Monoclonal antibody of cardiac troponin I (cTnI) and preparation method

Abstract

This invention belongs to the field of biotechnology. It provides a recombinant protein whose amino acid sequence consists of two dominant epitopes of cardiac troponin I (cTnI) repeated in tandem. The amino acid sequence is converted into a corresponding nucleotide sequence using E. coli-preferred codons. The nucleotide sequence is then chemically synthesized and a recombinant expression vector is constructed, thereby increasing the expression level of the recombinant protein in E. coli. This invention also involves using this recombinant protein to immunize mice to establish a phage library, obtaining the corresponding cardiac troponin I single-chain antibody scfv sequence through panning and screening, constructing a complete mouse IgG1 antibody sequence expression vector from the obtained scfv sequence, expressing the monoclonal antibody via transient transfection of HEK293F cells, purifying the monoclonal antibody, and determining the optimal monoclonal antibody pairing using an immunofluorescence orthogonal experiment.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology. Specifically, this invention expresses a novel recombinant protein, involves using the recombinant protein to immunize mice to establish a phage library, screening to obtain specific single-chain antibody scfv sequences, and constructing the obtained scfv sequences into a eukaryotic expression vector to express cardiac troponin I (cTnI) monoclonal antibodies, and applying them to the early diagnosis of acute myocardial infarction. Background Technology

[0002] Acute myocardial infarction is a common disease that seriously endangers human health. Cardiac troponin is a marker of myocardial injury and necrosis, and it has important clinical significance for the diagnosis and risk stratification of acute myocardial infarction (diagnostic sensitivity 100%, specificity 91%, and long duration). Elevated cardiac troponin levels indicate myocardial damage and can be seen in acute myocardial infarction, unstable angina, pulmonary infarction, heart failure shock, and other diseases that cause myocardial damage, such as pancreatitis, severe diabetic ketoacidosis, and connective tissue diseases. The higher the value, the wider the scope of damage. In patients with acute myocardial infarction, release begins 3–6 hours, peaks at 10–24 hours, and the recovery time to normal is 10–15 days for cTnT and 5–7 days for cTnI. Elevated levels can also be seen in some patients with renal insufficiency.

[0003] Currently, the diagnosis of myocardial infarction mainly relies on electrocardiogram, magnetic resonance imaging, coronary angiography, etc., which require special equipment and instruments, professional personnel to operate, and cannot be tested on-site. The preparation of cardiac troponin I (cTnI) monoclonal antibodies can be used for the early diagnosis of myocardial infarction. The conventional preparation of cardiac troponin I (cTnI) monoclonal antibodies involves preparing ascites from Balb / c mice using cardiac troponin I (cTnI) monoclonal cell lines, and purifying the monoclonal antibody using Protein A affinity chromatography. However, due to the uncertainty of ascites yield per mouse and the large individual differences, the batch-to-batch differences of the obtained anti-cardiac troponin I (cTnI) monoclonal antibodies are large, resulting in poor detection accuracy. Currently, cardiac troponin I (cTnI) monoclonal antibodies are mainly obtained through hybridoma technology. The disadvantages of obtaining monoclonal antibodies through hybridoma technology are: (1) It is not suitable to use hybridoma technology to prepare antibodies against animal toxic antigens, autoantigens, immune tolerance antigens, and weak immunogenic antigens because it cannot generate effective immunity. (2) After cell fusion, the number of candidate clones generated is small. Even with electrofusion techniques, the number of candidate clones typically reaches a maximum of 10. 4 The antibody library can have a capacity of 10. 10The above. (3) As heterozygous diploid cells, candidate hybridoma clones need to be subcloned to become stable. Candidate hybridoma cells that are not subcloned in time are unstable and are prone to losing positive clones. Selecting positive clones, especially complex functional screening, requires a certain time window. The longer the window, the more mature and reliable the functional screening. Micro-crystallization of candidate clones can extend the functional screening time window, but functional detection requires a sufficient amount of antibody protein, so batch subcloning still requires a large amount of work. The solution after hybridoma screening failure is re-immunization or re-fusion (which is also limited by the feeding cycle of mice after immunization), which inevitably incurs time costs. (4) Due to ethical considerations, the development and application of human hybridoma technology are limited. Usually, hybridoma monoclonal antibodies are animal antibodies, and humanization of antibodies is necessary for drug development. Summary of the Invention

[0004] To address the shortcomings of monoclonal antibodies in the prior art, a monoclonal antibody was prepared by designing and synthesizing recombinant cardiac troponin I (cTnI) protein and expressing it in eukaryotic cells through the establishment of a phage library. This significantly shortened the preparation time compared to traditional monoclonal antibody preparation, and the resulting monoclonal antibody exhibited high stability, good uniformity, and greatly reduced batch-to-batch variability.

[0005] To achieve the above objectives, this application provides:

[0006] A monoclonal antibody 2B6 specifically targeting cardiac troponin I (cTnI) comprises a light chain and a heavy chain, wherein the amino acid sequence of the light chain is shown in SEQ ID NO.1 and the amino acid sequence of the heavy chain is shown in SEQ ID NO.2.

[0007] A monoclonal antibody 5E3 specifically targeting cardiac troponin I (cTnI) comprises a light chain and a heavy chain, wherein the amino acid sequence of the light chain is shown in SEQ ID NO.3 and the amino acid sequence of the heavy chain is shown in SEQ ID NO.4.

[0008] Beneficial effects: Using an immune antibody library to screen cardiac troponin I (cTnI) monoclonal antibodies, under the same immunization conditions, the more candidate clones there are, the easier it is to screen for highly active antibodies, and once the antibody library is built, it can be stored indefinitely. The original phage library is sufficient for repeated screening, without the need to rebuild the library, and only a small amount of phage particles are needed when screening monoclonal antibodies. In addition, the affinity panning method can specifically enrich antibodies that bind to the antigen and also minimize the removal of antibodies that bind to the control protein, effectively improving the screening efficiency. The cardiac troponin I (cTnI) monoclonal antibodies produced by this method have small batch-to-batch differences, which improves the detection accuracy. (1) Using cardiac troponin I (cTnI) as the target antigen, two dominant antigenic epitopes of this antigen were analyzed and selected. The sequence comparison results showed that the two selected antigenic epitopes are common epitopes of all cardiac troponin I (cTnI) and have no obvious homology with other protein sequences. (2) In order to enhance the immunization effect and shorten the preparation time of monoclonal antibodies, the two selected dominant antigenic epitopes were tandemly linked and used to immunize mice. (3) To improve the expression level of the recombinant protein, the preferred codons of E. coli were used to convert the amino acid sequence of the recombinant protein into the corresponding nucleotide sequence. (5) The nucleotide sequence obtained in the previous step was chemically synthesized and ligated by enzyme digestion. The synthesized nucleotide fragment was inserted into the expression vector PET-32a(+) to construct the recombinant cardiac troponin I (cTnI) expression vector. (6) The recombinant cardiac troponin I (cTnI) expression vector was transformed into E. coli ER2566 competent cells, and the recombinant protein expression strain was screened. (7) After large-scale culture of the recombinant protein expression strain, the bacteria were destroyed by sonication and centrifuged at low temperature. The supernatant of the solution was taken and subjected to affinity chromatography on a nickel agarose column to elute and obtain purified recombinant cardiac troponin I (cTnI). (8) After repeatedly immunizing Balb / c mice with recombinant cardiac troponin I (cTnI) protein, lymphocytes were isolated from their spleens to establish a single-chain antibody scfv phage display library. The recombinant cardiac troponin I (cTnI) protein was used for multiple rounds of panning and screening to finally obtain the single-chain antibody scfv sequence that can bind to recombinant cardiac troponin I (cTnI). (9) The scfv sequence was used to construct a complete mouse IgG1 expression vector and expressed monoclonal antibodies using HEK293 cells. The monoclonal antibodies were purified using Protein A affinity chromatography and labeled with fluorescent microspheres. (10) Orthogonal experimental screening showed that the combination of 5E3 monoclonal antibody coating and 2B6 monoclonal antibody labeling was the best combination for detecting cardiac troponin I (cTnI). Detailed Implementation

[0009] Although the following embodiments provide a relatively detailed textual description of the design concept of the present invention, these textual descriptions are merely simple textual descriptions of the design concept of the present invention and are not intended to limit the design concept of the present invention. Any combination, addition, or modification that does not exceed the design concept of the present invention falls within the protection scope of the present invention.

[0010] Example 1: Selection of dominant antigenic epitopes of cardiac troponin I (cTnI)

[0011] Using cardiac troponin I (cTnI) as the target antigen, the hydrophilicity and antigenicity of its epitope sequences were analyzed using the biological software DNAssist 2.0, and dominant epitopes A and B were selected. Sequence comparison results showed that the selected dominant epitope sequences A and B have broad spectrum, being common epitopes of all cardiac troponin I (cTnI); furthermore, epitopes A and B showed no significant homology with other protein sequences, existing only within the cardiac troponin I (cTnI) sequence.

[0012] Example 2: Tandem of dominant antigenic epitopes of cardiac troponin I (cTnI)

[0013] To enhance the stimulation of the mouse immune system by the selected antigenic epitopes and facilitate subsequent experiments, the A and B dominant antigenic epitope sequences of cardiac troponin I (cTnI) were repeated and then linked by a flexible fragment (four consecutive glycine residues) to obtain the recombinant protein amino acid sequence.

[0014] Example 3: Optimization of the nucleotide sequence encoding recombinant cardiac troponin I (cTnI) protein

[0015] To improve the expression level of recombinant proteins in *E. coli*, the amino acid sequence encoding the recombinant protein was converted into the corresponding nucleotide sequence based on the codons preferred by *E. coli*, while maintaining the original amino acid sequence. Nucleotide sequences corresponding to the BamHI and EcoRI restriction enzyme sites were then added upstream and downstream of the transverse codons, respectively. The resulting protein was synthesized by General Biosystems Anhui Co., Ltd. The synthesized target gene was cloned into the pMD25-T vector (Takara Bio Engineering Dalian Co., Ltd.).

[0016] Example 4: Construction of a recombinant cardiac troponin I (cTnI) protein expression vector

[0017] The pMD25-T vector and PET-32a(+) vector (Novagen, Germany) containing the target gene were double-digested at 37°C for 12 hours with restriction endonucleases BamHI and EcoRI (Takara Bio Engineering, Dalian Co., Ltd.). The digestion products were subjected to 1% agarose gel electrophoresis, and the target gene and PET-32a(+) vector were recovered by gel extraction (the gel extraction kits used in this invention were all from Aisjin Biotechnology, Hangzhou Co., Ltd.). The recovered target gene and PET-32a(+) vector were ligated at a certain ratio at 4℃ for 12 hours using T4 ligase (Takara Bio Engineering Dalian Co., Ltd.). The ligation product was then transformed into DH5α competent cells (Hangzhou Xianzhi Biotechnology Co., Ltd.) and plated on LB agar containing ampicillin resistance (50 μg / mL). After incubation at 37℃ for 12 hours, single clones were picked from the agar and transferred to LB liquid medium containing ampicillin resistance (50 μg / mL). After incubation at 37℃ for 12 hours, plasmids were extracted using a plasmid purification kit (all plasmid extraction kits used in this invention were from Aisjin Biotechnology Hangzhou Co., Ltd.). The correct recombinant expression vector was obtained after double digestion with BamHI and EcoRI.

[0018] Example 5: Construction of a recombinant cardiac troponin I (cTnI) antigen-expressing strain

[0019] The constructed recombinant expression vector was transformed into E. coli ER2566 competent cells and plated on LB agar plates containing ampicillin-resistant (50 μg / mL) medium, and cultured overnight at 37°C. The next day, single clones from the plates were picked and cultured in LB liquid medium containing ampicillin-resistant (50 μg / mL) medium at 37°C for 8 hours using a shaker. Then, isopropyl thio-β-D-galactopyranoside (final concentration 1.0 mmol / L) was added to induce expression for 4 hours, followed by protein electrophoresis. 9% polyacrylamide gel electrophoresis results showed successful expression of the recombinant protein, yielding a strain expressing recombinant cardiac troponin I (cTnI) antigen.

[0020] Example 6: Purification of recombinant cardiac troponin I (cTnI) protein

[0021] Recombinant cardiac troponin I (cTnI) protein-expressing strains were inoculated into LB liquid medium, and ampicillin was added to a final concentration of 50 μg / mL. After incubation at 37°C in a shaker for 8 hours, the bacteria were diluted 1:100 with LB liquid medium containing 50 μg / mL ampicillin and aliquoted into bacterial culture flasks. The flasks were then incubated at 37°C in a shaker until OD600 = 0.8. Isopropyl thio-β-D-galactopyranoside was added to a final concentration of 1.0 mmol / L, and induction was continued for 4 hours. After centrifugation to collect the bacterial cells, the cells were lysed by sonication at 4°C. The supernatant was then passed through a nickel-agarose affinity chromatography column, and after washing and elution, purified recombinant cardiac troponin I (cTnI) protein was obtained.

[0022] Example 7: Construction of a single-chain antibody scfv phage library

[0023] Female Balb / c mice aged 4-6 weeks were used for the primary immunization. Each mouse received a subcutaneous injection of 100 μg of recombinant cardiac troponin I (cTnI) emulsified with Freund's complete adjuvant at multiple sites, totaling 400 μl per mouse. A second booster immunization was administered 20 days later, using 80 μg of recombinant cardiac troponin I (cTnI) emulsified with Freund's incomplete adjuvant, totaling 400 μl per mouse, injected subcutaneously at multiple sites. A third booster immunization was administered 15 days later, using the same method as the second booster. Twenty days later, a booster injection of 120 μg of recombinant cardiac troponin I (cTnI) antigen was administered intraperitoneally. 72 hours later, blood was collected from the orbital sinus, and the mice were sacrificed. Spleen lymphocytes were isolated using a mouse spleen lymphocyte isolation kit (Tianjin Haoyang Biological Products Technology Co., Ltd.). Total RNA was extracted from isolated lymphocytes using an RNA extraction kit (Tiangen Biotech Co., Ltd.). cDNA was synthesized by reverse transcription using a reverse transcription kit (Takara). The heavy chain variable region and light chain variable region genes were amplified using mouse single-chain antibody SCFV universal degenerate primers. The PCR products were subjected to 1% agarose gel electrophoresis, and the target genes were recovered by gel excision. The recovered target genes were then analyzed by overlap... PCR was used to ligate scfv. The PCR product was subjected to 1% agarose gel electrophoresis, and the target gene was recovered by gel excision. After digestion with NotI and SfiI enzymes, the ligation was carried out using T4 ligase and pCANTAB5e (Beijing Baokewei Food Safety Biotechnology Co., Ltd.) vector at a certain ratio at 4℃ for 12 hours. The ligation product was recovered using a gel recovery kit to remove enzymes and buffer substances. The recovered product was electroporated into E. coli TG1 electroporation competent cells multiple times using a bacterial electroporator (Biorad). The cells were then plated on 2×YT-AG plates containing ampicillin resistance (50 μg / mL) and 2% glucose. After incubation at 30℃ for 12 hours, an appropriate amount of 2×YT medium was taken and all colonies on the plate were scraped off with a sterile glass rod and the bacterial suspension was collected. This is the constructed phage antibody library.

[0024] Example 8: Panning and Screening of Single-Chain Antibody SCFV

[0025] A certain amount of bacterial culture was taken from the phage antibody library and inoculated into 2×YT-AG medium to achieve an OD600 of 0.3. The culture was incubated at 37°C with shaking at 250 rpm for approximately 1 hour until the OD600 reached 0.5. Then, helper phage M13K07 was added for superinfection at a ratio of M13K07 / TG1 = 20:1. After shaking at 37°C with shaking at 250 rpm for 1 hour, the bacteria were centrifuged at 3300 g for 10 minutes to precipitate the bacteria, and the supernatant was carefully discarded. The bacteria were resuspended in 2×YT-AK medium containing ampicillin-resistant (50 μg / mL) and kanamycin-resistant (50 μg / mL) medium and incubated overnight at 30°C with shaking at 250 rpm. The next day, the bacteria were centrifuged at 10800 g for 20 minutes to precipitate the bacteria. The supernatant was transferred to a clean centrifuge tube, and 1 / 5 volume of PEG / NaCl was added. After mixing, the mixture was incubated on ice for 2 hours. Centrifuge at 10800 g, 4℃ for 20 min to precipitate cells. Carefully discard the supernatant, remove excess water, and resuspend the precipitate in PBS. Filter through a 0.45 μm membrane to remove bacterial debris for panning. Dilute the purified recombinant cardiac troponin I (cTnI) antigen to 8 μg / ml with coating buffer and coat 4 ml of each immunotube (Thermo). Incubate overnight at 4℃. The next day, discard the coating buffer and unadsorbed antigen, wash three times with sterile PBST, add 5 ml of blocking buffer to each immunotube, and incubate at 37℃ for 2 h. Discard the blocking buffer, wash three times with sterile PBST, and add 4 ml of PEG-precipitated phage to each immunotube. Incubate at 37℃ for 1 h. Discard the liquid in the immunoassay tubes, wash 10 times with sterile PBST, then 10 times with sterile PBS. Add 1 ml of 100 mM triethylamine to elute the bound phages, and immediately add 500 μl of 1 M Tris-HCl to neutralize at pH 7.4. Introduce the neutralized phages into a certain amount of TG1 E. coli in the logarithmic growth phase for superinfection; this is the first round of panning and enrichment. After three rounds of panning, cardiac troponin I (cTnI)-specific scfvs were enriched. After the final round of elution and neutralization, the phages were infecting TG1 *E. coli* and plated onto 2×YT-AG plates. After incubation at 30°C for 12 hours, 400-600 single colonies were randomly selected and placed in 96-well deep-well plates. The plates were then incubated with 2×YT-AG medium at 37°C with shaking at 250 rpm for 2 hours. A certain amount of M13K07 helper phage was added for superinfection, followed by incubation at 37°C with shaking at 250 rpm for 1 hour. The supernatant was discarded by centrifugation, and 2×YT-AK medium containing ampicillin-resistant (50 μg / mL) and kanamycin-resistant (50 μg / mL) culture was added. The plates were incubated overnight at 30°C with shaking at 250 rpm. Single-clone ELISA screening was performed the following day:

[0026] Coating: Dilute the recombinant cardiac troponin I (cTnI) protein with coating buffer to a final concentration of 1 μg / mL, add 100 μL / well to the microplate (Shenzhen Jincanhua Industrial Co., Ltd.), incubate overnight at 4°C, and then wash once with washing buffer using a DEM-3 plate washer (Sun Yat-sen University Da An Gene Co., Ltd.).

[0027] Blocking: Add 200 μL of blocking solution to each well, block at 37°C for 2 hours, and wash once with washing solution using a plate washer;

[0028] Sample loading: Add overnight bacterial culture supernatant and control serum, 100 μL / well, incubate at 37°C for 1 h, and wash 3 times with washing buffer using a plate washer;

[0029] Add enzyme-labeled antibody: Add 100 μL / well of freshly diluted rabbit anti-M13 phage HRP enzyme-labeled secondary antibody (purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.), incubate at 37°C for 30 minutes, and then wash 4 times with washing buffer using a plate washer.

[0030] Add colorimetric reagents: Add 50 μL of colorimetric reagent A and 50 μL of colorimetric reagent B to each well, and develop the color at 37°C in the dark for 10 minutes.

[0031] To terminate the reaction, add 2M H2SO4 at a rate of 50 μL / well;

[0032] Result Interpretation: OD values ​​were read at 450 nm using a microplate reader after zeroing the blank wells. Immune mouse serum was used as a positive control. Results showed 16 positive clones with high OD values; sequencing yielded 5 SCFV sequences: 2B6, 3D7, 4G6, 5E3, and 2G8. The relevant solution formulations are as follows:

[0033] Coating solution: Na2CO3 1.5g, NaHCO3 2.9g, add ddH2O to make up to 1000mL (pH 9.6).

[0034] Blocking solution: Na2HPO4·12H2O 2.68g, NaH2PO4·2H2O 0.39g, NaCl 8.5g, 20g bovine serum albumin, add ddH2O to bring the volume to 1000mL (pH 7.4).

[0035] Washing solution: Na2HPO4·12H2O 2.68g, NaH2PO4·2H2O 0.39g, NaCl 8.5g, Tween-20 0.5mL, add ddH2O to make up to 1000mL (pH 7.4).

[0036] Colorimetric solution A: Dissolve 200 mg TMB in 100 mL of anhydrous ethanol, and add ddH2O to bring the volume to 1000 mL.

[0037] Colorimetric solution B: 2.1g citric acid, 71g Na2HPO4·12H2O, add ddH2O to make up to 1000mL.

[0038] When using: 1 mL of colorimetric solution A + 1 mL of colorimetric solution B + 0.4 μL of 30% H2O2

[0039] Stop solution: 2M H2SO4, 21.7mL concentrated H2SO4, add ddH2O to make up to 1000mL.

[0040] Example 9: Construction of eukaryotic expression vector and transient transduction expression and purification in HEK293F cells

[0041] Five single-chain antibody scfv sequences of cardiac troponin I (cTnI) were used to construct complete mouse IgG1 antibody sequences. The heavy and light chain variable regions of the scfv sequences were bridged to the heavy and light chain constant regions of mouse IgG1, respectively, by PCR, and then inserted into pcDNA3.1 (Novagen, Germany) plasmids. HEK293F cells were co-transfected with the constructed heavy and light chain plasmids via PEI. After 7 days of expression on a shaker at 37°C, 5% CO2, and 120 rpm, the cells were centrifuged, and the supernatant was collected and filtered through a 0.45 μm filter. An agarose affinity medium Protein A chromatography column (Nanjing Genscript Biotech Co., Ltd.) was equilibrated with 50 mL of PBS (pH 7.4) and sputtered onto a computer-controlled nucleic acid and protein analyzer (Shanghai Huxi Analytical Instrument Factory Co., Ltd.), where the absorbance was 0. After loading the supernatant, wash with PBS until the absorbance is 0, then elute with 0.1M glycine (pH 3.0), collect the eluent and add 500mM Tris-HCl (pH 8.5) buffer to neutralize to about pH 7.0 to obtain purified monoclonal antibodies 2B6, 3D7, 4G6, 5E3, and 2G8.

[0042] Example 10: Preparation of fluorescent microspheres labeled with cardiac troponin I (cTnI) monoclonal antibody

[0043] Fluorescent microspheres (Bangslab, Dragongreen) with a diameter of 190 nm were selected. The concentration of the microspheres was adjusted to 1% using 0.05 M pH 4.5 MES buffer. Cardiac troponin I (cTnI) monoclonal antibody was then labeled onto the fluorescent microspheres using covalent coupling of carbodiimide (EDC) and succinimide (NHS) at a concentration of 0.2 mg / ml. The prepared fluorescent microspheres were sprayed onto the fluorescent microsphere pads at a rate of 4 μl / cm using a quantitative spraying device and vacuum dried at 25 °C for 1–2 h. The microspheres were then stored in a dry environment for later use.

[0044] Example 11: Preparation of nitrocellulose membrane (NC membrane)

[0045] The concentrations of cardiac troponin I (cTnI) monoclonal antibodies (2B6, 3D7, 4G6, 5E3, 2G8) were adjusted to 0.4 mg / mL using 0.01 M pH 7.4 PBS (phosphate buffer, containing 5% sucrose and 0.05% Tween-20). The resulting solutions were then sprayed onto the NC membrane to form detection lines (T lines). Goat anti-mouse antibodies were adjusted to 0.5 mg / mL using 0.01 M pH 7.4 PBS (phosphate buffer, containing 5% sucrose and 0.05% Tween-20). This solution was then sprayed onto the NC membrane to form control zones (C lines). The spray volume for both zones was 1 μL / cm, with a 5 mm gap between them. The control zone was 2 mm from one end of the NC membrane. After drying at 37°C overnight, the membranes were stored at room temperature in a dry environment for later use.

[0046] Example 12: Preparation of Fluorescent Microsphere Immunoassay Card

[0047] Assemble the test strip: On the PVC base plate, overlap and paste the following in sequence: (1) Spray myoglobin monoclonal antibody (2B6, 3D7, 4G6, 5E3, 2G8) as the detection area and goat anti-mouse IgG as the quality control area of ​​the NC membrane; (2) Spray fluorescent microsphere pad with myoglobin monoclonal antibody (2B6, 3D7, 4G6, 5E3, 2G8) labeled with fluorescent microspheres; (3) Filter paper and sample pad, the sample pad is a glass fiber membrane treated with 2% Tween-20; (4) Absorbent paper, after assembly, cut to a width of 4mm, attach the reagent card strip shell and press it tightly to obtain the fluorescent microsphere immunochromatographic detection card.

[0048] Example 13: Screening of paired monoclonal antibodies

[0049] Clinical serum samples and normal serum samples of cardiac troponin I (cTnI) were loaded at 100 μL / well. After being incubated at room temperature for 15 min, the T and C line signals on the NC membrane were read by a fluorescence analyzer (BaseBio Biotechnology Co., Ltd.) and the measured value T / (T+C) was calculated. See Table 1 for details.

[0050]

[0051] As shown in the table above, the optimal antibody pairing for detecting cardiac troponin I (cTnI) is 5E3 monoclonal antibody coating and 2B6 monoclonal antibody-labeled fluorescent microspheres.

Claims

1. Anti-cardiac troponin I (cTnI) specific monoclonal antibody 2B6, comprising a light chain and a heavy chain, characterized in that: The light chain amino acid sequence is shown in SEQ ID NO.1; The heavy chain amino acid sequence is shown in SEQ ID NO.

2.

2. Anti-cardiac troponin I (cTnI) specific monoclonal antibody 5E3, comprising a light chain and a heavy chain, characterized in that: The light chain amino acid sequence is shown in SEQ ID NO.3; The heavy chain amino acid sequence is shown in SEQ ID NO.4.

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