CN114384249A - Detection method and kit for cardiac troponin I - Google Patents
Detection method and kit for cardiac troponin I Download PDFInfo
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- CN114384249A CN114384249A CN202011114995.6A CN202011114995A CN114384249A CN 114384249 A CN114384249 A CN 114384249A CN 202011114995 A CN202011114995 A CN 202011114995A CN 114384249 A CN114384249 A CN 114384249A
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Classifications
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- G—PHYSICS
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- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- G—PHYSICS
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G—PHYSICS
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- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
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- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
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- 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
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
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- G—PHYSICS
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- G01N2800/32—Cardiovascular disorders
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Abstract
The invention relates to a cardiac troponin I detection method and a kit, and relates to the field of protein detection. The kit comprises a first antibody and a second antibody for detecting cardiac troponin I, wherein the antibodies are selected from the group consisting of: the first antibody binds to an amino acid fragment 83-93 of cardiac troponin I; the second antibody is an antibody that binds to the cardiac troponin CTNI-CTNC complex.
Description
Technical Field
The present invention relates to the field of protein detection. In particular, the invention relates to a detection method and a kit for cardiac troponin I.
Background
The elevation of cardiac troponin I (cTnI: cardiac troponin I) has a very high sensitivity and specificity for diagnosing myocardial injury/necrosis (myocardiac necrosis). cTnI is an indispensable marker for clinical diagnosis, treatment and monitoring of myocardial infarction at present.
The cardiac troponin exists in trace amount in normal people, but when myocardial infarction occurs, cardiac muscle cells are damaged to release cTnI into blood, the cTnI is rapidly increased no matter whether ST segment is raised or not, the cTnI reaches a peak in about 24 hours and is reduced to a normal level after 7-10 days. The magnitude of the elevation of cTnI is correlated to the degree of cardiomyocyte necrosis to some extent. If the cTnI in the patient's blood rises again during the descent, it means that the myocardium is damaged/necrosed again. changes in the concentration of cTnI are clearly correlated with acs (acute corona syndrome) risk level, treatment decision and prognosis.
Disclosure of Invention
The inventor obtains the cardiac troponin I antigen region combination capable of being used for cardiac troponin I detection through a large number of theoretical researches and experimental grops, fully considers the structural characteristics of the cardiac troponin I, analyzes and researches various to-be-detected troponin antigen intervals and detection antibodies, and performs a large number of cross experiments and screens. The inventors have demonstrated that antibodies that bind to specific antigen fragments are unexpectedly capable of combining with each other for highly sensitive and specific detection of cTnI.
In some embodiments, the invention may include one or more of the following:
1. a cardiac troponin I detection kit comprising a first antibody and a second antibody for detecting cardiac troponin I, wherein the first antibody binds to an amino acid fragment of cardiac troponin I at positions 83-93; the second fragment binds the cardiac troponin CTNI-CTNC complex.
2. The kit according to item 1, wherein the first antibody is a labeled antibody and the second antibody is a coated antibody; or the second antibody is a labeled antibody and the first antibody is a coated antibody.
3. The kit of item 1, further comprising a third antibody that detects cardiac troponin I, wherein the third antibody binds to the fragment of cardiac troponin I from amino acid positions 41 to 49;
for example, antibodies for pairing are divided into a first set of antibodies and a second set of antibodies, wherein the first set of antibodies and the second set of antibodies are selected from the group consisting of:
1) the first set of antibodies comprises a first antibody and the second set of antibodies comprises a second antibody and a third antibody;
2) the first set of antibodies comprises a second antibody and the second set of antibodies comprises a first antibody and a third antibody;
3) the first set of antibodies comprises a third antibody and the second set of antibodies comprises a first antibody and a second antibody;
preferably, the first set of antibodies is labeled antibodies and the second set of antibodies is coated antibodies.
4. The kit of any one of items 1 to 3, wherein the reactivity of the antibody is OD evaluation by ELISA405≥0.5。
5. The kit of any of items 1-4, wherein the labeled antibody is labeled with a detectable label such as a metal particle, a fluorescent label, a chromophore label, an electron dense label, a chemiluminescent label, a radioactive label, or an enzyme label, and/or a binding partner; for example, the enzyme can be rhodamine, fluorescein, fluorescent microspheres, colloidal gold, acridinium esters, luciferase, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, oxidase, glucose oxidase, galactose oxidase or glucose-6-phosphate dehydrogenase labels; binding partners are, for example, biotin/avidin, biotin/streptavidin; for example, the labeled antibody is labeled with a binding partner and a detectable label.
6. The kit of any of items 1-5, wherein the coated antibody is attached to a solid phase such as a magnetic particle, a latex particle, a microtiter plate, a nitrocellulose membrane, or a microfluidic chip and/or a binding partner; binding partners are, for example, biotin/avidin, biotin/streptavidin; for example, the coated antibody is attached to a solid phase via a binding partner.
7. A combination of antibodies, including a first antibody and a second antibody, for detecting cardiac troponin I; optionally, a third antibody is also included; wherein the first antibody, the second antibody, the third antibody are defined according to any one of items 1-6.
8. Use of the antibody combination of item 7 in the preparation of a kit for the detection of cardiac troponin I.
9. The use according to item 8, wherein the kit is for 1) dynamic detection of the content of cardiac troponin, e.g. for real-time monitoring, and/or 2) diagnosis of early acute myocardial injury, including e.g. excluding or predicting early acute myocardial injury.
10. A method of making the antibody combination of item 7, the method comprising:
1) immunizing an animal with an antigen or hapten comprising an amino acid fragment 1-2 or protein selected from the group consisting of:
fragment 1: cardiac troponin I amino acid fragments 41 to 49;
fragment 2: cardiac troponin I amino acid fragments 83-93;
protein 1: a cardiac troponin CTNI-CTNC complex; and
2) obtaining antibodies from said animal that bind to said fragments 1-2 and protein 1, respectively.
In some embodiments, the kit of the invention comprises a first antibody and a second antibody for detecting cardiac troponin I, wherein the first antibody binds to an amino acid fragment of cardiac troponin I at positions 83-93; the second antibody binds to the cardiac troponin CTNI-CTNC complex; for example, the second set of antibodies are coated antibodies when the first antibody is a labeled antibody; or when the second antibody is a labeled antibody, the first antibody is a coated antibody, which can improve the detection specificity.
The kit comprises a cardiac troponin I detection reagent card (test strip). In some embodiments, cTnI in a subject, e.g., a patient with myocardial injury/necrosis, can be detected using the principle of double antibody sandwich, using immunofluorescence techniques, to label the antibody on a detectable label, e.g., a fluorescent microsphere. In some embodiments, the methods and kits of the invention can improve detection specificity and/or reduce missed detection.
In some embodiments, the invention may be performed by or include reagents that perform fluorescence immunochromatography. In some embodiments, immunochromatography can be achieved by a test strip by a double antibody sandwich method. In some embodiments, the reagents and apparatus for performing the double antibody sandwich method are not particularly limited, and any suitable reagents and/or apparatus may be employed. For example, the fluorescence immunochromatographic test strip may include a sample pad, a conjugate pad, an NC membrane, and a water absorbent pad. In some embodiments, at least one fluorophore-labeled antibody is immobilized on the conjugate pad; in some embodiments, the NC film may be coated with two lines, for example T and C lines, respectively; the T line can be coated with at least one antibody; can capture the fluorescent group labeled antibody from the binding pad to form a complex with the antigen in the sample, thereby forming a double antibody sandwich. In some embodiments, a sample to be detected is added into a sample adding port of the detection reagent card, and under the lateral capillary action, the sample to be detected firstly passes through the combination pad to be specifically and immunologically combined with a fluorescent group labeled antibody on the combination pad, and the antigen-antibody fluorescent complex is formed by combination of the combination pad and the fluorescent group labeled antibody, so that the sample to be detected is fixed in the T line. The C line is coated with a substance that reacts with the free fluorophore-labeled antibody, and when the free fluorophore-labeled antibody passes through the C line, it is immobilized in the C line by specific immunological binding to the substance on the C line. The control band (C) is inversely proportional to the fluorescence intensity of the test band (T), so that the individual differences of the samples can be corrected. The fluorescence intensity of the two bands detected by the fluorescence immunoassay instrument is represented by peak areas, a T/C value (T peak area/C peak area) is calculated by calculation software of the instrument, the T/C value is fitted to a set standard curve, and the fluorescence immunoassay instrument automatically converts the concentration value of cTnI in a corresponding sample to be detected.
In some embodiments, the invention can utilize antibodies that bind different amino acid fragments to recognize multiple positions on an antigen, reduce the risk of missed detection, and increase the detection rate.
In some embodiments, the antibody that binds to the 83-93aa fragment of cardiac troponin I is used as a labeled antibody, and the antibody that binds to the cardiac troponin I-C complex and the antibody that binds to the 41-49aa fragment of cardiac troponin I are used as coating antibodies for coating; in some embodiments, the antibody that binds to the cardiac troponin I-C complex is used as a labeled antibody, and the antibody that binds to the 83-93aa fragment of cardiac troponin I and the antibody that binds to the 41-49aa fragment of cardiac troponin I are used as coating antibodies for coating; in some embodiments, the antibody that binds to the 41-49aa fragment of cardiac troponin I is used as a labeled antibody, and the antibody that binds to the 83-93aa fragment of cardiac troponin I and the antibody that binds to the cardiac troponin I-C complex are used as coating antibodies for the coating.
In some embodiments, the invention provides a cardiac troponin I detection method, a detection kit and a preparation method. The reagent kit has improved sensitivity and specificity. In some embodiments, the antibodies of the invention may be monoclonal or polyclonal antibodies. In some embodiments, the antibodies of the invention can be prepared using methods known in the art. In some embodiments, an antibody of the invention can be prepared by immunizing an animal with an antigen comprising an amino acid fragment described herein. To increase immunogenicity, carrier proteins (including but not limited to BSA, ovalbumin, KLH, etc.) may be coupled to immunoreactive substances (e.g., epitope peptides). The carrier protein may comprise a protein or polypeptide, which may function as a carrier for the immunogen. These types of polypeptides include albumin, serum proteins, globulins, lens proteins, lipoproteins, and/or fragments thereof. In some embodiments, immunoreactive substances (e.g., cardiac troponin I amino acid fragments, cardiac troponin CTNI-CTNC complexes, but not limited thereto) can be used to generate antibodies with affinity for CTNI.
In some embodiments, binding of an antibody to a corresponding amino acid fragment of cardiac troponin may refer to the ability of the antibody to bind to the amino acid fragment, but the amino acid fragment is not necessarily the smallest binding fragment.
In some embodiments, the effect of the antibodies of the invention, such as binding activity and/or cross-reactivity, can be tested using any suitable in vitro assay, cell-based assay, in vivo assay, animal model, and the like. In some embodiments, the assay may include, for example, ELISA, FACS binding assay, Biacore, competitive binding assay, and the like. In some embodiments, the reactivity of the antibodies of the invention to antigen (antigenic peptide) binding is characterized, for example, in an ELISA, and better reactivity is determined, for example, by a peroxidase-labeled ELISA reading a response value ≧ 0.5 at 405 nm.
In some embodiments, a first antibody, a second antibody; in some embodiments, a third antibody is also included in the kit; in some embodiments, other antibodies may also be used as coating antibodies or labeled antibodies.
In some embodiments, the coated antibody is bound to a solid phase. In some embodiments, the coated antibody can be used to coat a solid support. In some embodiments, the solid support is not particularly limited and can be, for example, magnetic particles, latex particles, microtiter plates, nitrocellulose membranes, or microfluidic chips. In some embodiments, the coated antibody binds to a binding partner, such as biotin/avidin, biotin/streptavidin; in some embodiments, the coated antibody is attached to a solid phase via a binding partner.
In some embodiments, the labeled antibody is labeled with a detectable label, e.g., with a fluorescent label; in some embodiments, the labeled antibody is linked to a binding partner, such as biotin/avidin, biotin/streptavidin; in some embodiments, the labeled antibody is labeled with a binding partner and a detectable label.
In some embodiments, the term "antibody" herein is used in the broadest sense and can include full-length monoclonal antibodies, polyclonal antibodies, bispecific or multispecific antibodies, chimeric antibodies, and antigen-binding fragments of antibodies, so long as they exhibit the desired biological activity, e.g., binding to a cTnI antigen. "antibody fragments" include portions of full-length antibodies, preferably the antigen-binding or variable regions thereof. Examples of antibody fragments include Fab, Fab ', F (ab')2Fd, Fv, dAb, Complementarity Determining Region (CDR) fragment, single chain antibody (e.g., scFv), diabody or domain antibody.
In some embodiments, the detectable label used in the present invention is not particularly limited. In some embodiments, the label may include, but is not limited to, a metal particle, a fluorescent label, a chromophore label, an electron dense label, a chemiluminescent label, a radioactive label, or an enzymatic label, for example, for indirect detection by enzymatic reaction or molecular interaction. In some embodiments, exemplary labels include, but are not limited to, a label that can be rhodamine, fluorescein, fluorescent microspheres, colloidal gold, acridinium esters, luciferase, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, carbohydrate oxidase, glucose oxidase, galactose oxidase, or glucose-6-phosphate dehydrogenase, and the like.
In some embodiments, the kits of the invention comprise reagents suitable for performing an immunoassay. In some embodiments, the kits of the invention can be used to perform immunoassays, such as ELISA, indirect immunofluorescence assays IFA, radioimmunoassays RIA, and other non-enzyme-linked antibody binding assays or methods.
In this context, the sample to be tested for cTnI may comprise a biological tissue, cell or body fluid of a healthy or pathological state, such as a blood sample, e.g. plasma, serum, blood product, e.g. semen or vaginal secretions.
In some embodiments, the present invention provides antibody combinations for the detection of cardiac troponin I, and their use in the manufacture of a kit for the detection of cardiac troponin I. In some embodiments, the present invention provides a combination of an immunoreactive polypeptide comprising an amino acid fragment as described herein and a cardiac troponin CTNI-CTNC complex, and their use for the preparation of antibodies for the detection of cardiac troponin I. In some embodiments, the present invention provides a method for preparing an antibody for detecting cardiac troponin I, the method comprising immunizing an animal with an immunoreactive polypeptide comprising an amino acid fragment as described herein and the CTNI-CTNC complex as an antigen or a hapten, respectively, thereby preparing an antibody, such as a monoclonal antibody or a polyclonal antibody, for detecting cardiac troponin I. Monoclonal or polyclonal antibodies can be made by methods known in the art.
In some embodiments, the present invention provides the use of an immunoreactive polypeptide comprising an amino acid fragment as described herein and said CTNI-CTNC complex in the preparation of a reagent or kit for the detection of cardiac troponin I.
The invention has the following beneficial effects:
the invention provides a cardiac troponin (cTnI) detection method and a kit, wherein an antibody combined with an 83-93aa fragment of the cardiac troponin I and an antibody combined with a cardiac troponin CTNI-CTNC compound are adopted to improve the detection specificity, and an antibody combined with a 41-49aa fragment of the cardiac troponin I is further adopted to reduce the risk of missed detection.
Detailed Description
The present invention will be described in further detail with reference to specific examples. The following examples are provided to illustrate embodiments of the present invention and are not intended to limit the invention. The invention may optionally include embodiments that are not illustrated by the examples.
Preparation of myocardial troponin I monoclonal antibody
1. Immunizing animals: a BALB/c mouse which is 8-12 weeks old and has the same line with myeloma cells is taken, 100 mu g/cTnI antigen containing protein and the same amount of Freund's complete adjuvant are fully and uniformly mixed and injected into the abdominal cavity of the mouse, 100 mu g/cTnI antigen and the same amount of Freund's incomplete adjuvant are fully and uniformly mixed every 2 weeks, and the mixture is injected into the abdominal cavity of the mouse for multiple times to strengthen immunity. By detecting mouse serum (indirect ELISA method), the mice with titer of more than 1:2000 can be used for fusion, and 3 days before fusion, the mice are boosted again in the abdominal cavity, and the dosage is 50 mug/mouse.
2. Preparation of feeder cells
BALB/c mouse peritoneal macrophages were used as feeder cells. 1 day before fusion, BALB/c mouse neck-pulled to be killed, 75% alcohol soaked in whole body, in super clean bench, using scissors to cut off abdominal skin under aseptic operation, exposing peritoneum, using syringe to inject 5mL of RPMI1640 basic culture solution into abdominal cavity, repeatedly washing, recovering washing solution, 1000rpm, centrifuging for 5min, leaving precipitate, using RPMI1640 to screen culture solution (in complete culture solution of RPMI1640 containing HAT) to resuspend, regulating cell concentration to 1 × 105Add to 96-well plate at 150. mu.L/well, 37 ℃ with 5% CO2The culture was carried out overnight.
3. Preparation of immune spleen cells
Three days after the last immunization of the mice, the spleen is taken out under the aseptic condition, placed in a plate, washed once by RPMI1640 basic culture solution, placed on a nylon net of a small beaker, ground and filtered to prepare cell suspension. Centrifuging, discarding supernatant, resuspending RPMI1640 basic culture solution, repeating the steps three times, and counting.
4. Cell fusion
(1) Respectively putting 40mL HAT culture solution, 15mL DMEM serum-free culture solution and 1mL 50% PEG (M12000) in a water bath at 37 ℃ for pre-warming;
(2) separately, mouse myeloma cells Sp2/0 (stored by Roc Bio Inc.) (2-5X 10)7) The above immune spleen cell (10)8) The suspension is added into a 50mL centrifuge tube for even mixing, and DMEM serum-free culture solution is added to the centrifuge tube until the volume is 40 mL. Centrifuging for 10min, pouring out the supernatant, and mixing;
(3) the centrifuge tube was placed in pre-warmed water at 37 ℃ and 0.7mL of pre-warmed 50% PEG solution was allowed to stand for 90 seconds. Immediately dropwise adding 15mL of prewarmed serum-free culture solution at 37 ℃;
(4) DMEM serum-free culture solution is supplemented to 40mL, centrifugation is carried out for 10 minutes, and the supernatant is poured out. Adding 40mL HAT culture solution containing 15% -20% fetal calf serum. Mixing with a pipette, dropping 2 drops into small holes of 4 pieces of 96-well cell culture plate containing feeder cells, placing at 37 deg.C and 7% CO2Cultured in an incubator.
5. Selective culture of hybridoma cells
The immune mouse spleen cells and mouse myeloma cells are treated by PEG to form a mixture of various cell components, including unfused myeloma cells and immune spleen cells, a corebody of myeloma cells and a corebody of immune spleen cells, and a corebody of myeloma cells and immune spleen cells. Only the latter can form hybridoma cells. For this purpose, it is necessary to remove unfused cells and homologously fused coenckaryons from the various cell mixtures and to select for true hybrid cells. Therefore, the cells were cultured with the HAT medium on days 1, 3, 5, and 7 after cell fusion.
6. Detection of antibodies and hybridoma cell cloning
The supernatant of each culture well was aspirated, and the culture wells containing antibodies to cTnI in the culture were detected by indirect ELISA. Hybridoma cells were cloned by limiting dilution. After being cultured, the single cell can be proliferated into homologous cell clone; cell strains which have better reactivity and stably secrete anti-cTnI monoclonal antibodies are obtained through reactivity screening.
Method for reactivity screening: at room temperature, microtiter plates (Nunc, Maxisorb) were coated with 100. mu.L/well of coating buffer containing 2.5. mu.g/mL human cTnI (as antigen) for 1 hour while stirring. Post-coating treatments were incubated in PBS buffer and 1% monoclonal antibody for 30 min. Subsequently, washing was performed with a washing buffer. 100 μ L/well antibody samples were incubated for 1 hour while stirring at room temperature. Then washed 2 more times with the washing solution. Then, the goat anti-mouse IgG conjugated with the detection antibody peroxidase diluted with PBS buffer at 1:40000 was incubated with 100. mu.L/well for another 1 hour at room temperature while stirring. After washing again with the washing buffer, the peroxidase activity is measured in a conventional manner (for example, by reading the reaction value at 405nm with an ELISA reader) to obtain the OD405More than or equal to 0.5 (with better reactivity).
II, identification of antibody binding fragment
Respectively coating micropores with different cTnI antigen peptides, taking PBS + 20% NBS as a diluent, diluting the monoclonal antibody to 1 mu g/mL, taking goat anti-mouse IgG-HRP as a secondary antibody, and determining the binding fragment of the monoclonal antibody according to the reaction condition of each monoclonal antibody to different antigens. The results of identifying binding fragments of the cell lines used in the examples are given in the following table:
| binding fragments | Cell line |
| 18-29aa | 3D-7 |
| 41-49aa | 7B-1；10E-2；18F-2 |
| 83-93aa | 2C-7；5F-2；8E-2 |
| 167-178aa | 1B-6 |
| 180-190aa | 6E-7 |
| 190-196aa | 11C-6 |
| IC antibodies | 15A-6；16C-3 |
Wherein, the IC antibody is an antibody of CTNI-CTNC complex, and the preparation method refers to the above method, and the CTNI-CTNC complex is used for replacing cTnI antigen, and is immunized to obtain the IC antibody, wherein the CTNI-CTNC complex can be purchased from sea peptide organisms.
Third, screening by pairing
Selecting antibodies of different binding fragments for coating and marking respectively, and carrying out cross-pairing experiments, wherein the screening process is as follows:
1. marking
(1) Taking 100uL of fluorescent microspheres, centrifuging at 15000rpm/8min/4 ℃, removing supernatant after centrifugation, resuspending to 100uL, and performing ultrasonic treatment for 2-3 times;
(2) preparing 10mg/mL EDC and NHS;
(3) and (3) activation: activating fluorescent microspheres to a final volume of 200uL by using EDC and NHS, uniformly mixing for 18min in a dark place, centrifuging at 15000rpm/10min/4 ℃, and removing supernatant;
(4) washing: resuspending to 100uL, sonicating for 2-3 times at 15000rpm/8min/4 deg.C, removing supernatant, and repeating for 2 times;
(5) coupling: resuspending to 200uL, performing ultrasonic treatment for 2-3 times, adding the antibody into the fluorescent microspheres to a final volume of 300uL, uniformly mixing in a dark place, and reacting at 37 ℃ for 2 h;
(6) and (3) sealing: adding 100uL of sealant, uniformly mixing in dark, and reacting for 1h at 37 ℃;
(7) washing: after blocking, the cells were centrifuged at 15000rpm/12min/4 ℃ to remove the supernatant. Resuspend to 100uL, sonicate 2-3 times. Centrifuging at 15000rpm/8min/4 deg.C, and removing supernatant;
TABLE 1 Cross-Pair first round screening
TABLE 1
(8) And (3) storage: resuspending to 100uL, sonicating for 2-3 times, and storing at 4 ℃ for later use.
2. Coating quilt
(1) Assembling the nitrocellulose membrane and the fluorescent PVC base plate for later use;
(2) diluting the antibody to 1.0-2.0mg/mL, uniformly drawing a line on an NC film by using a gold spraying film drawing instrument, and then drying in a 37 ℃ thermostat for at least over 45 min. Assembling the cutting strips, and detecting the sample adding.
3. Detection of
(1) Quality control product: cTnI antigen, diluted to 50, 20, 5, 1.25ng/mL using PBS;
(2) the detection method comprises the following steps: detection was performed using a fluorometer. The reading value T/C of the instrument represents the ratio of the area of the T peak to the area of the C peak, the reaction pairing activity is high, and the higher the T/C is, the higher the activity is, under the condition of quality control and positive samples.
The results from table 1 (including table 1) show that the first round of cross-pairing screening resulted in pairs with superior activity and a gradient of test results, followed by specificity evaluation for these dominant pairs, and 100 negative samples of healthy human serum were tested (antibody self-pairing was not tested).
TABLE 2 Cross-Pair second round screening
As can be seen from the results in table 2, the cTnI antibody bound to 41-49aa was paired with the cTnI antibody bound to 83-93aa, and the cTnI antibody bound to 41-49aa was paired with the IC antibody, although the activity was better, but the specificity was poor (92% -98%), and the cTnI antibody bound to 83-93aa was paired with the IC antibody, which was clearly superior in specificity.
Antibodies that incorporate additional binding fragments are considered for testing in order to reduce the risk of missed detection. The activity was measured and the missing of each pair was examined with 100 positive fixed value samples. Wherein 2C-7, 5F-2 and 8E-2 are antibodies binding to the 83-93aa fragment, 7B-1, 18F-2 and 10E-2 are antibodies binding to the 41-49aa fragment, and 15A-6 and 16C-3 are antibodies binding to the CTNI-CTNC complex.
TABLE 3, 1+1 and 1+2 Pattern evaluation
Wherein, the T/C value is less than 0.1 when the quality control concentration is 50ng/mL, and no gradient is marked as basically no activity.
As can be seen from the results in Table 3, the combination of the superiority obtained in the 1+1 model, namely, the combination of the cTnI antibody combined with 83-93aa and the IC antibody, wherein the cTnI antibody combined with 41-49aa can supplement the respective detection deficiencies, and the combination has better pairing performance than the combination of the cTnI antibody combined with 18-29aa, the cTnI antibody combined with 180-190aa and different antibody strains with the same binding fragment as one of the antibodies; on the basis of good specificity (high negative coincidence rate) (table 2), the occurrence of missed detection is further reduced, and the accuracy of the detection result is improved.
The invention also provides the preparation scheme of the antibodyThe antibody which is combined with the 83-93aa fragment is prepared by immunizing and taking the cTnI amino acid fragment 83-93 coupled carrier protein as immunogen, for example, the OD is evaluated by ELISA through a reactivity screening reaction405The pairing specificity of the antibody I83A-9 and the like which are more than or equal to 0.5 to the IC antibodies 15A-6 and 16C-3 is higher than 99 percent; immunizing with cTnI amino acid fragment 41-49 coupled with carrier protein as immunogen to obtain antibody combined with 41-49aa fragment, such as ELISA to evaluate OD405The missed detection rate of the antibodies I41D-2 and the like which are more than or equal to 0.5 and matched with the former two antibodies is not more than 1 percent.
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 only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A cardiac troponin I detection kit comprising a first antibody and a second antibody for detecting cardiac troponin I, wherein the first antibody binds to an amino acid fragment of the cardiac troponin I at positions 83 to 93; the second antibody binds to the cardiac troponin CTNI-CTNC complex.
2. The kit of claim 1, wherein the first antibody is a labeled antibody and the second antibody is a coated antibody; or the second antibody is a labeled antibody and the first antibody is a coated antibody.
3. The kit of claim 1, further comprising a third antibody that detects cardiac troponin I, wherein the third antibody binds to the fragment of amino acids 41 to 49 of cardiac troponin I;
for example, antibodies for pairing are divided into a first set of antibodies and a second set of antibodies, wherein the first set of antibodies and the second set of antibodies are selected from the group consisting of:
1) the first set of antibodies comprises a first antibody and the second set of antibodies comprises a second antibody and a third antibody;
2) the first set of antibodies comprises a second antibody and the second set of antibodies comprises a first antibody and a third antibody;
3) the first set of antibodies comprises a third antibody and the second set of antibodies comprises a first antibody and a second antibody;
preferably, the first set of antibodies is labeled antibodies and the second set of antibodies is coated antibodies.
4. The kit of any one of claims 1 to 3, wherein the reactivity of the antibody is an ELISA-assessed OD405≥0.5。
5. The kit of any one of claims 1 to 4, wherein the labeled antibody is labeled with a detectable label such as a metal particle, a fluorescent label, a chromophore label, an electron dense label, a chemiluminescent label, a radioactive label, or an enzyme label, and/or a binding partner; for example, the enzyme can be rhodamine, fluorescein, fluorescent microspheres, colloidal gold, acridinium esters, luciferase, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, oxidase, glucose oxidase, galactose oxidase or glucose-6-phosphate dehydrogenase labels; binding partners are, for example, biotin/avidin, biotin/streptavidin; for example, the labeled antibody is labeled with a binding partner and a detectable label.
6. The kit of any one of claims 1 to 5, wherein the coated antibody is attached to a solid phase such as a magnetic particle, a latex particle, a microtiter plate, a nitrocellulose membrane or a microfluidic chip and/or a binding partner; binding partners are, for example, biotin/avidin, biotin/streptavidin; for example, the coated antibody is attached to a solid phase via a binding partner.
7. A combination of antibodies for detecting cardiac troponin I, comprising a first antibody and a second antibody; optionally, a third antibody is also included; wherein the first antibody, the second antibody, the third antibody are as defined in any one of claims 1 to 6.
8. Use of the antibody combination according to claim 7 for the preparation of a kit for the detection of cardiac troponin I.
9. Use according to claim 8, wherein the kit is for 1) dynamic detection of the content of cardiac troponin, e.g. for real-time monitoring, and/or 2) diagnosis of early acute myocardial injury, including e.g. excluding or predicting early acute myocardial injury.
10. A method of making the antibody combination of claim 7, comprising:
1) immunizing an animal with an antigen or hapten comprising an amino acid fragment 1-2 or protein selected from the group consisting of:
fragment 1: cardiac troponin I amino acid fragments 41 to 49;
fragment 2: cardiac troponin I amino acid fragments 83-93;
protein 1: a cardiac troponin CTNI-CTNC complex; and
2) obtaining antibodies from said animal that bind to said fragments 1-2 and protein 1, respectively.
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| CN109239361A (en) * | 2018-09-29 | 2019-01-18 | 迈克生物股份有限公司 | A kind of detection kit of cardiac muscle troponin I and preparation method thereof |
| CN110836973A (en) * | 2019-09-09 | 2020-02-25 | 浙江博实生物科技有限公司 | Double-labeling kit for detecting troponin and compound, and preparation and detection methods thereof |
| CN111308084A (en) * | 2019-12-30 | 2020-06-19 | 菲鹏生物股份有限公司 | Detection method and kit for hypersensitive cardiac troponin I |
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| CN110836973A (en) * | 2019-09-09 | 2020-02-25 | 浙江博实生物科技有限公司 | Double-labeling kit for detecting troponin and compound, and preparation and detection methods thereof |
| CN111308084A (en) * | 2019-12-30 | 2020-06-19 | 菲鹏生物股份有限公司 | Detection method and kit for hypersensitive cardiac troponin I |
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