Myocardial Myosin Binding Protein C Detection Kit and Its Application

Abstract

This text provides a myosin-binding protein C detection kit, which includes 1) magnetic beads conjugated with a first antibody and 2) a second antibody conjugated with acridinium ester. Both the first antibody and the second antibody are human myosin-binding protein C antibodies, but bind to different epitopes. This myosin-binding protein C detection kit highly sensitively detects the content of myosin-binding protein C in serum through the double antibody sandwich method and the chemiluminescent properties of acridinium ester.

Description

Technical Field

[0001] The present invention relates to a myosin binding protein C detection kit, in particular to a detection kit that can highly sensitively detect the content of myosin binding protein C in serum by a sandwich ELISA method. Background Art

[0002] Acute myocardial infarction (AMI) is an acute and critical disease of the cardiovascular system caused by unstable ischemic syndrome, with a high incidence, poor prognosis, and high mortality. However, for patients with myocardial infarction without typical chest pain and no obvious changes in electrocardiogram, it is difficult to make an accurate diagnosis relying solely on electrocardiogram, echocardiogram, and cardiac magnetic resonance. Currently, the globally recognized "gold standard" for the diagnosis of myocardial infarction is the myocardial injury marker cardiac troponin (cTn). cTn begins to be released 4 - 6 hours after the occurrence of AHI (acute myocardial injury) / AMI, can be detected in the blood, reaches the peak at 18 - 24 hours, and disappears two weeks later. High-sensitivity cTnT products appear 2.5 hours after the onset of myocardial injury, with low specificity and a clinical false positive rate as high as 30% - 50%, which is prone to misdiagnosis. The European Heart Guidelines clearly state that it is not recommended to use high-sensitivity cTn as a diagnostic standard within 3 hours.

[0003] Myocardial myosin binding protein C (cMyBP-C) is the latest AHI / AMI cardiac marker studied at home and abroad, with high specificity and can specifically reflect myocardial lesions. In the early stage of AMI, cMyBP-C and its metabolites are released into the blood in large amounts, and the increase in its content can be detected in the circulatory system as early as 30 minutes after myocardial injury. Therefore, it can be effectively applied to the detection of early myocardial injury; and compared with other markers, myocardial myosin binding protein C is cleared from the blood circulation earliest, so it also has good prognostic value.

[0004] For the detection method of myocardial myosin binding protein C (cMyBP-C), existing ones such as some four-in-one myocardial injury detection cards (colloidal gold method) kits, enzyme-linked immunosorbent assay kits, etc., are all limited by methodology, with insufficient sensitivity, unable to perform quantitative detection, and unable to make a good judgment on the trend and risk assessment of the disease. Moreover, there is a non-specific situation with biomarkers, often presenting false positive cases. The biomarker indicators of patients with renal insufficiency, pulmonary embolism, stroke but without myocardial injury will all increase, which can cause various misdiagnoses, delays, or over-treatments.

[0005] As a new type of detection marker, there is currently a lack of a method for accurately quantifying human cardiac myosin binding protein C (cMyBP-C, MYBPC3, cMyC). Currently, the only authorized patent related to the detection of this marker is the nano-gold cage colloidal gold method cMyBP-C detection kit of Nanjing Botian Kezhi Biotechnology Co., Ltd. However, the principle of the colloidal gold method is based on the massive aggregation of antibody-antigen complexes on the detection line, so it can only be applied to qualitative or semi-quantitative determination, and the detection values often have large errors. On the other hand, the HOOK effect of the colloidal gold method is relatively significant, which limits the linear range of the detection kit, making it unable to be well applied to the discrimination and evaluation of weakly positive and strongly positive patients in clinical applications. Generally, current human cardiac myosin binding protein C products can only provide qualitative results for users with 50 pg / ml as the reference cut-off value (positive judgment value), which to a certain extent limits the application of this marker in the grading determination of extremely early myocardial injury degree and the prognosis evaluation of cardiovascular-related surgeries in clinical practice. Summary of the Invention

[0006] In one aspect, the present invention provides a myosin binding protein C detection kit, which comprises:

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

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

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

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

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

[0014] In some embodiments, the myosin-binding protein C detection kit further comprises myosin-binding protein C standard solutions at different concentrations.

[0015] In one aspect, the present disclosure provides the use of the above-mentioned myosin-binding protein C detection kit in the preparation of a diagnostic kit for diagnosing acute myocardial infarction.

[0016] Compared with the existing manual myosin-binding protein C detection, the myosin-binding protein C detection kit provided herein has advantages such as high sensitivity, good specificity, wide linear range, and simple operation. Brief Description of the Drawings

[0017] Figure 1Shows the detection results of the cMyBP-C detection kit of the present invention for cMyBP-C antigen standards in a large concentration range.

[0018] Figure 2 Shows the detection results of the cMyBP-C detection kit of the present invention for cMyBP-C antigen standards at low concentrations. Detailed implementation manners

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

[0020] The present invention is based on the sandwich-specific binding of self-developed antibodies to human cMyBP-C antigen fragments and the direct chemiluminescence principle of AE (acridinium ester and its derivatives). Among them, the first antibody is a self-developed recombinant antibody of human cMyBP-C. The antibody is bound to the activated carboxyl magnetic bead solid-phase carrier through an amide bond to form a magnetic bead-first antibody conjugate, which is used to capture human cMyBP-C antigen fragments in the test sample in chemiluminescent immunoassay; the second antibody is a self-developed recombinant antibody of human cMyBP-C, and its reactivity is roughly the same as that of the first antibody. The antibody is bound to the AE compound through an amide bond. AE reacts instantaneously to emit light under the action of hydrogen peroxide and hydroxide. The relative light emission intensity (RLU) follows a quantitative relationship with the concentration of the magnetic bead-first antibody-human cMyBP-C antigen-second antibody-AE conjugate, so as to realize the quantitative detection of human cMyBP-C antigen fragments in the sample.

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

[0022] Example 1 Preparation of antibodies

[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 VH and VL genes by RT-PCR, constructed a phage display library in the form of ScFv, and performed four rounds of screening and enrichment with cMyBP-C antigen. Multiple clones with high affinity for cMyBP-C antigen were identified. Among them, two clones with high affinity for cMyBP-C antigen (EC determined by ELISA 50The values are all less than 30 ng / mL), and the clones binding to different epitopes of cMyBP-C antigen are used in the present invention, and their variable region sequences are as follows.

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

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

[0026] QIQLVQSGPELKKPGETVKISCKASGYTFR NYGMN WVKQAPGKGLKWMG WINTYSGVIYAKDFKG RFAFSLDTSASTAFLQINNLKNEDTATYFCAR DDYGYAMDF WGQGTSVTVSS (HCDR sequences are numbered as SEQ ID NO: 3, 4, and 5 in sequence)

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

[0028] DVVMTQIPLSLPVSLGDQASISC RSQSLGVHSYGITYH WYLQKPGQSPKLLTY RVSNSFS GVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC SQSTHVPIT FGGGTKLEIK (LCDR sequences are numbered as SEQ ID NO: 7, 8, and 9 in sequence)

[0029] Second antibody:

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

[0031] QIQLVQSGPELKKPGETVKISCKASGYNFRQ YLGMH WVKQRPGQGLEWIG HINPYGSLLNQNAKFKN RVTITRDTSASTAYMELSSLRSEDTAVYYCAR SGSFHRRMDY WGLGTSVTVSS (HCDR sequences are numbered as SEQ ID NO: 11, 12, and 13 in sequence)

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

[0033] DVVMTQTPLSLPVSLSDQASISC RSQQSLVHSNGTYLH WYLQKPGQSPKLLIY KLSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC SQSTIVSYTFGGGTKLEIR (LCDR sequences are numbered as SEQ ID NO: 15, 16, and 17 in sequence)

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

[0035] Example 2 Preparation of magnetic bead-antibody conjugate

[0036] The preparation process is briefly described as follows.

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

[0038] Activation of magnetic beads: Add 100 μL of 5 mg / mL NHS (N-hydroxysuccinimide), and then add 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 are used as activators of magnetic beads. Among them, NHS can react with carboxyl groups to form esters containing NHS structures, thereby directly activating carbonyl groups, and can also stabilize the reaction intermediates formed by EDC and carboxyl groups, improving the cross-linking efficiency of EDC; EDC contains a carbodiimide structure and can form amine-reactive acyl isourea intermediates with carboxyl groups on magnetic beads. Amino groups react with this intermediate to finally form stable amide compounds. The two jointly participate in the activation of carboxyl magnetic beads and can effectively improve the coupling of magnetic beads and antibody proteins.

[0039] Washing of magnetic beads: The activated magnetic beads were resuspended with MES buffer for washing purposes, and the washing step was repeated 3 times.

[0040] Coupling reaction of magnetic beads and antibody: Resuspend the magnetic beads with 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 coupling reaction and preservation of the conjugate: After the reaction, take out the magnetic bead-antibody conjugate solution, wash it 3 times with TBST buffer, and add an appropriate amount of CE510 blocking agent to block overnight. The next day, resuspend it with DMSO preservation solution and adjust the concentration of the magnetic bead conjugate solution to 1 mg / mL (the concentration of the first antibody is about 20 μg / mL).

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

[0043] The preparation process is briefly described as follows.

[0044] Antibody labeling reaction: Dilute the secondary antibody to a concentration of 1 mg / mL (pH 9.0) with 0.2M NaHCO 3 solution. Add 5 μL of 5 mmol / L AE solution (Helison (Xiamen) Biotechnology Co., Ltd., HS-13016011) to 100 μL of the secondary antibody solution, centrifuge the reaction slowly for 1 h (20 °C, 500 rpm), then add 25 μL of the termination solution containing 10% lysine, and oscillate the reaction for 1 h.

[0045] Purification and preservation of the AE-antibody labeling complex: Use PBS as the eluent to elute the AE-antibody labeling complex through a chromatography column. Store the collected AE-antibody labeling complex in PBS, adjust the concentration to 10 μg / mL, and store it in the dark at 2-8 °C.

[0046] Example 4 Standard curve drawing

[0047] 4.1 Prepare MYBPC3 antigen standard products with concentrations of 10 pg / mL, 25 pg / mL, 50 pg / mL, 100 pg / mL, 250 pg / mL, and 1000 pg / mL respectively with PBS. The test instrument is the Kosmile SMART 6500 chemiluminescence detection platform. The sample loading method is the one-step method. Add 50 μL of the magnetic bead-antibody conjugate prepared in Example 2, 50 μL of the MYBPC3 antigen standard product, and 50 μL of the AE-antibody labeling complex prepared in Example 3 in sequence, incubate at 37 °C for 30 min, wash with PBS, then add 100 μL of acidic excitation solution (nitric acid, hydrogen peroxide) and 100 μL of basic excitation solution (NaOH) in sequence, record the luminescence value results at 470 nm, and draw a standard curve. The results are as Figure 1 shown. In a quite large concentration range, there is a good linear relationship between the antigen concentration and the detected luminescence value (RLU).

[0048] 4.2 To determine whether there is also a linear relationship between the antigen concentration and the detected luminescence value in the low concentration range (20 pg / mL - 100 pg / mL), we prepared MYBPC3 antigen standards of 20 pg / mL, 40 pg / mL, 60 pg / mL, 80 pg / mL, and 100 pg / mL, detected them according to the above method, and plotted a standard curve. The results are as Figure 2 shown. In the case of extremely low antigen concentrations, the double antibody sandwich method of the present invention combined with acridinium ester chemiluminescence can still quantitatively give the detection results.

[0049] Example 5 Detection of Serum Samples

[0050] Preparation of serum samples: Dilute the 200 pg / mL MYBPC3 antigen standard with healthy human serum 2-fold, 5-fold, and 10-fold to obtain 2-fold, 5-fold, and 10-fold serum dilution samples. Detect these serum dilution samples according to the method described in Example 4, and the detection results obtained based on Figure 2 the standard curve are shown in Table 1.

[0051] Table 1 Detection Results of Serum Dilution Samples

[0052]

[0053] 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 detection kit, including: 1) magnetic beads coupled to a first antibody, wherein the heavy chain variable region of the first antibody comprises HCDR1 with a sequence of SEQ ID NO: 3, HCDR2 with a sequence of SEQ ID NO: 4, and HCDR3 with a sequence of SEQ ID NO: 5, and the light chain variable region of the first antibody comprises LCDR1 with a sequence of SEQ ID NO: 7, LCDR2 with a sequence of SEQ ID NO: 8, and LCDR3 with a sequence of SEQ ID NO: 9; and 2) a second antibody coupled to an acridinium ester, wherein the heavy chain variable region of the second antibody comprises HCDR1 with a sequence of SEQ ID NO: 11, HCDR2 with a sequence of SEQ ID NO: 12, and HCDR3 with a sequence of SEQ ID NO: 13, and the light chain variable region of the first antibody comprises LCDR1 with a sequence of SEQ ID NO: 15, LCDR2 with a sequence of SEQ ID NO: 16, and LCDR3 with a sequence of SEQ ID NO: 17, or 1) magnetic beads coupled to a first antibody, wherein the heavy chain variable region of the first antibody comprises HCDR1 with a sequence of SEQ ID NO: 11, HCDR2 with a sequence of SEQ ID NO: 12, and HCDR3 with a sequence of SEQ ID NO: 13, and the light chain variable region of the first antibody comprises LCDR1 with a sequence of SEQ ID NO: 15, LCDR2 with a sequence of SEQ ID NO: 16, and LCDR3 with a sequence of SEQ ID NO: 17; and 2) a second antibody coupled to an acridinium ester, the heavy chain variable region of the second antibody comprising HCDR1 with a sequence of SEQ ID NO: 3, HCDR2 with a sequence of SEQ ID NO: 4, and HCDR3 with a sequence of SEQ ID NO: 5, and the light chain variable region of the first antibody comprising LCDR1 with a sequence of SEQ ID NO: 7, LCDR2 with a sequence of SEQ ID NO: 8, and LCDR3 with a sequence of SEQ ID NO:

9.

2. The myosin binding protein C detection kit according to 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 comprising 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 to 3 in the preparation of a diagnostic kit for diagnosing acute myocardial infarction.

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