KR20230110262A - Myocardial troponin I detection methods and kits - Google Patents

Abstract

A method and kit for detecting myocardial troponin I in the field of protein detection. The kit includes a first antibody and a second antibody for detecting myocardial troponin I, wherein the antibody is selected from a first antibody that binds to the amino acid fragment at positions 27 to 40 of myocardial troponin I and a second antibody that binds to the myocardial troponin CTNI-CTNC complex.

Description

Myocardial troponin I detection methods and kits

This application claims the priority of a Chinese patent application filed with the Chinese Intellectual Property Office on October 16, 2020, application number 202011114994.1, titled 'Method and kit for detecting myocardial troponin I', all of which are incorporated into the content of this application by reference.

This application relates to the field of protein detection. Specifically, the present application relates to a method and kit for detecting myocardial troponin I.

An increase in myocardial troponin I (cTnI) has very high sensitivity and specificity in the diagnosis of myocardial necrosis. Currently, cTnI has become an indispensable marker for clinical diagnosis, treatment, and measurement of myocardial infarction.

Myocardial troponin is present in a small amount in normal humans, but when myocardial infarction occurs, myocardial cells are damaged and cTnI is released into the blood. Regardless of ST-segment elevation, cTnI increases rapidly, peaks at about 24 hours, and falls to normal levels after 7 to 10 days. The extent of increase in cTnI is related to the degree of necrosis of cardiomyocytes. If cTnI in the patient's blood increases again while decreasing, it means re-injury/necrosis of the myocardium. Changes in cTnI concentrations are clearly associated with the degree of risk, treatment decision-making, and prognosis of Acute Coronary Syndrome (ACS).

The inventors have thoroughly considered the structural characteristics of myocardial troponin I through many theoretical studies and experimental explorations, conducted analysis and research on various troponin antigen sections and detection antibodies to be tested, and obtained a combination of myocardial troponin I antigen regions that can be used for myocardial troponin I detection through many crossover experiments and screenings. The inventors have already demonstrated that antibodies that bind to specific antigen fragments can unexpectedly cross-couple and can be used for highly sensitive detection of cTnI.

In one aspect, the present application provides a myocardial troponin I detection kit, comprising a first antibody and a second antibody for detecting myocardial troponin I, the first antibody binding to the amino acid fragment at positions 27 to 40 of myocardial troponin I, and the second antibody binding to myocardial troponin CTNI-CTNC complex.

In one or several embodiments, the second antibody is a coating antibody when the first antibody is a labeled antibody, or the first antibody is a coating antibody when the second antibody is a labeled antibody.

In one or several embodiments, the kit further comprises a third antibody for detecting myocardial troponin I, wherein the third antibody binds to the amino acid fragment at positions 83 to 93 of myocardial troponin I.

For example, the antibodies are divided into a first antibody group and a second antibody group for pairing, and the first antibody group and the second antibody group are

1) the first antibody group includes a first antibody and the second antibody group includes a second antibody and a third antibody; or

2) the first antibody group comprises a second antibody, and the second antibody group comprises a first antibody and a third antibody; or

3) the first antibody group includes a third antibody, and the second antibody group includes a first antibody and a second antibody;

selected from the group.

In one or several embodiments, the reactivity of the antibody is OD ₄₀₅ ≧0.5 as assessed by ELISA.

In one or several embodiments, the labeled antibody is labeled with at least one of a detectable label and a binding partner. For example, the detectable label may be, for example, a metal particle, a fluorescent label, a chromophore label, an electron high density label, a chemiluminescent label, a radioactive label, or an enzyme label. Specifically, the detectable label is rhodamine, fluorescein, fluorescent microspheres, colloidal gold, acridinium ester, luciferase, mustard peroxidase, alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, glycooxidase, glucose oxidase, galactose oxidase or glucose hexaphosphate dehydrogenase label. The binding partner can be, for example, a biotin/avidin protein or biotin/streptavidin.

In one or several embodiments, the coated antibody is linked to a solid phase and/or binding partner, and the solid phase can be, for example, magnetic particles, latex particles, microtiter plates, nitrocellulose membranes, or microfluidic chips; Binding partners can be, for example, biotin/avidin proteins, biotin/streptavidin; For example, the coated antibody is linked to the solid phase through a binding partner.

In another aspect, the present application provides an antibody combination for detecting myocardial troponin I, comprising a first antibody and a second antibody; optionally further comprising a third antibody; Here, the first antibody, the second antibody and the third antibody are as defined above.

In another aspect, there is provided a use of such an antibody combination for preparing a kit for detecting myocardial troponin I.

In one or several embodiments, the kit is used for diagnosis of early acute myocardial injury, including 1) dynamic detection of myocardial troponin content, eg for real-time monitoring, and/or 2) exclusion or prediction of early acute myocardial injury.

In another aspect, by providing a method for preparing such an antibody combination,

One) immunizing an animal using an amino acid fragment or complex selected from cardiac troponin I amino acid fragments 27 to 40, myocardial troponin I amino acid fragments 83 to 93, and myocardial troponin CTNI-CTNC complex as an antigen or hapten; and

2) and obtaining antibodies that bind to the myocardial troponin I amino acid fragments 27 to 40, the myocardial troponin I amino acid fragments 83 to 93, and the myocardial troponin CTNI-CTNC complex, respectively, from the animal.

In one or several embodiments, the kit of the present application includes a first antibody and a second antibody for detecting myocardial troponin I, wherein the first antibody binds to an amino acid fragment at positions 27 to 40 of myocardial troponin I; the second antibody binds to the myocardial troponin CTNI-CTNC complex; For example, when the first antibody is a labeled antibody, the second antibody is a coating antibody, or when the second antibody is a labeled antibody, since the first antibody is a coating antibody, detection sensitivity can be improved.

The kit of the present application includes a myocardial troponin I detection reagent card (test strip). In one or several embodiments, an immunofluorescence technique is used to label the antibody to a detectable marker, such as a fluorescent microsphere, and a double antibody sandwich method can be used to detect cTnI in a subject, such as a patient with myocardial injury/necrosis. In one or several embodiments, the methods and kits of the present application may realize improved detection sensitivity and/or reduced detection misses.

In one or several embodiments, the present application may provide a method for detecting myocardial troponin I by fluorescence immunochromatography or a reagent comprising fluorescence immunochromatography. In one or several embodiments, immunochromatography may be achieved by a double antibody sandwich method through a test strip. In one or several embodiments, reagents and devices for performing the double antibody sandwich method are not particularly limited and any appropriate reagents and/or devices may be used. For example, a fluorescence immunochromatography test strip may include a sample pad, a bonding pad, a nitrocellulose (NC) film, and an absorbent pad. In one or several embodiments, an antibody labeled with at least one fluorescent group is immobilized on a binding pad. In one or several embodiments, the NC film may be coated with two lines, such as test (T) and control (C) lines, respectively. The T-line may be coated with at least one antibody, and the fluorescent group-labeled antibody of the binding pad may be captured and complexed with the antigen in the sample to form a double antibody sandwich. In one or several embodiments, the sample to be detected is added to the sample port of the detection reagent card, and under the action of the lateral capillary, the sample to be detected first passes through the binding pad to generate a specific immune binding with the fluorescent group-labeled antibody on the binding pad, thereby binding to each other to form an antigen-antibody fluorescent complex and immobilized on the T line. The C line is coated with a material that reacts with the free fluorescent group-labeled antibody, and when the free fluorescent group-labeled antibody passes through the C line, it can generate a specific immune binding with the material on the C line and is fixed to the C line. Individual differences in samples can be corrected by contrasting the fluorescence intensities of the C line and the T line. The fluorescence intensities of the two lines detected by the fluorescence immunoassay are expressed as peak areas, and the T/C value (T peak area/C peak area) is calculated through the calculation software of the detector itself, and fitted to a preset standard curve, and the fluorescence immunoassay automatically converts the corresponding cTnI concentration value in the sample for detection.

In one or several embodiments, the present application can use antibodies bound to different amino acid fragments to identify different locations of the antigen, reduce the risk of missing detection and improve detection rates.

In one or several embodiments, an antibody bound to the 27 to 40 aa fragment of myocardial troponin I is used as a labeling antibody, and an antibody bound to the myocardial troponin I-C complex and an antibody bound to the 83 to 93 aa fragment of myocardial troponin I are used as coating antibodies to mix and coat. In one or several embodiments, an antibody bound to the myocardial troponin I-C complex is used as a labeling antibody, and an antibody bound to the myocardial troponin 27 to 40 aa fragment and an antibody bound to the myocardial troponin 83 to 93 aa fragment are used as coating antibodies to perform mixed coating. In one or several embodiments, an antibody bound to the 83 to 93 aa fragment of myocardial troponin I is used as a labeling antibody, and an antibody bound to the myocardial troponin 27 to 40 aa fragment and an antibody bound to the myocardial troponin I-C complex are used as coating antibodies to perform mixed coating.

In one or several embodiments, the present application provides myocardial troponin I detection methods, detection kits, and manufacturing methods. The kit of this application has improved sensitivity and specificity. In one or several embodiments, the antibodies of the present application may be monoclonal antibodies, polyclonal antibodies. In one or several embodiments, antibodies of the present application may be prepared using methods known in the art. In one or several embodiments, antibodies of the present application may be prepared by immunizing an animal with an antigen comprising an amino acid fragment described herein. Carrier proteins (including but not limited to BSA, ovalbumin, KLH, etc.) can be coupled to immunoreactive substances (eg epitope peptides) to increase immunogenicity. Carrier proteins may include proteins or polypeptides and may serve as immunogen carriers. Polypeptides of this type include albumin, serum proteins, globulins, lens proteins, lipoproteins and/or fragments thereof. In one or several embodiments, an immunoreactive substance (eg, but not limited to, myocardial troponin I amino acid fragment, myocardial troponin CTNI-CTNC complex) can be used to generate antibodies that are friendly to cTnI.

In one or several embodiments, binding of an antibody to an amino acid fragment of myocardial troponin may indicate that the antibody is capable of binding to the amino acid fragment, but the amino acid fragment need not be the minimum binding fragment.

In one or several embodiments, any suitable in vitro assay, cell-based assay, in vivo assay, or animal model may be used to detect effects of an antibody of the present application, such as binding activity and/or cross-reactivity. In one or several embodiments, the assay may include, for example, ELISA, FACS binding assay, Biacore, competitive binding assay, and the like. In one or several embodiments, the binding reactivity of the antibody of the present application to an antigen (antigenic peptide) is expressed, for example, in an ELISA, and a response value ≧0.5 is read at 405 nm by, for example, a peroxidase-labeled ELISA method to confirm this as a relatively good reactivity.

In one or several embodiments, the kit comprises a first antibody and a second antibody as described above, and in one or several embodiments, the kit further comprises a third antibody as described above; In one or several embodiments, other antibodies may be used as coating antibodies or labeling antibodies.

In one or several embodiments, the coating antibody is bound to a solid phase. In one or several embodiments, the coating antibody can be used to coat a solid phase support. In one or several embodiments, the solid support is not particularly limited and may be, for example, magnetic particles, latex particles, microtiter plates, nitrocellulose membranes, or microfluidic chips. In one or several embodiments, the coating antibody binds to a binding partner, which binding partner is, for example, a biotin/avidin protein, biotin/streptavidin; In one or several embodiments, the coated antibody is linked to the solid phase via a binding partner.

In one or several embodiments, the labeled antibody is labeled with a detectable label such as a fluorescent label; In one or several embodiments, the labeled antibody is linked to a binding partner, and the binding partner is, for example, biotin/avidin protein, biotin/streptavidin; In one or several embodiments, the labeled antibody proceeds to label a detectable label via a binding partner.

In one or several embodiments, the term 'antibody' in this application is used in the broadest sense and may include full-length monoclonal antibodies, polyclonal antibodies, bispecific or multispecific antibodies, chimeric antibodies and antigen-binding fragments of antibodies as long as they exhibit the requisite biological activity, such as binding to the cTnI antigen. An 'antibody fragment' comprises a portion of a full-length antibody, preferably the antigen-binding region or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab') ₂ , Fd, Fv, dAb, complementarity determining region (CDR) fragments, single-stranded antibodies (eg scFv), bivalent antibodies or domain antibodies.

In one or several embodiments, the detectable label used in this application is not particularly limited. In one or several embodiments, the label may include, but is not limited to, a metal particle, a fluorescent label, a chromophore label, an electron high density label, a chemiluminescent label, a radioactive label, or an enzyme label. For example, indirect detection through enzymatic reactions or molecular interactions. In one or several embodiments, exemplary labels may include, but are not limited to, labels such as rhodamine, fluorescein, fluorescent microspheres, colloidal gold, acridinium esters, luciferase, mustard peroxidase, alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, glycooxidase, glucose oxidase, galactose oxidase, or glucose hexaphosphate dehydrogenase. don't In one or several embodiments, the detectable label used in this application is colloidal gold.

In one or several embodiments, the kit of the present application includes reagents suitable for immunoassay. In one or several embodiments, the kits of the present application may be used in immunoassays such as ELISA, indirect immunofluorescence assay (IFA), radioimmunoassay (RIA), and other non-enzyme-linked antibody binding assays or methods.

A sample of cTnI to be detected in the present application may include a biological tissue, cell, or body fluid in a healthy or pathological state, and includes, for example, blood samples such as plasma, serum, and blood products, and, for example, semen or vaginal secretion.

In one or several embodiments, the present application provides antibody combinations for detecting myocardial troponin I and their use in making a kit for detecting myocardial troponin I. In one or several embodiments, the application provides combinations of myocardial troponin CTNI-CTNC complexes with immunoreactive polypeptides comprising the amino acid fragments described herein and their use in preparing antibodies for myocardial troponin I detection. In one or several embodiments, the present application provides methods of making antibodies for detecting myocardial troponin I. The method includes preparing an antibody for detecting myocardial troponin I, such as a monoclonal antibody or a polyclonal antibody, by immunizing an animal using an immunoreactive polypeptide comprising an amino acid fragment described herein and a CTNI-CTNC complex as an antigen or a hapten, respectively. Monoclonal antibodies or polyclonal antibodies can be prepared by methods known in the art.

In one or several embodiments, the present application provides use of a CTNI-CTNC complex and an immunoreactive polypeptide comprising an amino acid fragment described herein in the manufacture of a reagent or kit for detecting myocardial troponin I.

This application has the following advantageous effects.

The present application provides a myocardial troponin (cTnI) detection method and kit that improve detection sensitivity using an antibody bound to the 27 to 40 aa fragment of myocardial troponin I and an antibody bound to the myocardial troponin CTNI-CTNC complex, and reduce the risk of missing detection by using an antibody additionally bound to the 83 to 93 aa fragment of myocardial troponin I.

Hereinafter, the present application will be described in more detail, mainly with specific examples. The following examples are provided to illustrate embodiments of the present application and are not intended to limit the scope of the present application. This application may arbitrarily select and include embodiments not presented by examples.

Example 1, Preparation of Myocardial Troponin I Monoclonal Antibody

1. Immunized animals: Take BALB/c mice of the same species as 8-12 week-old myeloma cells, mix 100μg/mouse cTnI antigen and the same amount of Freund's complete adjuvant (FCA) sufficiently, and inject into the mouse peritoneal cavity, mix 100μg/mouse cTnI antigen and the same amount of Freund's incomplete adjuvant (FIA) at 2-week intervals, and inject into the mouse peritoneal cavity several times to immunize. strengthen After measuring mouse serum (indirect ELISA method), a titer of 1:2000 or higher can be used for fusion, and 3 days before fusion, a dose of 50 μg/mouse is injected intraperitoneally to enhance immunity again.

2. Preparation of feeder cells

Abdominal macrophages from BALB/c mice were used as feeder cells. One day before fusion, BALB/c mice were killed by pulling the cervix, immersed in 75% alcohol, cut the abdominal skin with scissors under aseptic operation on a super clean bench, exposed the peritoneum, injected 5 mL of RPMI 1640 basic culture into the abdominal cavity using a syringe, washed repeatedly, recovered the lavage, centrifuged at 1000 rpm for 5 minutes, retained the precipitate, and replaced the RPMI 1640 screening culture medium (HAT-containing RPMI 16 40 complete culture medium), adjust the cell concentration to 1×10 ⁵ cell/mL, add to a 96-well plate, and incubate overnight under conditions of 150 μL/well, 37° C., 5% CO ₂ .

3. Preparation of immune spleen cells

3 days after the last immunization of the mouse, the spleen was taken out under aseptic conditions, placed on a plate, washed once with RPMI 1640 basic culture medium, placed on a nylon mesh placed in a small beaker, ground and filtered, and a cell suspension was prepared. After centrifugation and removal of the supernatant, the cells were resuspended in RPMI 1640 basic culture medium, repeated three times in this manner, and then counted.

4. Fusion of cells

(1) Take 40mL of HAT culture medium, 15mL of DMEM serum-free culture medium, and 1mL of 50% PEG (M12000) and put them in a 37℃ water bath to preheat.

(2) A suspension of mouse myeloma cells Sp2/0 (preserved by Fapon Biotech Inc.) (2-5×10 ⁷ cells) and the immune splenocytes (10 ⁸ cells) are taken and placed in a 50mL centrifuge tube, sufficiently diluted, and DMEM serum-free culture medium is added up to 40mL. After centrifugation for 10 minutes, the supernatant was completely discarded and diluted sufficiently.

(3) Place the centrifuge tube in preheated water at 37°C, take 0.7mL of the preheated 50% PEG solution, and leave it for 90 seconds. 15 mL of preheated 37°C serum-free culture medium is immediately added dropwise. (4) Add up to 40 mL of DMEM serum-free culture medium and centrifuge for 10 minutes, completely discarding the supernatant. Add 40 mL of HAT broth containing 15-20% fetal bovine serum. After thoroughly mixing with a pipette, 2 drops per well were dropwise added to the small wells of four 96-well cell culture plates containing feeder cells, and cultured in an incubator at 37°C and 7% CO ₂ .

5. Selective culture of hybridoma cells

Immune mouse splenocytes and mouse myeloma cells are treated with PEG to form a mixture of various cellular components, including unfused myeloma cells and immune splenocytes, syncytes of myeloma cells and immune splenocytes, and binuclear bodies of myeloma cells and immune splenocytes. Only the latter can form hybridoma cells. To do this, it is necessary to remove unfused cells and homologous syncytia from this diverse cell mixture and select true hybrid cells. Therefore, on the 1st, 3rd, 5th, and 7th days after cell fusion, the solution was changed to the HAT culture medium described above and cultured.

6. Antibody Detection and Hybridoma Cell Cloning

The supernatant from each culture well is aspirated and culture wells containing the cTnI antibody are detected in the culture medium using an indirect ELISA method. Using the limiting dilution method, hybridoma cells were cloned. After culturing, single cells can proliferate into homologous cell clones, and cell lines that are highly reactive and stably secrete anti-cTnI monoclonal antibodies were obtained through reactive screening.

Reactive screening method: Coat microtiter plates (Nunc, Maxisorb) with coating buffer containing 2.5 μg/mL human cTnI (used as antigen) in 100 μL/well at room temperature for 1 hour. After coating, incubation was performed for 30 minutes in PBS buffer and 1% monoclonal antibody. It was then washed with wash buffer. 100 μL/well antibody samples were incubated for 1 hour at room temperature. It was then washed twice more with the washing solution. It was then incubated for 1 hour at room temperature with detection antibody peroxidase-conjugated goat anti-mouse IgG diluted 1:40,000 in PBS buffer in 100 μL/well. After washing again with the washing buffer, the peroxidase activity is measured by a conventional method (e.g., reading the response value at 405 nm with an ELISA plate reader), and an antibody with an OD ₄₀₅ ≧0.5 (which has relatively good reactivity) is obtained.

Example 2, evaluation of antibody-binding fragments

Different cTnI antigen peptides are used to coat the microholes, and the concentration of the primary antibody of the monoclonal antibody is diluted to 1 μg/mL using PBS+20% NBS as a diluent, and goat anti-mouse IgG-HRP is used as the secondary antibody to determine the binding fragment of the monoclonal antibody according to the reaction situation of each monoclonal antibody to different antigens. The binding fragment evaluation results of the cell lines used in the embodiments are shown in the table below.

Here, the IC antibody is an antibody bound to the CTNI-CTNC complex, and the production method was obtained by replacing the CTNI-CTNC complex with the cTnI antigen and immunizing with reference to the above-mentioned information to obtain an IC antibody. The CTNI-CTNC complex can be purchased from HyTest.

Example 3, pairing screening

Antibodies of different binding fragments are selected for coating and labeling, cross-pairing experiments are performed, and the screening process is as follows.

1. Cover

(1) 100uL of fluorescent microspheres were centrifuged at 15000rpm/8min/4°C, the supernatant was removed after centrifugation, resuspended in 100uL, and ultrasonically vibrated 2-3 times;

(2) EDC and NHS at 10 mg/mL were prepared;

(3) Activation: Activate the fluorescent microspheres with EDC and NHS to a final volume of 200uL, mix thoroughly for 18 minutes in the dark, and centrifuge at 15000rpm/10min/4°C to remove the supernatant;

(4) washing: resuspend in 100 uL, vibrate 2-3 times with ultrasonic waves, centrifuge at 15000 rpm/8 min/4° C., remove supernatant, repeat twice;

(5) Coupling: after resuspending in 200uL and vibrating with ultrasonic waves 2-3 times, the antibody was added to the fluorescent microspheres until the final volume was 300uL, thoroughly mixed in the dark, and reacted at 37° C. for 2 hours;

(6) Blocking: 100uL of blocking agent was added, thoroughly mixed in a dark room, and reacted at 37° C. for 1 hour;

(7) Washing: After blocking, centrifugation at 15000 rpm/12 min/4° C., removing the supernatant, resuspending in 100 uL, vibrating 2-3 times with ultrasonic wave, and centrifuging at 15000 rpm/8 min/4° C. to remove the supernatant;

(8) Preservation: Resuspend at 100uL and vibrate 2-3 times with ultrasonic waves to preserve at 4°C for use.

2. Coating

(1) Assemble and prepare a nitrocellulose membrane and a fluorescent PVC substrate.

(2) Dilute the antibody to 1.0 to 2.0 mg/mL, draw a line uniformly on the NC film with a sputter coater, put it in a 37°C incubator, and dry it for at least 45 minutes. Assemble, cut strips, add samples and measure.

3. Detection

(1) Quality Control: cTnI antigen diluted to 50, 20, 5, 1.25 ng/mL using PBS.

(2) Detection method: Detect using a fluorescence meter. The instrument reading T/C represents the ratio of the T peak area to the C peak area and the reaction pairing activity, the higher the T/C in quality control and positive samples, the higher the activity.

From the results in Table 1 (including subsequent Table 1), the first cross-pairing selection resulted in pairings that were relatively dominant in activity and whose test results had a constant slope, and then the minimum detection limit for these dominant pairings was tested and tested with 80 fixed value samples (antibody self-pairing was not tested).

As can be seen from the results in Table 2, pairing of the cTnI antibody bound to 27 to 40 aa and the cTnI antibody bound to 83 to 93 aa can be obtained in the second screening using the minimum detection limit as an indicator, and the pairing of the cTnI antibody bound to 83 to 93 aa and the IC antibody does not reach the pairing of the cTnI antibody bound to 27 to 40 aa and the IC antibody at the minimum detection limit, and the minimum detection limit is Since it indicates the sensitivity of the pairing, the corresponding dominant combination is maintained in the following 1+2 mode and the optimization of the pairing continues.

In addition, to reduce the risk of missing detection, the next step may consider the introduction of antibodies of different binding fragments for testing. We first measure the activity and then use 100 positive fixed value samples to investigate the missing detection situation of each pairing. Among them, 12F-3, 1C-2, and 4F-8 are antibodies that bind to the 27 to 40 aa fragment, 2C-7, 8E-2, and 5F-2 are antibodies that bind to the 83 to 93 aa fragment, and 15A-6 and 16C-3 are all antibodies that bind to the CTNI-CTNC complex.

When the quality control concentration is 50 ng/mL, the T/C value is less than 0.1, there is no gradient, and it is basically recorded as inactive.

As can be seen from the results in Table 3, when the cTnI antibody bound to 83 to 93 aa is introduced into the dominant combination obtained in the 1+1 mode, that is, the cTnI antibody bound to 27 to 40 aa and the IC antibody combination, several binding fragments of the cTnI antigen can be identified, each detection defect can be compensated, and the cTnI antibody bound to 18 to 29 aa and the cTnI antibody bound to 180 to 190 aa can be identified. Pairing performance is better than introducing a cTnI antibody and an antibody of a different strain bound to the same fragment; On the basis of high sensitivity (minimum detection limit) (Table 2), missing detection is further reduced and the accuracy of detection results is improved through complementation of binding fragments.

The present application also obtained antibodies bound to the 27 to 40 aa fragments by immunization using the cTnI amino acid fragment 27 to 40 coupling vector protein as an immunogen through the above-mentioned antibody production method, and the minimum detection limit of pairing of the IC antibodies 15A-6 and 16C-3 with the antibody I27C-3 with an OD ₄₀₅ ≥ 0.5 is all less than 0.4 ng/mL (in the above, evaluated by the aforementioned fluorescent microsphere detection method); Antibodies bound to the 83 to 93aa fragments were obtained through immunization using the cTnI amino acid fragment 83 to 93 coupling vector protein as an immunogen, and, for example, antibody I83A-9 having an OD ₄₀₅ ≧0.5 through a reactive screening reaction, as a result of pairing with both, the omission rate did not exceed 1% (evaluated by the fluorescent microsphere detection method mentioned above).

4. Colloidal Gold Platform Testing

The dominant combinations obtained in the 1+2 mode, i.e. cTnI antibody bound to 27 to 40 aa, cTnI antibody bound to 83 to 93 aa and IC antibody were mixed using 12F-3 as the labeling antibody and 2C-7 and 15A-6 as the coating antibody, applied to a colloidal gold platform as an example, and tested with 80 fixed value samples and 500 healthy human sera (negative samples).

1. cover

(1) Preparation of colloidal gold: using the traditional sodium citrate reduction method, first heat the chloroauric acid solution to boiling, quickly add a certain proportion of the trisodium citrate solution, stir evenly, stop heating when the color of the solution becomes wine-colored and no longer changes, and cool to room temperature to obtain a colloidal gold solution with a concentration of 4/only.

(2) Labeling: A 0.2MK ₂ CO ₃ solution is added to the colloidal gold solution to adjust the pH to 7.0 to 10.0.

(3) Centrifugation: The labeled antibody is added to the pH-adjusted colloidal gold solution, mixed thoroughly, a blocking agent is added, labeling is terminated, and the supernatant is removed by centrifugation at 10000 rpm/7min/4°C.

(4) Dissolution again: resuspend to 100 uL and sonicate 2 to 3 times.

(5) Gold application: After diluting the concentrated gold obtained by resuspending and applying it to a glass cellulose film, put it in a freeze dryer and freeze-dry it (1 to 2 hours) or put it in a drying room at 37 ° C and dry it overnight.

2. Coating

(1) Assemble and prepare a nitrocellulose membrane and a colloidal gold PVC substrate.

(2) Dilute the antibody to 1.0 to 2.0 mg/mL, draw a line uniformly on the NC film with a sputter coater, put it in a 37°C incubator, and dry it for at least 45 minutes. Assemble, cut strips, add samples and measure.

The detection results are as follows.

1) Minimum detection limit (colloidal gold platform):

The number after the letter C in Table 4 indicates the intensity of color development, and the larger the number, the weaker the color development.

Eighty fixed-value samples were measured, and the color development of C9 and C9 or higher was considered detected, and the minimum detection limit reached 0.3 ng/mL.

1) Negative agreement rate

As a result of measuring 500 voice samples, the voice concordance rate is 98.2%.

The above specific embodiments have been described in more detail for the purpose, technical solutions, and beneficial effects of the present application, and the above-mentioned are only specific embodiments of the present application, and do not limit the present application. All modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of this application shall be included in the protection scope of this application.

Claims (16)

A myocardial troponin I detection kit comprising a first antibody and a second antibody for detecting myocardial troponin I,
A kit for detecting myocardial troponin I, wherein the first antibody binds to the amino acid fragment at positions 27 to 40 of myocardial troponin I, and the second antibody binds to myocardial troponin CTNI-CTNC complex. According to claim 1,
When the first antibody is a labeled antibody, the second antibody is a coating antibody, or
When the second antibody is a labeled antibody, the first antibody is a coating antibody, myocardial troponin I detection kit. According to claim 1,
A kit for detecting myocardial troponin I, further comprising a third antibody for detecting myocardial troponin I, wherein the third antibody binds to an amino acid fragment at positions 83 to 93 of myocardial troponin I. According to claim 3,
The antibodies are divided into a first antibody group and a second antibody group for pairing,
1) the first antibody group includes a first antibody and the second antibody group includes a second antibody and a third antibody; or
2) the first antibody group comprises a second antibody, and the second antibody group comprises a first antibody and a third antibody; or
3) The myocardial troponin I detection kit, wherein the first antibody group includes a third antibody, and the second antibody group includes a first antibody and a second antibody. According to claim 1,
The reactivity of the antibody is OD ₄₀₅ ≥ 0.5 according to ELISA evaluation, myocardial troponin I detection kit. According to any one of claims 1 to 5,
The myocardial troponin I detection kit, wherein the labeled antibody is labeled with at least one of a detectable label and a binding partner. According to claim 6,
The myocardial troponin I detection kit, wherein the detectable label is a metal particle, a fluorescent label, a chromophore label, an electron high-density label, a chemiluminescent label, a radioactive label, or an enzyme label. According to claim 6,
The detectable label is rhodamine, fluorescein, fluorescent microspheres, colloidal gold, acridinium ester, luciferase, mustard peroxidase, alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, glycooxidase, glucose oxidase, galactose oxidase or glucose hexaphosphate dehydrogenase label, myocardial troponin I detection kit. According to claim 6,
A myocardial troponin I detection kit, wherein the binding partner is biotin/avidin protein or biotin/streptavidin. According to any one of claims 1 to 5,
The coated antibody is linked to a solid phase and/or binding partner, the solid phase being magnetic particles, latex particles, microtiter plates, nitrocellulose membranes or microfluidic chips; A myocardial troponin I detection kit, wherein the binding partner is biotin/avidin protein or biotin/streptavidin. According to claim 10,
A myocardial troponin I detection kit, wherein the coating antibody is linked to the solid phase via a binding partner. As an antibody combination for detecting myocardial troponin I,
An antibody combination for detecting myocardial troponin I comprising a first antibody and a second antibody, wherein the first antibody binds to an amino acid fragment at positions 27 to 40 of myocardial troponin I, and the second antibody binds to myocardial troponin CTNI-CTNC complex. According to claim 12,
An antibody combination for detecting myocardial troponin I, further comprising a third antibody that binds to an amino acid fragment at positions 83 to 93 of myocardial troponin I. Use of the antibody combination according to claim 12 or 13 for preparing a kit for detecting myocardial troponin I. According to claim 14,
Wherein the kit is used for diagnosis of early acute myocardial injury, including 1) dynamic detection of myocardial troponin content for real-time monitoring and/or 2) exclusion or prediction of early acute myocardial injury. A method for producing the antibody combination according to claim 12 or 13,
1) Immunizing an animal using an amino acid fragment or complex selected from cardiac troponin I amino acid fragments 27 to 40, myocardial troponin I amino acid fragments 83 to 93, and myocardial troponin CTNI-CTNC complex as an antigen or hapten; and 2) obtaining antibodies that bind to the myocardial troponin I amino acid fragments 27 to 40, the myocardial troponin I amino acid fragments 83 to 93, and the myocardial troponin CTNI-CTNC complex, respectively, from the animal.