Cardiac myosin-binding protein c detection kit and use thereof

Abstract

Provided herein is a myosin binding protein C detection kit, comprising: 1) a magnetic bead coupled with a first antibody, and 2) a second antibody coupled with an acridinium ester, the first antibody and the second antibody both being human myosin binding protein C antibodies, but binding to different antigenic epitopes. By means of a double antibody sandwich method and the chemiluminescence characteristics of the acridinium ester, the myosin binding protein C detection kit detects the content of myosin binding protein C in serum with high sensitivity.

Description

Cardiac myosin-binding protein C detection kit and its application Technical Field

[0001] This invention relates to a myosin-binding protein C detection kit, and more particularly to a detection kit that can detect the content of myosin-binding protein C in serum with high sensitivity using a double antibody sandwich method. Background Technology

[0002] Acute myocardial infarction (AMI) is a critical cardiovascular event caused by unstable ischemic syndrome, characterized by high incidence, poor prognosis, and high mortality. However, for patients with AMI who do not present with typical chest pain and show no significant changes on electrocardiogram (ECG), accurate diagnosis cannot be made solely based on ECG, echocardiography, and cardiac MRI. Currently, the globally recognized "gold standard" for diagnosing AMI uses cardiac troponin (cTn) as a marker of myocardial injury. cTn begins to be released 4–6 hours after the onset of AHI / AMI, is detectable in the blood, peaks at 18–24 hours, and disappears after two weeks. High-sensitivity cTnT products appear 2.5 hours after the onset of myocardial injury, but have low specificity, with a clinical false positive rate as high as 30%-50%, easily leading to misdiagnosis. European cardiac guidelines explicitly state that high-sensitivity cTn should not be used as a diagnostic criterion within 3 hours.

[0003] Cardiac myosin-binding protein C (cMyBP-C) is currently the most recently studied cardiac biomarker for acute myocardial infarction (AHI) / acute myocardial infarction (AMI) both domestically and internationally. It exhibits high specificity and can specifically reflect myocardial lesions. In the early stages of AMI, large amounts of cMyBP-C and its metabolites are released into the bloodstream. Its elevated levels can be detected in the circulatory system as early as 30 minutes after myocardial injury, making it effective for early detection of myocardial injury. Furthermore, compared to other biomarkers, cardiac myosin-binding protein C is the first to be cleared from the bloodstream, thus possessing good prognostic value.

[0004] For the detection of cardiac myosin-binding protein C (cMyBP-C), existing methods such as some myocardial injury quadruple test cards (colloidal gold method) and enzyme-linked immunosorbent assay (ELISA) kits are limited by methodological constraints. Their sensitivity is insufficient, and they cannot achieve quantitative detection. They cannot accurately assess the trend and risk of the disease. Furthermore, biomarkers are nonspecific, often resulting in false positives. Patients with renal insufficiency, pulmonary embolism, or stroke but without myocardial injury may have elevated biomarker indicators, leading to various misdiagnoses, delays, or overtreatment.

[0005] As a novel biomarker, there is currently a lack of accurate quantitative detection methods for human cardiac myosin-binding protein C (cMyBP-C, MYBPC3, cMyC). Currently, the only authorized patent related to this biomarker detection is the cMyBP-C detection kit using a colloidal gold nanocage method developed by Nanjing Botiankezhi Biotechnology Co., Ltd. However, the colloidal gold method relies on the large aggregation of antibody-antigen complexes on the detection line, thus limiting its application to qualitative or semi-quantitative determination, often resulting in significant errors. Furthermore, the colloidal gold method exhibits a significant hook effect, restricting the linear range of the kit and hindering its clinical application in differentiating between weakly and strongly positive cases. Generally, current human cardiac myosin-binding protein C products only provide qualitative results with a reference cutoff value of 50 pg / ml, which limits the clinical application of this biomarker in grading early myocardial injury and assessing the prognosis of cardiovascular surgeries. Summary of the Invention

[0006] On one hand, this article provides a myosin-binding protein C detection kit, which includes:

[0007] 1) Magnetic beads conjugated with a first antibody, wherein the heavy chain variable region of the first antibody includes HCDR1 (SEQ ID NO:3), HCDR2 (SEQ ID NO:4), and HCDR3 (SEQ ID NO:5), and the light chain variable region of the first antibody includes LCDR1 (SEQ ID NO:7), LCDR2 (SEQ ID NO:8), and LCDR3 (SEQ ID NO:9); and

[0008] 2) A second antibody conjugated with acridinium ester, wherein the heavy chain variable region of the second antibody includes HCDR1 (SEQ ID NO:11), HCDR2 (SEQ ID NO:12), and HCDR3 (SEQ ID NO:13), and the light chain variable region of the first antibody includes LCDR1 (SEQ ID NO:15), LCDR2 (SEQ ID NO:16), and LCDR3 (SEQ ID NO:17).

[0009] or

[0010] 1) Magnetic beads conjugated with a first antibody, wherein the heavy chain variable region of the first antibody includes HCDR1 (SEQ ID NO: 11), HCDR2 (SEQ ID NO: 12), and HCDR3 (SEQ ID NO: 13), and the light chain variable region of the first antibody includes LCDR1 (SEQ ID NO: 15), LCDR2 (SEQ ID NO: 16), and LCDR3 (SEQ ID NO: 17); and

[0011] 2) A second antibody conjugated with acridinium ester, wherein the heavy chain variable region of the second antibody includes HCDR1 with sequence SEQ ID NO:3, HCDR2 with sequence SEQ ID NO:4, and HCDR3 with sequence SEQ ID NO:5, and the light chain variable region of the first antibody includes LCDR1 with sequence SEQ ID NO:7, LCDR2 with sequence SEQ ID NO:8, and LCDR3 with sequence SEQ ID NO:9.

[0012] In some embodiments, the heavy chain variable region sequence of the first antibody is shown in SEQ ID NO:2, and the light chain variable region sequence of the first antibody is shown in SEQ ID NO:6; the heavy chain variable region sequence of the second antibody is shown in SEQ ID NO:10, and the light chain variable region sequence of the second antibody is shown in SEQ ID NO:14.

[0013] In some embodiments, the heavy chain variable region sequence of the first antibody is shown in SEQ ID NO:10, and the light chain variable region sequence of the first antibody is shown in SEQ ID NO:14; the heavy chain variable region sequence of the second antibody is shown in SEQ ID NO:2, and the light chain variable region sequence of the first antibody is shown in SEQ ID NO:6.

[0014] In some embodiments, the myosin-binding protein C assay kit further includes myosin-binding protein C standard solutions of varying concentrations.

[0015] On the one hand, this article provides the use of the above-mentioned myosin-binding protein C detection kit in the preparation of diagnostic kits for the diagnosis of acute myocardial infarction.

[0016] Compared with existing manual methods for detecting myosin-binding protein C, the myosin-binding protein C detection kit provided in this paper has advantages such as high sensitivity, good specificity, wide linear range, and simple operation. Attached Figure Description

[0017] Figure 1 shows the detection results of the cMyBP-C detection kit of the present invention for a wide range of cMyBP-C antigen standards.

[0018] Figure 2 shows the detection results of the cMyBP-C detection kit of the present invention on low concentrations of cMyBP-C antigen standards. Detailed Implementation

[0019] Unless otherwise stated, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art.

[0020] This invention is based on the sandwich-specific binding of a self-developed antibody to the human cMyBP-C antigen fragment and the direct chemiluminescence principle of AE (acridinium ester and its derivatives). The first antibody is a self-developed recombinant human cMyBP-C antibody. The antibody binds to an activated carboxyl magnetic bead solid-phase carrier via an amide bond, forming a magnetic bead-first antibody conjugate, which is used in chemiluminescent immunoassay to capture the human cMyBP-C antigen fragment in the test sample. The second antibody is also a self-developed recombinant human cMyBP-C antibody with reactivity largely similar to the first antibody. This antibody binds to an AE compound via an amide bond. The AE reacts instantaneously with hydrogen peroxide and hydroxide ions, emitting light. The relative luminescence intensity (RLU) follows a quantitative relationship with the concentration of the magnetic bead-first antibody-human cMyBP-C antigen-second antibody-AE conjugate, thus enabling the quantitative detection of the human cMyBP-C antigen fragment in the sample.

[0021] The present invention will be further illustrated by the following examples.

[0022] Example 1: Preparation of Antibody

[0023] We immunized mice with the N-terminal fragment of the cMyBP-C antigen (amino acids 1-159, MPEPGKKPVSAFSKKPRSVEVAAGSPAVFEAETERAGVKVRWQRGGSDISASNKYGLATEGTRHTLTVREVGPADQGSYAVIAGSSKVKFDLKVIEAEKAEPMLAPAPAPAEATGAPGEAPAPAAELGESAPSPKGSSSAALNGPTPGAPDDPIGLFVM, SEQ ID NO: 1), collected mouse spleen cells, amplified the VH and VL genes by RT-PCR, constructed a phage display library in the form of ScFv, and performed four rounds of enrichment using the cMyBP-C antigen, identifying several clones with high affinity for the cMyBP-C antigen. Among them, two clones with high affinity for the cMyBP-C antigen (ECG as measured by ELISA) were selected. 50The values ​​are all less than 30 ng / mL), and clones with different epitopes of cMyBP-C antigen are used in this invention, and their variable region sequences are as follows.

[0024] First antibody (underlined sequences are CDR sequences, according to the Kabat numbering scheme, the same below):

[0025] VH sequence (SEQ ID NO: 2)

[0026] QIQLVQSGPELKKPGETVKISCKASGYTFRNYGMNWVKQAPGKGLKWMGWINTYSGVIYAKDFKGRFAFSLDTSASTAFLQINNLKNEDTATYFCARDDYGYAMDFWGQGTSVTVSS (HCDR sequences are numbered sequentially as SEQ ID NO: 3, 4 and 5)

[0027] VL sequence (SEQ ID NO: 6)

[0028] The LCDR sequences are numbered sequentially as SEQ ID NO: 7, 8, and 9.

[0029] Second antibody:

[0030] VH sequence (SEQ ID NO: 10)

[0031] QIQLVQSGPELKKPGETVKISCKASGYNFRQYLGMHWVKQRPGQGLEWIGHINPYGSLLNQNAKFKNRVTITRDTSASTAYMELSSLRSEDTAVYYCARSGSFHRRMDYWGLGTSVTVSS (HCDR sequences are numbered sequentially as SEQ ID NO: 11, 12, and 13)

[0032] VL sequence (SEQ ID NO: 14)

[0033] The LCDR sequences are numbered sequentially as SEQ ID NO: 15, 16, and 17.

[0034] After codon optimization, DNA fragments of the variable region were synthesized and fused with the constant regions of the human IgG1 heavy chain and light chain κ constant region, respectively. The heavy and light chain expression constructs were cloned into plasmid expression vectors and secreted in HEK293 cells. The antibody was purified using magnetic beads coated with protein A. The stock solution concentration of the purified antibody was generally 1.5-4.5 mg / mL, which could be adjusted to 5 mg / mL by ultrafiltration through a 30 kDa ultrafiltration membrane.

[0035] Example 2: Preparation of magnetic bead-antibody conjugates

[0036] The preparation process is briefly described below.

[0037] Preparation of magnetic bead suspension: Take 100 μL of JSR MS300C magnetic beads (JSR Life Science, hydrophilic carboxylated magnetic beads, 3 μm particle size) and suspend them in 900 μL of MES (2-(N-morpholino)ethanesulfonic acid, potassium 2-(N-morpholino)ethanesulfonate) buffer (0.5 mol / L pH=5.0).

[0038] Activation of magnetic beads: Add 100 μL of 5 mg / mL NHS (N-hydroxysuccinmide), followed by 100 μL of 7 mg / mL EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide), and react at room temperature for 1 h. Here, both NHS and EDC act as activators for the magnetic beads. NHS reacts with the carboxyl group to form an ester containing the NHS structure, directly activating the carbonyl group. It also stabilizes the reaction intermediate formed between EDC and the carboxyl group, improving the crosslinking efficiency of EDC. EDC contains a carbodiimide structure, which can form an amine-reactive acyl isourea intermediate with the carboxyl group on the magnetic beads. The amino group reacts with this intermediate to ultimately form a stable amide compound. Both NHS and EDC jointly participate in the activation of carboxyl magnetic beads, effectively improving the coupling between magnetic beads and antibody proteins.

[0039] Cleaning of magnetic beads: The activated magnetic beads are resuspended in MES buffer to achieve the purpose of washing. The washing step is repeated 3 times.

[0040] Magnetic bead-antibody coupling reaction: Resuspend the magnetic beads in the first antibody solution, adjust the antibody concentration to 3 mg / mL, and incubate the coupling reaction in an incubator for 5 h.

[0041] Termination of the conjugation reaction and preservation of the conjugate: After the reaction, remove the magnetic bead-antibody conjugate solution, wash it three times with TBST buffer, and block it overnight with an appropriate amount of CE510 blocking agent. The next day, resuspend it in DMSO preservation solution and adjust the concentration of the magnetic bead conjugate solution to 1 mg / mL (the primary antibody concentration is approximately 20 μg / mL).

[0042] Example 3 Preparation of AE-antibody labeled complex

[0043] The preparation process is briefly described below.

[0044] Antibody labeling reaction: Dilute the second antibody with 0.2M NaHCO3 solution to a concentration of 1 mg / mL (pH 9.0). Add 5 μL of 5 mmol / L AE solution (Helison (Xiamen) Biotechnology Co., Ltd., HS-13016011) to 100 μL of the second antibody solution, centrifuge slowly for 1 h (20℃, 500 rpm), then add 25 μL of stop solution containing 10% lysine, and shake for 1 h.

[0045] Purification and preservation of AE-antibody labeled complex: The AE-antibody labeled complex was eluted by column chromatography using PBS as the elution buffer. The collected AE-antibody labeled complex was then stored in PBS at a concentration of 10 μg / mL, and stored at 2-8℃ in the dark.

[0046] Example 4: Standard Curve Plotting

[0047] 4.1 MYBPC3 antigen standards with concentrations of 10 pg / mL, 25 pg / mL, 50 pg / mL, 100 pg / mL, 250 pg / mL, and 1000 pg / mL were prepared using PBS. The testing instrument was a Cosmetic SMART 6500 chemiluminescence detection platform. The sample loading method was a one-step method. 50 μL of the magnetic bead-antibody conjugate prepared in Example 2, 50 μL of the MYBPC3 antigen standard, and 50 μL of the AE-antibody-labeled complex prepared in Example 3 were added sequentially and incubated at 37°C for 30 min. After washing with PBS, 100 μL of acidic excitation solution (nitric acid, hydrogen peroxide) and 100 μL of alkaline excitation solution (NaOH) were added sequentially. The luminescence value at 470 nm was recorded, and a standard curve was plotted. The results are shown in Figure 1. Within a considerable concentration range, the antigen concentration showed a good linear relationship with the detected luminescence value (RLU).

[0048] 4.2 To determine whether there is a linear relationship between antigen concentration and the detected luminescence value within a low concentration range (20 pg / mL-100 pg / mL), we prepared MYBPC3 antigen standards at concentrations of 20 pg / mL, 40 pg / mL, 60 pg / mL, 80 pg / mL, and 100 pg / mL, and performed detection according to the above method. A standard curve was plotted, and the results are shown in Figure 2. Even at extremely low antigen concentrations, the double-antibody sandwich method combined with acrid ester chemiluminescence of this invention can still provide quantitative detection results.

[0049] Example 5: Serum Sample Detection

[0050] Serum sample preparation: 200 pg / mL of MYBPC3 antigen standard was diluted 2-, 5-, and 10-fold with healthy human serum to obtain 2-, 5-, and 10-fold serum dilutions. These serum dilutions were tested according to the method described in Example 4, and the test results based on the standard curve in Figure 2 are shown in Table 1.

[0051] Table 1. Detection results of serum diluted samples

[0052] The results in Table 1 show that serum does not significantly interfere with the detection results of the method of the present invention, and the method of the present invention can be used for the detection of MYBPC3 content in serum.

Claims

1. Myosin-binding protein C assay kit, including: 1) Magnetic beads conjugated with a first antibody, wherein the heavy chain variable region of the first antibody includes HCDR1 (SEQ ID NO:3), HCDR2 (SEQ ID NO:4), and HCDR3 (SEQ ID NO:5), and the light chain variable region of the first antibody includes LCDR1 (SEQ ID NO:7), LCDR2 (SEQ ID NO:8), and LCDR3 (SEQ ID NO:9); and 2) A second antibody conjugated with acridinium ester, wherein the heavy chain variable region of the second antibody includes HCDR1 (SEQ ID NO:11), HCDR2 (SEQ ID NO:12), and HCDR3 (SEQ ID NO:13), and the light chain variable region of the first antibody includes LCDR1 (SEQ ID NO:15), LCDR2 (SEQ ID NO:16), and LCDR3 (SEQ ID NO:17). or 1) Magnetic beads conjugated with a first antibody, wherein the heavy chain variable region of the first antibody includes HCDR1 (SEQ ID NO: 11), HCDR2 (SEQ ID NO: 12), and HCDR3 (SEQ ID NO: 13), and the light chain variable region of the first antibody includes LCDR1 (SEQ ID NO: 15), LCDR2 (SEQ ID NO: 16), and LCDR3 (SEQ ID NO: 17); and 2) A second antibody conjugated with acridinium ester, wherein the heavy chain variable region of the second antibody includes HCDR1 with sequence SEQ ID NO:3, HCDR2 with sequence SEQ ID NO:4, and HCDR3 with sequence SEQ ID NO:5, and the light chain variable region of the first antibody includes LCDR1 with sequence SEQ ID NO:7, LCDR2 with sequence SEQ ID NO:8, and LCDR3 with sequence SEQ ID NO:

9.

2. The myosin-binding protein C detection kit as described in claim 1, wherein: 1) The heavy chain variable region sequence of the first antibody is shown in SEQ ID NO:2, and the light chain variable region sequence of the first antibody is shown in SEQ ID NO:6; the heavy chain variable region sequence of the second antibody is shown in SEQ ID NO:10, and the light chain variable region sequence of the second antibody is shown in SEQ ID NO:14; or 2) The heavy chain variable region sequence of the first antibody is shown in SEQ ID NO:10, and the light chain variable region sequence of the first antibody is shown in SEQ ID NO:14; the heavy chain variable region sequence of the second antibody is shown in SEQ ID NO:2, and the light chain variable region sequence of the first antibody is shown in SEQ ID NO:

6.

3. The myosin-binding protein C detection kit as described in claim 1 further includes myosin-binding protein C standard solutions of different concentrations.

4. Use of the myosin-binding protein C detection kit according to any one of claims 1-3 in the preparation of a diagnostic kit for diagnosing acute myocardial infarction.

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