticles of example 1 were uniformly dispersed without agglomeration, and comparative examples 1 to 2 all had some degree of agglomeration.
1.4 precision
1.4.1 the kit of example 1 was evaluated for the precision and the difference between the batches, and serum samples of 2ng/ml and 200ng/ml were tested, respectively, and the precision was calculated, the results of which are shown in Table 2.
TABLE 2
Figure BDA0002906470160000122
Figure BDA0002906470160000131
The kit of example 1 has excellent intra-batch difference and inter-batch difference, both of which can be controlled within 2%.
1.4.2 the kits of examples 1-4 and comparative examples 1-5 were each tested for a cytokeratin 19 fragment series calibrator, and the luminescence intensity was counted and 2ng/ml serum samples were each tested 10 times, and the precision was calculated, the results are shown in Table 3.
TABLE 3
Figure BDA0002906470160000132
The magnetic particles of the kit in example 1 have excellent dispersibility, and the luminescence signal and the precision are obviously improved compared with those of examples 2-3 and comparative examples 1-5.
1.5 stability
The kits of examples 1 to 4 and comparative examples 1 to 5 were stored at 37 ℃ and tested for 3 days, 7 days and 9 days, respectively, and the stability results are shown in Table 4.
Figure BDA0002906470160000141
Figure BDA0002906470160000151
The kit of example 1 showed excellent stability, the deviation remained within 8% when stored at 37 ℃ for 9 days, and the magnetic particles of the kits of comparative examples 1 to 5 were not uniformly dispersed and easily agglomerated during storage, resulting in poor stability.
The specific embodiments described herein are merely illustrative of the spirit of the invention and do not limit the scope of the invention. Various modifications or additions may be made to the described embodiments or alternatives may be employed by those skilled in the art without departing from the spirit or ambit of the invention as defined in the appended claims.

Claims (10)

1. A kit for measuring the content of cytokeratin 19 fragments by a magnetic particle chemiluminescence method is characterized by comprising a magnetic particle reagent marked by an antibody of the cytokeratin 19 fragments and an alkaline phosphatase reagent marked by an antibody of the cytokeratin 19 fragments;
in the magnetic particle reagent marked by the cytokeratin 19 fragment antibody, the concentration of the magnetic particles marked by the cytokeratin 19 fragment antibody is 0.05-1 mg/ml; the concentration of the cytokeratin 19 fragment antibody-labeled alkaline phosphatase in the cytokeratin 19 fragment antibody-labeled alkaline phosphatase reagent is 0.05-2. mu.g/ml.
2. The kit according to claim 1, wherein the preparation method of the magnetic microparticle reagent labeled with the cytokeratin 19 fragment antibody comprises the following steps: adding the cytokeratin 19 fragment antibody after the magnetic particles are resuspended, adding a reaction catalyst, carrying out suspension reaction for 4-24h at 24-42 ℃, then adding a sealant, carrying out suspension reaction for 6-24 h at 24-42 ℃, carrying out sealing, washing, and adding a magnetic particle preservation solution for resuspension to obtain the magnetic particle reagent marked by the cytokeratin 19 fragment antibody.
3. The kit according to claim 1 or 2, characterized in that the mass ratio of the cytokeratin 19 fragment antibody to the magnetic microparticle is (0.1-2): 100.
4. the kit according to claim 2, wherein the blocking agent is a bovine serum albumin solution of 0.5-3 wt%, a casein solution of 0.5-3 wt%, a BlockmasterTM CE210、BlockmasterTM CE510、BlockmasterTM DB1130、BlockmasterTMOne or more of PA 1080.
5. The kit according to claim 2 or 4, wherein the blocking agent is a 0.5-3 wt% casein solution, a BlockmasterTM CE210、BlockmasterTMCE510 was measured at a volume ratio of 1: (0.8-1.5): (0.8-1.5) to form a mixed solution.
6. The kit according to claim 2, wherein the magnetic microparticle preservation solution has a pH of 6 to 9 and comprises: 0.01-0.2mol/L Tris buffer solution, 0.1-0.5mol of sodium chloride, 0.1-10 wt% of one or more of bovine serum albumin, casein and gamma globulin (cattle), 1.0-5 wt% of Triton X-405, 0.01-5 wt% of surfactant S9, 0.5-5 wt% of one or two of surfactant RPE1740 and surfactant RPE2520, 0.5-10 wt% of one or more of PEG2000, PEG4000, PEG6000, PEG8000, PEG10000 and PEG20000, 0.5-10 wt% of dextran, 0.5-10 wt% of gelatin, 0.1-5.0 wt% of hexadecyl trimethyl ammonium bromide and/or lauryl trimethyl ammonium bromide, and 0.05-5.0 wt% of Proclin series preservative and/or sodium azide.
7. The kit according to claim 1, wherein the cytokeratin 19 fragment antibody-labeled alkaline phosphatase reagent is prepared by a method comprising the steps of:
s1, activated antibody: passing cytokeratin 19 fragment antibody through desalting column, and concentrating eluate to 2-4 mg/ml; adding dithiothreitol or tris (2-carbonylethyl) phosphate, and reacting at 20-30 ℃ for 10-30 min; adding glycine solution with pH of 7.0-7.4 and 0.5-2mol/L, reacting for 5-10min, passing through desalting column, and concentrating eluate to 2-4mg/ml to obtain activated cytokeratin 19 fragment antibody solution;
s2, activated alkaline phosphatase: passing alkaline phosphatase through desalting column, and concentrating eluate to 2-4 mg/ml; adding LC-SMCC solution, and reacting at 20-30 deg.C for 10-30 min; adding glycine solution with pH of 7.0-7.4 and 0.5-2mol/L, reacting for 10-20min, passing through desalting column, and concentrating eluate to 2-4mg/ml to obtain activated alkaline phosphatase solution;
s3, mixing the cytokeratin 19 fragment antibody and alkaline phosphatase in a mass ratio of 1: (0.5-2) mixing the activated cytokeratin 19 fragment antibody solution and the activated alkaline phosphatase solution, adding 0.1-0.5mol/L magnesium chloride solution, and reacting at 2-8 ℃ for 8-20 h;
s4, purifying the connection object, and adding buffer solution for dilution.
8. The kit of claim 1, further comprising an antibody buffer solution, wherein the antibody buffer solution has a pH of 7.0-9.0 and comprises one or more of bovine serum albumin, casein, trehalose, and blocking agents.
9. The kit of claim 1, further comprising a cytokeratin 19 fragment series calibrator, wherein said cytokeratin 19 fragment series calibrator is obtained by diluting cytokeratin 19 fragments to a plurality of series concentrations with a calibrator buffer.
10. The kit of claim 9, wherein the calibrator buffer comprises 1-10g/L HEPES, 20-60g/L bovine serum albumin, 5-10g/L sodium chloride, 0.05-0.2ml of 0.5-2mol/L magnesium chloride solution, 0.05-0.2ml of 0.1-0.5mol/L zinc chloride solution, 1-4g/L preservative, and water, and the pH of the buffer is 7.0-8.0.
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