WO2022077931A1 - Cardiac troponin i detection method and kit - Google Patents

Abstract

A cardiac troponin I detection method and a kit, which belong 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 following antibodies: a first antibody binding to an amino acid fragment at positions 83-93 of cardiac troponin I; and a second antibody, which is an antibody binding to the cardiac troponin CTNI-CTNC complex.

Description

Cardiac troponin I detection method and kit

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority of the Chinese Patent Application No. 202011114995.6 and entitled "A Cardiac Troponin I Detection Method and Kit" filed with the China Patent Office on October 16, 2020, the entire contents of which are combined by application in this application.

technical field

The present application relates to the field of protein detection. Specifically, the present application relates to a detection method and kit for cardiac troponin I.

Background technique

Elevation of cardiac troponin I (cTnI) has very high sensitivity and specificity for the diagnosis of myocardial injury/necrosis (myocardial necrosis). At present, cTnI has become an indispensable marker for clinical diagnosis, treatment and monitoring of myocardial infarction.

Cardiac troponin exists in trace amounts in normal human body, but when myocardial infarction occurs, the damage of myocardial cells results in the release of cTnI into the blood. Regardless of ST segment elevation, cTnI rises sharply, peaking around 24 hours, and falling to normal levels after 7-10 days. The magnitude of cTnI elevation was related to the degree of myocardial cell necrosis to a certain extent. If the cTnI in the patient's blood rises again during the descending process, it means that the myocardium is damaged/necrotic again. Changes in cTnI concentrations are clearly related to the risk of ACS (Acute Coronary Syndrome), treatment decisions and prognosis.

SUMMARY OF THE INVENTION

After a lot of theoretical research and experimental exploration, the inventors have fully considered the structural properties of cardiac troponin I, analyzed and studied various troponin antigen ranges and detection antibodies to be tested, and through a large number of cross-experiments and screenings, obtained can be used. A combination of cardiac troponin I antigen regions for cardiac troponin I detection. The inventors have demonstrated that antibodies that bind specific antigenic fragments are unexpectedly able to combine with each other for highly sensitive and specific detection of cTnI.

In one aspect, the present application provides 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 the first antibody of cardiac troponin I Amino acid fragment at positions 83-93; secondary antibody binds to the cardiac troponin CTNI-CTNC complex.

In one or more embodiments, the second antibody is a coated antibody when the first antibody is a labeled antibody; or the first antibody is a coated antibody when the second antibody is a labeled antibody.

In one or more embodiments, the kit further comprises a third antibody that detects cardiac troponin I, the third antibody binding to amino acid fragment 41-49 of cardiac troponin I;

For example, the antibodies are divided into a first set of antibodies and a second set of antibodies for pairing, wherein the first set of antibodies and the second set of antibodies are selected from the group consisting of:

1) the first group of antibodies includes a first antibody, and the second group of antibodies includes a second antibody and a third antibody;

2) the first group of antibodies includes the second antibody, and the second group of antibodies includes the first antibody and the third antibody;

3) The first set of antibodies includes the third antibody, and the second set of antibodies includes the first antibody and the second antibody.

In one or more embodiments, the reactivity of the antibody is _OD405 > 0.5 as assessed by ELISA.

In one or more embodiments, the labeled antibody is labeled with at least one of a detectable label and a binding partner. Detectable labels can be, for example, metal particles, fluorescent labels, chromophore labels, electron dense labels, chemiluminescent labels, radioactive labels, or enzymatic labels. Specifically, the detectable label can be, for example, rhodamine, fluorescein, fluorescent microspheres, colloidal gold, acridine ester, luciferase, horseradish peroxidase, alkaline phosphatase, β-galactosidase, Labeled with glucoamylase, lysozyme, sugar oxidase, glucose oxidase, galactose oxidase or glucose-6-phosphate dehydrogenase. The binding partner can be, for example, biotin/avidin or biotin/streptavidin.

In one or more embodiments, the coated antibody is attached to a solid phase and/or a binding partner, which may be, for example, magnetic particles, latex particles, microtiter plates, nitrocellulose membranes, or microfluidic chips ; the binding partner can be, for example, biotin/avidin, biotin/streptavidin; for example, the coated antibody is attached to the solid phase through the binding partner.

In another aspect, the application provides an antibody combination for detecting cardiac troponin I, including a first antibody and a second antibody; optionally, also includes a third antibody; wherein the first antibody, the second antibody, and the third antibody are as above definition.

In another aspect, the present application provides the use of the antibody combination as described above in the preparation of a kit for detecting cardiac troponin I.

In one or more embodiments, the kits are used for 1) dynamic detection of cardiac troponin levels, eg, for real-time monitoring, and/or 2) diagnosis of early acute myocardial injury, including, eg, excluding or Prediction of early acute myocardial injury.

In another aspect, the application provides a method for preparing the antibody combination as described above, the method comprising:

1) using amino acid fragments or complexes selected from cardiac troponin I amino acid fragments 41-49, cardiac troponin I amino acid fragments 83-93 and cardiac troponin CTNI-CTNC complexes as antigens or haptens to immunize animals; and

2) Obtain from the animal antibodies that bind to the cardiac troponin I amino acid fragments 41-49, the cardiac troponin I amino acid fragments 83-93, and the cardiac troponin CTNI-CTNC complex, respectively.

In one or more embodiments, the kit of the present application includes a first antibody and a second antibody for detecting cardiac troponin I, wherein the first antibody binds to positions 83-93 of cardiac troponin I Amino acid fragment; the secondary antibody binds to the cardiac troponin CTNI-CTNC complex; for example, when the primary antibody is a labeled antibody, the secondary antibody is a coating antibody; or when the secondary antibody is a labeled antibody, the primary antibody is a coating antibody, and can be Improve detection specificity.

The kit of the present application includes a cardiac troponin I detection reagent card (test strip). In one or more embodiments, immunofluorescence techniques can be used to label antibodies on detectable labels such as fluorescent microspheres, and a double antibody sandwich method can be used to detect cTnI in subjects such as patients with myocardial injury/necrosis. In one or more embodiments, the methods and kits of the present application can improve detection specificity and/or reduce missed detections.

In one or more embodiments, the present application may provide methods for the detection of cardiac troponin I by fluorescent immunochromatography or provide reagents comprising performing fluorescent immunochromatography. In one or more embodiments, immunochromatography can be accomplished by a double-antibody sandwich method with test strips. In one or more embodiments, the reagents and devices for performing the double-antibody sandwich method are not particularly limited, and any suitable reagents and/or devices may be employed. For example, fluorescent immunochromatographic test strips can include sample pads, binding pads, nitrocellulose (NC) membranes, and absorbent pads. In one or more embodiments, at least one fluorophore-labeled antibody is immobilized on the binding pad. In one or more embodiments, the NC membrane can be coated with two lines, eg, a test (T) line and a control (C) line, respectively; the T line can be coated with at least one antibody and can capture the The fluorophore-labeled antibody on the pad forms a complex with the antigen in the sample, thereby forming a double-antibody sandwich. In one or more embodiments, the sample to be tested is added to the sample loading port of the detection reagent card, and under the action of the lateral capillary, the sample to be tested first passes through the binding pad, and the fluorophore-labeled antibody on the binding pad undergoes specific binding. Immunobinding, each binds to form an antigen-antibody fluorescent complex, which is immobilized in the T-line. The C line is coated with a substance that reacts with the free fluorophore-labeled antibody. When the free fluorophore-labeled antibody passes through the C line, it can specifically immunocombine with the substance on the C line, thereby being immobilized on the C line. in line. The fluorescence intensities of the C-line and T-line are contrasted, so that individual differences between samples can be corrected. The fluorescence intensity of the two lines detected by the fluorescence immunoassay analyzer is reflected in the peak area, and the T/C value (T peak area/C peak area) is calculated by the instrument's own calculation software and fitted to the set standard curve. The fluorescence immunoassay analyzer automatically converts the concentration value of cTnI in the corresponding sample to be tested.

In one or more embodiments, the present application can use antibodies that bind different amino acid fragments to recognize multiple positions on the antigen, thereby reducing the risk of missed detection and improving the detection rate.

In one or more embodiments, an antibody that binds to the 83-93aa fragment of cardiac troponin I is used as the labeling antibody, and an antibody that binds to the cardiac troponin I-C complex and 41-49aa that binds to cardiac troponin I is used Fragmented antibodies are mixed as coating antibodies. In one or more embodiments, an antibody that binds to the cardiac troponin I-C complex is used as the labeling antibody, and an antibody that binds the 83-93aa fragment of cardiac troponin I and 41-49aa that binds cardiac troponin I Fragmented antibodies are mixed as coating antibodies. In one or more embodiments, an antibody that binds to the 41-49aa fragment of cardiac troponin I is used as the labeling antibody, and an antibody that binds to the 83-93aa fragment of cardiac troponin I is complexed with an antibody that binds to the cardiac troponin I-C The antibody of the substance was mixed as the coating antibody.

In one or more embodiments, the present application provides a cardiac troponin I detection method, detection kit and preparation method. The kit of the present application has improved sensitivity and specificity. In one or more embodiments, the antibodies of the present application may be monoclonal or polyclonal antibodies. In one or more embodiments, the antibodies of the present application can be prepared using methods known in the art. In one or more embodiments, the antibodies of the present application can be prepared by immunizing animals with antigens comprising the amino acid fragments described herein. To increase immunogenicity, carrier proteins (including but not limited to BSA, ovalbumin, KLH, etc.) can be conjugated to immunoreactive substances (eg, epitope peptides). Carrier proteins can include proteins or polypeptides that can function as immunogenic carriers. These types of polypeptides include albumin, serum protein, globulin, crystallin, lipoprotein and/or fragments thereof. In one or more embodiments, immunoreactive substances (eg, cardiac troponin I amino acid fragments, cardiac troponin CTNI-CTNC complex, but not limited thereto) can be used to generate antibodies with affinity for cTnI.

In one or more embodiments, binding of an antibody to an amino acid fragment of cardiac troponin may refer to the ability of the antibody to bind to the amino acid fragment, although the amino acid fragment is not necessarily the smallest binding fragment.

In one or more embodiments, any suitable in vitro assay, cell-based assay, in vivo assay, animal model, etc., can be used to test the effects of the antibodies of the present application, such as binding activity and/or cross-reactivity. In one or more embodiments, the assay can include, for example, ELISA, FACS binding assay, Biacore, competitive binding assay, and the like. In one or more embodiments, the reactivity of the antibodies of the present application in binding to an antigen (antigenic peptide) is characterized, for example, in an ELISA, for example, as determined by reading a reaction value ≥ 0.5 at 405 nm in a peroxidase-labeled ELISA method for better reactivity.

In one or more embodiments, the kit includes the first antibody and the second antibody as described above; in one or more embodiments, the kit also includes the third antibody as described above; in In one or more embodiments, other antibodies may also be used as coating or labeling antibodies.

In one or more embodiments, the coated antibody is bound to a solid phase. In one or more embodiments, the coated antibody can be used to coat a solid support. In one or more embodiments, the solid support is not particularly limited, and it can be, for example, magnetic particles, latex particles, microtiter plates, nitrocellulose membranes, or microfluidic chips. In one or more embodiments, the coated antibody binds to a binding partner such as biotin/avidin, biotin/streptavidin; in one or more In embodiments, the coated antibody is attached to the solid phase via a binding partner.

In one or more embodiments, the labeled antibody is labeled with a detectable label, such as a fluorescent label; in one or more embodiments, the labeled antibody is linked to a binding partner, the binding partner Examples are biotin/avidin, biotin/streptavidin; in some embodiments, the labeled antibody is labeled with a detectable label by a binding partner.

In one or more embodiments, the term "antibody" is used herein in the broadest sense, which may 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, such as binding to 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') ₂ , Fd, Fv, dAb, complementarity determining region (CDR) fragments, single chain antibodies (eg, scFv), diabodies or domain antibodies.

In one or more embodiments, the detectable labels used in this application are not particularly limited. In one or more embodiments, the labels may include, but are not limited to, metal particles, fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, radiolabels, or enzymatic labels, eg, by enzymatic reactions or molecular interactions for indirect detection. In one or more embodiments, exemplary labels include, but are not limited to, can be rhodamine, fluorescein, fluorescent microspheres, colloidal gold, acridine esters, luciferase, horseradish peroxidase, alkaline phosphate Enzymes, beta-galactosidase, glucoamylase, lysozyme, sugar oxidase, glucose oxidase, galactose oxidase or glucose-6-phosphate dehydrogenase label, among others.

In one or more embodiments, the kits of the present application include reagents suitable for performing immunoassays. In one or more embodiments, the kits of the present application can be used to perform immunoassays, such as ELISA, indirect immunofluorescence assay (IFA), radioimmunoassay (RIA), and other non-enzyme-linked antibody binding assays or methods.

In this context, a sample of cTnI to be examined may include biological tissues, cells or body fluids in healthy or pathological states, eg blood samples, eg plasma, serum, blood products, eg semen or vaginal secretions.

In one or more embodiments, the present application provides antibody combinations for the detection of cardiac troponin I, and their use in the manufacture of kits for the detection of cardiac troponin I. In one or more embodiments, the application provides combinations of immunoreactive polypeptides comprising amino acid fragments described herein and cardiac troponin CTNI-CTNC complexes, and their use in the preparation of antibodies for the detection of cardiac troponin I the use of. In one or more embodiments, the present application provides a method of making an antibody that detects cardiac troponin I, the method comprising adding an immunoreactive polypeptide comprising an amino acid fragment described herein and the CTNI-CTNC complex as Animals are immunized with the antigen or hapten, respectively, to prepare antibodies such as monoclonal or polyclonal antibodies that detect cardiac troponin I. Monoclonal or polyclonal antibodies can be prepared by methods known in the art.

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

This application has the following beneficial effects:

The present application provides a cardiac troponin (cTnI) detection method and kit, using an antibody that binds to the 83-93aa fragment of cardiac troponin I and an antibody that binds to the cardiac troponin CTNI-CTNC complex to improve detection specificity , and further reduce the risk of missed detection by using an antibody that binds to the 41-49aa fragment of cardiac troponin I.

Detailed ways

The present application will be described in further detail below mainly in conjunction with specific embodiments. The following examples are provided to illustrate embodiments of the present application and are not intended to limit the present application. The application may optionally include embodiments not shown by way of example.

Example 1. Preparation of Cardiac Troponin I Monoclonal Antibody

1. Immune animals: Take 8-12-week-old BALB/c mice of the same strain as myeloma cells, mix well with 100 μg/mouse cTnI antigen and an equal amount of Freund's complete adjuvant, and inject them into the abdominal cavity of mice. Every 2 weeks, 100 μg/mouse cTnI antigen was thoroughly mixed with the same amount of incomplete Freund's adjuvant, and injected into the mouse intraperitoneally for multiple times for boosting immunization. After testing mouse serum (indirect ELISA method), those with a titer above 1:2000 can be used for fusion. Three days before fusion, the mice were boosted by intraperitoneal immunization again at a dose of 50 μg/mouse.

2. Preparation of Feeder Cells

BALB/c mouse peritoneal macrophages were used as feeder cells. One day before fusion, BALB/c mice were sacrificed by pulling their necks, immersed in 75% alcohol, and in a clean bench, the abdominal skin was cut open with scissors under aseptic operation, the peritoneum was exposed, and 5 mL of RPMI 1640 basal medium was injected into the abdominal cavity with a syringe. Rinse repeatedly, recover the rinse solution, centrifuge at 1000rpm for 5 minutes, leave the precipitate, resuspend in RPMI 1640 screening medium (complete RPMI 1640 medium containing HAT), adjust the cell concentration to 1×10 ⁵ cells/mL, add to 96 wells Plate, 150 μL/well, incubated overnight at 37°C, 5% CO ₂ .

3. Preparation of Immune Splenocytes

Three days after the last immunization of the mice, the spleen was taken out under sterile conditions, placed in a petri dish, washed once with RPMI 1640 basal medium, and placed on a nylon mesh in a small beaker to grind and filter to prepare a cell suspension. Centrifuge, discard the supernatant, resuspend in RPMI 1640 basal medium, repeat this three times, and count.

4. Cell Fusion

(1) Take 40 mL of HAT medium, 15 mL of DMEM serum-free medium and 1 mL of 50% PEG (M12000) and place them in a 37°C water bath to pre-warm;

(2) Take mouse myeloma cell Sp2/0 (preserved by Philips Biotech Co., Ltd.) (2-5×10 ⁷ cells) and the above immune spleen cells (10 ⁸ cells) suspension and add them to a 50 mL centrifuge tube Mix well, and add DMEM serum-free medium to 40 mL. Centrifuge for 10 minutes, pour off the supernatant, and mix.

(3) Place the centrifuge tube in pre-warmed water at 37°C, take 0.7 mL of the pre-warmed 50% PEG solution, and let it stand for 90 seconds. Immediately add 15 mL of pre-warmed serum-free medium at 37°C dropwise;

(4) Supplement DMEM serum-free medium to 40 mL, centrifuge for 10 minutes, and pour off the supernatant. Add 40 mL of HAT medium containing 15% to 20% fetal bovine serum. Mix well with a pipette, add dropwise to the wells of 4 96-well cell culture plates containing feeder cells, 2 drops per well, and culture in an incubator at 37°C and 7% CO ₂ .

5. Selective Culture of Hybridoma Cells

Immune mouse spleen cells and mouse myeloma cells, after PEG treatment, form a mixture of various cellular components, including unfused myeloma cells and immune spleen cells, myeloma cell synkaryon and immune spleen cells , and heterokaryons of myeloma cells and immune splenocytes. Only the latter can form hybridoma cells. For this purpose, unfused cells and homofused synkaryons must be removed from this multi-cell mixture and true hybrid cells selected. Therefore, on the 1st, 3rd, 5th, and 7th days after cell fusion, the culture medium was exchanged with the aforementioned HAT medium.

6. Detection of antibodies and cloning of hybridoma cells

The supernatant of each culture well was aspirated, and the culture wells containing cTnI antibody in the culture medium were detected by indirect ELISA method. Hybridoma cells were cloned by limiting dilution. After culturing, a single cell can be proliferated into a homologous cell clone; a cell line with better reactivity and stable secretion of anti-cTnI monoclonal antibody is obtained through reactivity screening.

Method for reactivity screening: Microtiter plates (Nunc, Maxisorb) were coated with 100 μL/well of coating buffer containing 2.5 μg/mL human cTnI (as antigen) for 1 hour at room temperature. Post-coating treatments were incubated in PBS buffer and 1% monoclonal antibody for 30 minutes. Then, wash with wash buffer. 100 μL/well of antibody samples were incubated for 1 hour at room temperature. Then wash 2 more times with washing solution. It was then incubated with 100 μL/well of detection antibody peroxidase-conjugated goat anti-mouse IgG diluted 1:40000 in PBS buffer for 1 hour at room temperature. After washing with washing buffer again, the peroxidase activity is measured by conventional methods (eg, the reaction value at 405 nm is read with an ELISA plate reader), and an antibody with OD ₄₀₅ ≥ 0.5 (with good reactivity) is obtained.

Example 2. Identification of Antibody Binding Fragments

Different cTnI antigen peptides were used to coat the microwells, PBS+20% NBS was used as the diluent, the primary antibody was diluted to a concentration of 1 μg/mL, and the goat anti-mouse IgG-HRP was used as the secondary antibody. The reaction of the antigen to determine the binding fragment of the monoclonal antibody. The identification results of the binding fragments of the cell lines used in the embodiments are as follows:

binding fragment	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

Among them, the IC antibody is the antibody of the CTNI-CTNC complex, and its preparation method is referred to above. The CTNI-CTNC complex is replaced by the cTnI antigen, and the IC antibody is obtained by immunization, wherein the CTNI-CTNC complex can be purchased from Haipeptide Biology.

Example 3, paired screening

Antibodies with different binding fragments were selected for coating and labeling, and cross-pairing experiments were performed. The screening process was as follows:

1. Mark

(1) Take 100uL of fluorescent microspheres, centrifuge at 15000rpm/8min/4°C, remove the supernatant after centrifugation, resuspend to 100uL, and sonicate for 2-3 times;

(2) Prepare 10mg/mL of EDC and NHS;

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

(4) Washing: resuspend to 100uL, ultrasonically shake for 2-3 times, centrifuge at 15000rpm/8min/4°C, remove the supernatant, repeat twice;

(5) Coupling: resuspend to 200uL, sonicate for 2-3 times, add the antibody to the fluorescent microspheres to a final volume of 300uL, mix well in the dark, and react at 37°C for 2h;

(6) Blocking: Add 100uL of blocking agent, mix well in the dark, and react at 37°C for 1h;

(7) Washing: After blocking, centrifuge at 15000rpm/12min/4°C and remove the supernatant. Resuspend to 100uL and sonicate 2-3 times. Centrifuge again at 15000rpm/8min/4℃, and remove the supernatant;

(8) Storage: resuspend to 100uL, ultrasonically shake for 2-3 times, and store at 4°C for later use.

Table 1. The first round of cross-matching screening

Continued from Table 1

2. Coat

(1) Assemble the nitrocellulose membrane and the fluorescent PVC bottom plate for subsequent use;

(2) Dilute the antibody to 1.0-2.0 mg/mL, and evenly draw a line on the NC film with a gold-spraying scriber, and then place it in a 37°C incubator for drying for at least 45 minutes. Assemble and cut strips, add samples for testing.

3. Detection

(1) Quality control substance: cTnI antigen, diluted to 50, 20, 5, 1.25ng/mL with PBS;

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

The results from Table 1 (including Continued Table 1) show that in the first round of cross-pairing screening, pairs with superior activity and a certain gradient of test results were obtained. Negative samples were tested (antibody self-pairing was not tested).

Table 2. The second round of cross-matching screening

It can be seen from the results in Table 2 that in the second round of screening with specificity as an indicator, the cTnI antibody that binds 41-49aa is paired with the cTnI antibody that binds 83-93aa, and the cTnI antibody that binds 41-49aa is paired with the IC antibody. The activity is better, but the specificity is poor (92%-98%). The pairing of cTnI antibody with 83-93aa and IC antibody has obvious advantages in specificity.

To reduce the risk of missed detection, consider introducing antibodies with other binding fragments for testing. The activity was measured first, and then 100 positive samples were used to examine the missed detection of each pair. Among them, 2C-7, 5F-2, 8E-2 are antibodies that bind the 83-93aa fragment, 7B-1, 18F-2, 10E-2 are antibodies that bind the 41-49aa fragment, and 15A-6 and 16C-3 are both is an antibody that binds to the CTNI-CTNC complex.

Table 3. Evaluation of 1+1 and 1+2 modes

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

It can be seen from the results in Table 3 that the introduction of cTnI antibodies that bind 41-49aa to the combination of cTnI antibodies that bind 83-93aa and IC antibodies to the advantageous combination obtained in the 1+1 model can supplement the shortcomings of the respective detections, which is better than introducing cTnI antibodies that bind to 41-49aa. The cTnI antibody binding to 18-29aa, the cTnI antibody binding to 180-190aa, and the introduction of different antibody strains with the same binding fragment as one of the antibodies have better pairing performance; on the basis of good specificity (high negative coincidence rate) (Table 1) 2), further reducing the occurrence of missed detection and improving the accuracy of detection results.

The present application also uses the above-mentioned antibody preparation scheme, using cTnI amino acid fragment 83-93 coupled to carrier protein as an immunogen to prepare an antibody that binds to the 83-93aa fragment, for example, an OD ₄₀₅ ≥ 0.5 is evaluated by ELISA after reactivity screening reaction The pairing specificity of antibody I83A-9 and IC antibodies 15A-6 and 16C-3 are all higher than 99%; using cTnI amino acid fragment 41-49 coupled to carrier protein as immunogen to obtain antibodies that bind to 41-49aa fragment, for example The reactive screening reaction was ELISA to evaluate the antibody I41D-2 with OD ₄₀₅ ≥ 0.5, and the missed detection rate of pairing with the former two did not exceed 1%.

The specific embodiments described above further describe the purpose, technical solutions and beneficial effects of the present application in detail. It should be understood that the above are only specific embodiments of the present application, and are not intended to limit the present application. Within the spirit and principle of this application, any modifications, equivalent replacements, improvements, etc. made shall be included within the protection scope of this application.

Claims (16)

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 the 83-93rd amino acid fragment of cardiac troponin I; The secondary antibody binds to the cardiac troponin CTNI-CTNC complex.
2. The kit of claim 1, wherein when the first antibody is a labeled antibody, the second antibody is a coated antibody; or when the second antibody is a labeled antibody, the first antibody is a coated antibody.
3. The kit of claim 1, wherein the kit further comprises a third antibody for detecting cardiac troponin I, the third antibody binding to the amino acid fragment at positions 41-49 of cardiac troponin I.
4. The kit of claim 3, wherein the antibodies are divided into a first set of antibodies and a second set of antibodies for pairing, and

   1) the first group of antibodies includes a first antibody, and the second group of antibodies includes a second antibody and a third antibody;

   2) the first set of antibodies includes the second antibody, and the second set of antibodies includes the first antibody and the third antibody; or

   3) The first set of antibodies includes the third antibody, and the second set of antibodies includes the first antibody and the second antibody.
5. The kit according to any one of claims 1-4, wherein the reactivity of the antibody is OD ₄₀₅ ≥ 0.5 as assessed by ELISA.
6. The kit of any one of claims 1-5, wherein the labeled antibody is labeled with at least one of a detectable label and a binding partner.
7. The kit of claim 6, wherein the detectable label is a metal particle, a fluorescent label, a chromophore label, an electron dense label, a chemiluminescent label, a radioactive label, or an enzymatic label.
8. The kit of claim 6, wherein the detectable label is rhodamine, fluorescein, fluorescent microspheres, colloidal gold, acridine ester, luciferase, horseradish peroxidase, alkaline phosphatase, beta -Galactosidase, glucoamylase, lysozyme, sugar oxidase, glucose oxidase, galactose oxidase or glucose-6-phosphate dehydrogenase label.
9. The kit of claim 6, wherein the binding partner is biotin/avidin or biotin/streptavidin.
10. The kit of any one of claims 1-5, wherein the coated antibody is attached to a solid phase and/or a binding partner, the solid phase being magnetic particles, latex particles, microtiter plates, nitrocellulose membranes or Microfluidic chip; binding partner is biotin/avidin or biotin/streptavidin.
11. The kit of claim 10, wherein the coated antibody is attached to the solid phase via a binding partner.
12. An antibody combination for detecting cardiac troponin I, including a first antibody and a second antibody; wherein the first antibody binds to the 83-93rd amino acid fragment of cardiac troponin I; the second antibody binds to cardiac troponin CTNI-CTNC Complex.
13. The antibody combination of claim 12, further comprising a third antibody that binds to amino acid fragment 83-93 of cardiac troponin I.
14. Use of the antibody combination according to any one of claims 12-13 in the preparation of a kit for detecting cardiac troponin I.
15. The use according to claim 14, wherein the kit is used for 1) dynamic detection of cardiac troponin content for real-time monitoring, and/or 2) diagnosis of early acute myocardial injury, including To rule out or predict early acute myocardial injury.
16. A method of preparing the antibody combination of any one of claims 12-13, comprising:

    1) using amino acid fragments or complexes selected from cardiac troponin I amino acid fragments 41-49, cardiac troponin I amino acid fragments 83-93 and cardiac troponin CTNI-CTNC complexes as antigens or haptens to immunize animals; and

    2) Obtain from the animal antibodies that bind to the cardiac troponin I amino acid fragments 41-49, the cardiac troponin I amino acid fragments 83-93, and the cardiac troponin CTNI-CTNC complex, respectively.