CN114487442A - Mouse monoclonal antibody coated magnetic bead, preparation method and kit for determining high-sensitivity cardiac troponin I - Google Patents
Mouse monoclonal antibody coated magnetic bead, preparation method and kit for determining high-sensitivity cardiac troponin I Download PDFInfo
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- G01N33/531—Production of immunochemical test materials
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Abstract
The invention discloses a mouse monoclonal antibody coated magnetic bead, a preparation method and a kit for measuring high-sensitivity cardiac troponin I.A mouse anti-human cTnI monoclonal antibody is diluted and labeled by biotin and then coated by naked magnetic beads to obtain the mouse anti-human cTnI monoclonal antibody coated magnetic bead; the dilution concentration is 0.1 mg/mL-0.6 mg/mL; two mouse anti-human cTnI monoclonal antibodies aiming at different cTnI epitope are adopted to prepare and obtain two magnetic beads coated by the mouse anti-human cTnI monoclonal antibodies, and the two magnetic beads are mixed to prepare the kit for measuring the high-sensitivity cardiac troponin I. The kit prepared by the invention has high sensitivity, high accuracy and good stability.
Description
Technical Field
The invention relates to the technical field of biomedical detection, in particular to a mouse monoclonal antibody coated magnetic bead, a preparation method and a kit for measuring high-sensitivity cardiac troponin I.
Background
cTnI belongs to a complex subunit of cardiac troponin and is mainly present in cardiac myocytes. In heart failure, myocardial cell apoptosis, ventricular muscle reconstruction, ventricular cavity enlargement and endothelial cell dysfunction appear in myocardial cells under the conditions of ischemia and hypoxia, so that the integrity of the myocardial cells is damaged, the permeability of cell membranes is increased, and the cTnI is released into blood, so that the cTnI level in the blood is obviously increased. The cTnI level begins to rise within 3-4 h after the myocardial damage occurs, reaches a peak value within 10-24 h, is a specific serological marker reflecting the myocardial damage and necrosis, is a 'gold standard' for diagnosing the myocardial damage in serology, and the more serious the myocardial ischemia, hypoxia and damage condition is, the higher the cTnI level of a patient is. The detection of cTnI has great clinical value for prognosis of acute coronary syndrome, diagnosis and dynamic detection of acute myocardial infarction, curative effect observation, prognosis judgment of unstable angina, evaluation of thrombolytic treatment effect and the like, and has very key clinical significance.
In recent years, a great progress in the detection technology of cardiac troponin has been the emergence of highly sensitive cardiac troponin. The high-sensitivity cardiac troponin has lower detection limit than that of the traditional cardiac troponin and can detect the cardiac troponin with extremely low level. The high-sensitivity cardiac troponin (cTnI) immunoassay can be used for diagnosis within 3-4 h, so that early diagnosis and treatment are really realized in the acute myocardial infarction course, and valuable time is won for reducing myocardial infarction and saving the life of a patient.
At present, common methods for clinically detecting cTnI comprise enzyme-linked immunosorbent assay (ELISA), colloidal gold immunochromatography, electrochemiluminescence and other immunodiagnosis technologies, due to the limitation of methodology and antibody pairing, the sensitivity and the detection quantitative accuracy of the colloidal gold immunochromatography are difficult to meet the requirements, the ELISA method consumes a long time, the steps are more, the sensitivity and the accuracy of the electrochemiluminescence are higher, but the cost is high, most markets are occupied by foreign reagents such as Siemens Roche and the like, and the method is not suitable for primary hospitals. In addition, some detection products mostly adopt an antibody of a certain section of antigen epitope to detect the antigen, and the detection omission rate of normal people is more than or equal to 50 percent. Therefore, the screening can identify cTnI and simultaneously prevent the pairing of antibodies of false detection, the invention provides a detection kit which is high in sensitivity and rapid, monitors the development trend of the disease condition in time and meets the requirement of detecting the content change of the myocardial troponin in the blood of patients at different time, and is particularly important.
Disclosure of Invention
Based on the technical background, the invention provides a mouse monoclonal antibody coated magnetic bead, a preparation method and a kit for measuring high-sensitivity cardiac troponin I, aiming at obtaining an hs-cTnI immunoassay reagent with high sensitivity, accuracy and stability and lower cost.
The invention is realized by the following technical scheme:
a magnetic bead coated by a mouse monoclonal antibody is a mixed magnetic bead, and the mixed magnetic bead is a magnetic bead coated by two mouse anti-human cTnI monoclonal antibodies aiming at different cTnI antigen epitopes and is used for preparing a kit for measuring high-sensitivity cardiac troponin I.
Two antibodies that are preferably employed in the present invention are: antibody 1: a fragment of amino acids 41-49 that specifically binds cTnI; antibody 2: specifically binds to amino acid fragments 86-92 of cTnI.
A preparation method of a magnetic bead coated with a murine monoclonal antibody comprises the following steps: diluting a mouse anti-human cTnI monoclonal antibody, labeling with biotin, and then carrying out naked magnetic bead coating to obtain magnetic beads coated with the mouse anti-human cTnI monoclonal antibody; the dilution concentration is 0.1 mg/mL-0.6 mg/mL; two mouse anti-human cTnI monoclonal antibodies aiming at different cTnI epitope are adopted to prepare and obtain two magnetic beads coated by the mouse anti-human cTnI monoclonal antibodies, and the two magnetic beads are mixed to prepare the kit for measuring the high-sensitivity cardiac troponin I.
According to the invention, before the mouse anti-human cTnI monoclonal antibody is marked, the mouse anti-human cTnI monoclonal antibody is diluted to a specific concentration, so that the stability of the kit can be improved, and the functions of the kits in different batches are similar. When the mouse anti-human cTnI monoclonal antibody is diluted to the concentration within the range, the kit has high titer and good stability. In addition, the invention aims to improve the sensitivity and the accuracy of the hs-cTnI immunoassay reagent in the prior art by simultaneously coating and marking two antibodies capable of identifying different epitopes.
The specific steps are as follows:
respectively diluting two mouse anti-human cTnI monoclonal antibodies aiming at different cTnI epitopes to set concentrations, wherein the dilution concentration is 0.1-0.6 mg/mL;
respectively carrying out biotin labeling on the diluted two mouse anti-human cTnI monoclonal antibodies to obtain two biotin antibodies;
respectively coating the two biotin antibodies on naked magnetic beads to obtain magnetic beads coated by two mouse anti-human cTnI monoclonal antibodies;
the two magnetic beads are mixed to prepare a kit for measuring the hypersensitive cardiac troponin I.
Further preferably, the biotin labeling is carried out by the following method: dialyzing the mouse anti-human cTnI monoclonal antibody by PBS (phosphate buffer solution), and reacting the dialyzed mouse anti-human cTnI monoclonal antibody with a crosslinking agent BNHS (BNHS) to obtain a biotin antibody; dialyzing the biotin antibody by using PBS (phosphate buffer solution); glycerol was added to the dialyzed biotin antibody for storage.
Further preferably, the coating is performed by the following method: coating streptavidin magnetic beads with the biotin-labeled mouse anti-human cTnI monoclonal antibody according to the corresponding coating amount; and diluting the magnetic beads coated with the mouse anti-human cTnI monoclonal antibody to a set concentration by using a buffer solution of 0.08-0.12% BSA.
A kit for determining high-sensitivity cardiac troponin I comprises mixed magnetic beads, wherein the mixed magnetic beads are coated by two mouse anti-human cTnI monoclonal antibodies aiming at different cTnI epitopes; more preferably, the mixed magnetic beads are prepared based on the above-mentioned method for preparing murine monoclonal antibody-coated magnetic beads.
Further preferably, the kit further comprises an enzyme working solution and an analysis buffer solution.
Further preferably, the assay buffer is calf serum diluent.
Further preferably, the enzyme working solution is an enzyme working solution labeled by a goat anti-human cTnI monoclonal antibody.
Further preferably, the volume ratio of the mixed magnetic beads, the enzyme working solution and the analysis buffer solution is 1:1: 1.
Preferably, the goat anti-human cTnI monoclonal antibody-labeled enzyme working solution is prepared from two alkaline phosphatase-labeled anti-cTnI goat monoclonal antibodies aiming at different epitopes of cTnI.
Further preferably, the preparation method of the goat anti-human cTnI monoclonal antibody labeled enzyme working solution comprises the following steps: and mixing two alkaline phosphatase-labeled anti-cTnI sheep monoclonal antibodies aiming at different epitopes of cTnI according to the mass ratio of 1:1, and diluting to a set concentration.
The invention has the following advantages and beneficial effects:
the invention provides a mouse monoclonal antibody coated magnetic bead, a preparation method and a kit for measuring high-sensitivity cardiac troponin I.
Drawings
The accompanying drawings, which are included to provide a further understanding of the embodiments of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the principles of the invention. In the drawings:
FIG. 1 shows the result of the detection of the reagents corresponding to Beckmann and magnetic bead I in application example I.
FIG. 2 is the result of the detection of the reagents corresponding to Beckmann and magnetic bead II in application example I.
FIG. 3 shows the result of the application of the first embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples and accompanying drawings, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not meant to limit the present invention.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, it will be apparent to one of ordinary skill in the art that: it is not necessary to employ these specific details to practice the present invention. In other instances, well-known structures, circuits, materials, or methods have not been described in detail so as not to obscure the present invention.
Example 1
The embodiment provides a kit for determining high-sensitivity cardiac troponin I, which comprises magnetic beads coated by a mouse anti-human cTnI monoclonal antibody, an analysis buffer solution and an enzyme working solution, wherein biotin is marked on the magnetic beads coated by the mouse anti-human cTnI monoclonal antibody.
Firstly, preparing magnetic beads coated with a mouse anti-human cTnI monoclonal antibody:
A. two mouse anti-human cTnI monoclonal antibodies (two antibodies are antibody 1: the amino acid fragment at the 41 st to 49 th positions of the cTnI specifically bound, and antibody 2: the amino acid fragment at the 86 th to 92 th positions of the cTnI specifically bound) are respectively diluted to 1.0mg/mL by PBS buffer.
B. Taking 0.25mg of each of the two diluted mouse anti-human cTnI monoclonal antibodies, dialyzing by PBS dialysate, and reacting the two dialyzed mouse anti-human cTnI monoclonal antibodies with 217 mu L of cross-linking agent BNHS for 30min to obtain two biotin antibodies; dialyzing the two biotin antibodies by using PBS (phosphate buffer solution); glycerol was added to each of the dialyzed biotin antibodies for storage.
C. Naked magnetic beads are respectively coated with two biotin-labeled biotin antibodies, the coating amount is 10 mug/mg, and magnetic beads coated with two mouse anti-human cTnI monoclonal antibodies are obtained. And respectively diluting the magnetic beads with Buffer solution of 1% BSA for later use to obtain magnetic beads I and II.
II, preparing an enzyme working solution marked by the goat anti-human cTnI monoclonal antibody:
two alkaline phosphatase-labeled goat anti-human cTnI monoclonal antibodies directed against different epitopes of cTnI were mixed at a mass ratio of 1:1, and diluted to 0.05mg/mL with 0.5% BSA buffer to serve as an enzyme working solution.
Thirdly, preparing an analysis buffer solution:
commercial calf serum purchased was diluted with purified water to a concentration of 100 mL/L.
Fourthly, preparing a calibrator and a quality control product:
commercially available cTnI antigens were dissolved and mixed as required by the manufacturer and diluted with 1% casein buffer to 6 concentrations of calibrator dots and two appropriate concentrations of quality control dots.
Fifthly, the using method of the kit is as follows:
the kit for the high-sensitivity cardiac troponin I in the embodiment adopts a chemiluminescence method, is provided with a full-automatic chemiluminescence immunoassay analyzer, can realize full automation of the test process, is internally provided with a device for measuring the project parameters of the high-sensitivity cardiac troponin I, is used for calibration on a computer, is used for quality control test after calibration is finished, is used for testing a patient sample after passing the verification, can automatically detect according to the reaction principle of the chemiluminescence method and the built-in reaction parameters, automatically performs fitting analysis according to a calibration curve, and quickly obtains a quantitative result, so that the cTnI content of early-stage AMI patients is monitored, and a basis is provided for eliminating and predicting early-stage acute myocardial injury patients.
Application case 1
And (3) taking the magnetic bead I and the magnetic bead II obtained by the preparation method in the embodiment 1 and a kit prepared by two mixed magnetic beads, and carrying out a kit sensitivity and specificity comparison experiment under the same condition.
Experimental materials: crosslinking agents BNHS, glycerol, PBS Buffer solution, two mouse anti-human cTnI monoclonal antibodies, Buffer solution of 1% BSA, Buffer solution of 0.5% BSA, calf serum, 1% casein Buffer solution, two sheep anti-human cTnI monoclonal antibodies, alkaline phosphatase, naked magnetic beads, calibrators Cal-1-Cal-6 prepared from hs-cTnI antigen (the concentration of Cal-1 to Cal-6 is 0pg/mL, 25pg/mL, 150pg/mL, 825pg/mL, 4950/mL, 24750pg/mL), samples S1-S40, substrate solution, cleaning solution, and a Wwinter biological full-automatic chemiluminescence immunoassay analyzer LA 2000.
The method of example 1 was used to combine the three beads (bead i, bead ii, and mixed beads of bead i and bead ii) with the enzyme working solution and the assay buffer to form a kit, to measure hs-cTnI calibrators Cal-1 to Cal-6, to observe the measurement curves and sensitivities of the different beads, and to measure samples S1-S40, to compare them with the beckmann values.
The reaction mode is as follows: adding 20 μ L of calibrator, 50 μ L of enzyme working solution, 50 μ L of analysis buffer solution, 50 μ L of magnetic bead, incubating at 37 deg.C for 5min, adding cleaning solution, washing and separating in magnetic field for three times, adding substrate solution, incubating at 37 deg.C for 5min, and detecting with full-automatic chemiluminescence determinator.
The results of the experiments are shown in table 1, table 2 and fig. 1-3.
TABLE 1 RLU and SNR detection results of three magnetic beads corresponding to kit for calibrator
TABLE 2 Beckmann value measurement results of the three kinds of magnetic beads corresponding to the reagents
And (4) experimental conclusion: it can be known from the combination of tables 1-2 and fig. 1-3 that when the mixed magnetic beads are used, the measured sample value is closer to Beckmann, which indicates that the anti-interference capability of the detection kit is enhanced, the clinical coincidence rate is higher, the sensitivity and specificity of the kit paired by the multi-epitope antibody are greatly improved, and the kit curve meets the use requirement.
Application case two
The stability of the three kits was observed using the magnetic beads i and ii obtained in the preparation method of example 1 and the two kits prepared from the mixed magnetic beads.
Experimental materials: crosslinking agents BNHS, glycerol, PBS Buffer solution, two mouse anti-human cTnI monoclonal antibodies, Buffer solution of 1% BSA, Buffer solution of 0.5% BSA, calf serum, 1% casein Buffer solution, two sheep anti-human cTnI monoclonal antibodies, alkaline phosphatase, naked magnetic beads, calibrators Cal-1-Cal-6 prepared from hs-cTnI antigen (the concentration of Cal-1 to Cal-6 is 0pg/mL, 25pg/mL, 150pg/mL, 825pg/mL, 4950/mL, 24750pg/mL), samples S1-S40, substrate solution, cleaning solution, and a Wwinter biological full-automatic chemiluminescence immunoassay analyzer LA 2000.
The method of example 1 was used to combine the three beads (bead I, bead II, and mixed beads of bead I and bead II) with the enzyme working solution and the assay buffer to form a kit.
The experimental method comprises the following steps: the kit is prepared by the method of example 1, specifically, three magnetic beads are divided into two parts in equal volume and put into the kit, one part is stored at 2-8 ℃, one part is stored at 37 ℃ in a constant temperature incubator, and the three parts are taken out after 6 days. And (3) respectively matching the three magnetic beads with an analysis buffer solution and an enzyme working solution to determine the hs-cTnI calibrator. And (4) observing the luminescent signals of the calibrator on the third day and the sixth day respectively, calculating the signal retention rate, and verifying the stability of the kit.
The reaction mode is shown in application case one.
The results of the experiments are shown in tables 3 and 4.
TABLE 3
TABLE 4
And (4) experimental conclusion: when the mixed magnetic beads are used, the signal retention rate is better after the mixed magnetic beads are stored at 37 ℃, and the stability of the kit is better.
Two experiments can be combined to obtain: the hs-cTnI determination kit can identify a plurality of epitopes of an antigen by using the multi-epitope antibody, the sensitivity and the specificity are effectively improved, the anti-interference capability of the kit is enhanced when a clinical sample is tested, the clinical compliance rate is greatly improved, the accuracy is higher, the risk of missed detection is reduced, the detection rate is improved, the stability is good, the kit can be stored for a long time, and the kit has more advantages in the market.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are merely exemplary embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (10)
1. A magnetic bead coated by a mouse monoclonal antibody is characterized by being a mixed magnetic bead, wherein the mixed magnetic bead is a magnetic bead coated by two mouse anti-human cTnI monoclonal antibodies aiming at different cTnI antigen epitopes and is used for preparing a kit for measuring high-sensitivity cardiac troponin I.
2. A preparation method of a magnetic bead coated with a murine monoclonal antibody comprises the following steps: diluting the mouse anti-human cTnI monoclonal antibody, labeling with biotin, and then coating with naked magnetic beads to obtain magnetic beads coated with the mouse anti-human cTnI monoclonal antibody,
the dilution concentration is 0.1 mg/mL-0.6 mg/mL;
two mouse anti-human cTnI monoclonal antibodies aiming at different cTnI epitope are adopted to prepare and obtain two magnetic beads coated by the mouse anti-human cTnI monoclonal antibodies, and the two magnetic beads are mixed to prepare the kit for measuring the high-sensitivity cardiac troponin I.
3. The method of claim 2, wherein the biotin labeling is performed by the following steps: dialyzing the mouse anti-human cTnI monoclonal antibody by PBS (phosphate buffer solution), and reacting the dialyzed mouse anti-human cTnI monoclonal antibody with a crosslinking agent BNHS (BNHS) to obtain a biotin antibody; dialyzing the biotin antibody by using PBS (phosphate buffer solution); glycerol was added to the dialyzed biotin antibody for storage.
4. The method of claim 2, wherein the coating is performed by the following steps: coating streptavidin magnetic beads with the biotin-labeled mouse anti-human cTnI monoclonal antibody according to the corresponding coating amount; and (3) diluting the magnetic beads coated with the mouse anti-human cTnI monoclonal antibody to a set concentration by using a buffer solution of 0.08-0.12% BSA.
5. A kit for determining high-sensitivity cardiac troponin I is characterized by comprising mixed magnetic beads, wherein the mixed magnetic beads are magnetic beads coated by two mouse anti-human cTnI monoclonal antibodies aiming at different cTnI epitopes.
6. The kit for assaying hypersensitive cardiac troponin I according to claim 5, further comprising an enzyme working solution and an assay buffer.
7. The kit for detecting hypersensitive cardiac troponin I according to claim 5, wherein the analysis buffer solution is calf serum diluent, and the enzyme working solution is goat anti-human cTnI monoclonal antibody labeled enzyme working solution.
8. The kit for detecting highly sensitive cardiac troponin I according to claim 6 or 7, wherein the volume ratio of the mixed magnetic beads, the enzyme working solution and the analysis buffer is 1:1: 1.
9. The kit for detecting hypersensitive cardiac troponin I according to claim 7, wherein the goat anti-human cTnI monoclonal antibody-labeled enzyme working solution is prepared from two alkaline phosphatase-labeled anti-cTnI goat monoclonal antibodies aiming at different epitopes of cTnI.
10. The kit for detecting hypersensitive cardiac troponin I according to claim 9, wherein the preparation method of the sheep anti-human cTnI monoclonal antibody labeled enzyme working solution comprises the following steps: and mixing two alkaline phosphatase-labeled anti-cTnI sheep monoclonal antibodies aiming at different epitopes of cTnI according to the mass ratio of 1:1, and diluting to a set concentration.
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| CN115453130A (en) * | 2022-09-30 | 2022-12-09 | 四川沃文特生物技术有限公司 | Kit for determining interferon-gamma and application thereof |
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