Cardiac myosin binding protein C detection kit and application thereof

Abstract

The invention provides a myosin binding protein C detection kit which comprises 1) a magnetic bead coupled with a first antibody and 2) a second antibody coupled with acridinium ester, and the first antibody and the second antibody are human myosin binding protein C antibodies but are combined with different epitopes. The myosin binding protein C detection kit can detect the content of myosin binding protein C in serum with high sensitivity through a double-antibody sandwich method and the chemiluminescence characteristic of acridinium ester.

Description

Technical Field

[0001] The invention relates to a myosin binding protein C detection kit, in particular to a detection kit which can detect the content of myosin binding protein C in serum with high sensitivity through a double antibody sandwich method. Background Art

[0002] Acute myocardial infarction (AMI) is an acute and critical cardiovascular disease caused by unstable ischemic syndrome, with high morbidity, poor prognosis and high mortality. However, for patients with myocardial infarction who do not have typical chest pain and no obvious changes in the electrocardiogram, it is difficult to make an accurate diagnosis based solely on electrocardiogram, echocardiogram and cardiac magnetic resonance imaging. The currently recognized global "gold standard" for the diagnosis of myocardial infarction is cardiac troponin (cTn), a marker of myocardial injury. cTn begins to be released 4 to 6 hours after the onset of AHI (acute myocardial injury) / AMI, can be detected in the blood, reaches a peak in 18 to 24 hours, and disappears after two weeks. High-sensitivity cTnT products appear 2.5 hours after the onset of myocardial injury, have low specificity, and have a clinical false positive rate of up to 30%-50%, which can easily lead to misdiagnosis. The European Heart Guidelines clearly state that high-sensitivity cTn is not recommended as a diagnostic standard within 3 hours.

[0003] Cardiac myosin binding protein C (cMyBP-C) is the latest AHI / AMI cardiac marker studied at home and abroad. It has 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 quantities. The increase in their content can be monitored in the circulation system as early as 30 minutes after myocardial injury occurs. Therefore, it can be effectively used for early myocardial injury detection. Compared with other markers, cardiac myosin binding protein C is cleared from the blood circulation the earliest, so it has 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) kits and enzyme-linked immunosorbent assay kits are limited by methodology, and their sensitivity is insufficient to achieve quantitative detection. They cannot make good judgments on the trend of the disease and risk assessment, and biomarkers are non-specific, and false positives often occur. The biomarker indicators of patients with renal insufficiency, pulmonary embolism, and stroke without myocardial injury will increase, which will cause various misdiagnoses, delays or excessive treatments.

[0005] As a new detection marker, human cardiac myosin binding protein C (cMyBP-C, MYBPC3, cMyC) currently lacks a method that can achieve accurate quantitative detection. At present, the only authorized patent involving the detection of this marker is the nano-gold cage colloidal gold cMyBP-C detection kit of Nanjing Botian Kezhi Biotechnology Co., Ltd. However, the principle of the colloidal gold method is based on the large-scale aggregation of antibody-antigen complexes on the detection line, so it can only be applied to qualitative or semi-quantitative determination, and the detection value often has a large error; on the other hand, the HOOK effect (hook effect) of the colloidal gold method is more significant, thereby limiting the linear range of the detection kit, so that the detection kit cannot be well used in the clinical application of weak positive and strong positive patients. In general, the current human cardiac myosin binding protein C product can only provide users with qualitative results with 50pg / ml as the reference cut off value (positive judgment value), which limits the application of this marker in the clinical classification of very early myocardial injury and the prognosis evaluation of cardiovascular-related surgery to a certain extent. Summary of the invention

[0006] In one aspect, the present invention provides a myosin binding protein C detection kit, comprising: 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 of SEQ ID NO: 3, HCDR2 of SEQ ID NO: 4 and HCDR3 of SEQ ID NO: 5, and the light chain variable region of the first antibody comprising LCDR1 of SEQ ID NO: 7, LCDR2 of SEQ ID NO: 8 and LCDR3 of SEQ ID NO: 9.

[0007] In some embodiments, the heavy chain variable region sequence of the first antibody is shown as SEQ ID NO: 2, and the light chain variable region sequence of the first antibody is shown as SEQ ID NO: 6; the heavy chain variable region sequence of the second antibody is shown as SEQ ID NO: 10, and the light chain variable region sequence of the second antibody is shown as SEQ ID NO: 14.

[0008] In some embodiments, the heavy chain variable region sequence of the first antibody is shown as SEQ ID NO: 10, and the light chain variable region sequence of the first antibody is shown as SEQ ID NO: 14; the heavy chain variable region sequence of the second antibody is shown as SEQ ID NO: 2, and the light chain variable region sequence of the first antibody is shown as SEQ ID NO: 6.

[0009] In some embodiments, the myosin binding protein C detection kit further comprises myosin binding protein C standard solutions of different concentrations.

[0010] In one aspect, the present invention 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.

[0011] Compared with the existing manual myosin binding protein C detection method, the myosin binding protein C detection kit provided in this article has the advantages of high sensitivity, good specificity, wide linear range and simple operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1The results show the detection results of the cMyBP-C detection kit of the present invention on cMyBP-C antigen standards with a wide concentration range.

[0013] Figure 2 The results of the detection of low-concentration cMyBP-C antigen standards by the cMyBP-C detection kit of the present invention are shown. DETAILED DESCRIPTION

[0014] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0015] The present invention is based on the sandwich specific binding of self-developed antibody pairs with 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 human cMyBP-C recombinant antibody, which is bound to an activated carboxyl magnetic bead solid phase carrier through an amide bond to form a magnetic bead-first antibody conjugate, which is used to capture the human cMyBP-C antigen fragment in the sample to be tested in the chemiluminescent immunoassay; the second antibody is a self-developed human cMyBP-C recombinant antibody, which has roughly the same reactivity as the first antibody, and the antibody is bound to the AE compound through an amide bond. AE reacts and emits light instantaneously under the action of hydrogen peroxide and hydroxide, and its 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, thereby realizing the quantitative detection of the human cMyBP-C antigen fragment in the sample.

[0016] The present invention is further illustrated by the following examples.

[0017] Example 1 Preparation of Antibodies We immunized mice with the N-terminal fragment of 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 ScFv phage display library, and carried out four rounds of screening and enrichment with cMyBP-C antigen, identifying multiple clones with high affinity to cMyBP-C antigen. Among them, two clones with high affinity to cMyBP-C antigen (EC determined by ELISA) were selected. 50The values ​​were all less than 30 ng / mL), and clones binding to different epitopes of cMyBP-C antigen were used in the present invention, and their variable region sequences were as follows.

[0018] First antibody (the underlined ones are CDR sequences, according to the Kabat numbering scheme, the same below): VH sequence (SEQ ID NO: 2) QIQLVQSGPELKKPGETVKISCKASGYTFR NYGMN WVKQAPGKGLKWMG WINTYSGVIYAKDFKG RFAFSLDTSASTAFLQINNLKNEDTATYFCAR DDYGYAMDF WGQGTSVTVSS (HCDR sequences are numbered as SEQ ID NOs: 3, 4 and 5) VL sequence (SEQ ID NO: 6) DVVMTQIPLSLPVSLGDQASISC RSQSLGVHSYGITYH WYLQKPGQSPKLLTY RVSNSFS GVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC SQSTHVPIT FGGGTKLEIK (LCDR sequences are numbered as SEQ ID NOs: 7, 8 and 9) Secondary Antibody: VH sequence (SEQ ID NO: 10) QIQLVQSGPELKKPGETVKISCKASGYNFRQ YLGMH WVKQRPGQGLEWIG HINPYGSLLNQNAKFKN RVTITRDTSASTAYMELSSLRSEDTAVYYCAR SGSFHRRMDY WGLGTSVTVSS (HCDR sequences are numbered as SEQ ID NOs: 11, 12 and 13) VL sequence (SEQ ID NO: 14) DVVMTQTPLSLPVSLSDQASISC RSQQSLVHSNGTYLH WYLQKPGQSPKLLIY KLSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC SQSTIVSYT FGGGTKLEIR (LCDR sequences are numbered as SEQ ID NOs: 15, 16 and 17) After codon optimization, the DNA fragments of the variable regions were synthesized and fused to the heavy chain constant region and light chain kappa constant region of human IgG1, respectively. After the heavy chain and light chain expression constructs were cloned into plasmid expression vectors, they were secreted and expressed in HEK293 cells, and the antibodies were purified by magnetic beads coated with protein A. The concentration of the purified antibody stock solution is generally 1.5-4.5 mg / mL, and can be ultrafiltered through a 30Kda ultrafiltration membrane to adjust the antibody concentration to 5 mg / mL.

[0019] Example 2 Preparation of magnetic bead-antibody conjugates The preparation process is briefly described as follows.

[0020] Preparation of magnetic bead suspension: Take 100 μL JSR MS300C magnetic beads (JSR Life Science, hydrophilic carboxyl magnetic beads, magnetic bead particle size 3 μm) and suspend them in 900 μL MES (2-(N-morpholino)ethanesulfonic acid, 2-(N-morpholino)ethanesulfonic acid potassium) buffer (0.5 mol / L pH = 5.0).

[0021] Activation of magnetic beads: Add 100μL of 5 mg / mL NHS (N-Hydroxysuccinmide) and 100μL of 7 mg / ml EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide, 1-(3-Dimethylaminopropyl)3-ethylcarbodiimide) and react at room temperature for 1 h. Here, both NHS and EDC are used as activators of magnetic beads. NHS reacts with carboxyl groups to form esters containing NHS structures, thereby directly activating carbonyl groups. It can also stabilize the reaction intermediates formed by EDC and carboxyl groups, thereby improving the cross-linking efficiency of EDC. EDC contains a carbodiimide structure, which can form an amine-reactive acylisourea intermediate with the carboxyl groups on the magnetic beads. The amino group reacts with the intermediate to finally form a stable amide compound. The two participate in the activation of carboxyl magnetic beads together, which can effectively improve the coupling of magnetic beads and antibody proteins.

[0022] Washing of magnetic beads: The activated magnetic beads were resuspended in MES buffer to achieve the purpose of washing, and the washing step was repeated 3 times.

[0023] Magnetic bead-antibody coupling reaction: resuspend the magnetic beads with the primary antibody solution, adjust the antibody concentration to 3 mg / mL, and incubate the coupling reaction in a constant temperature box for 5 h.

[0024] Termination of coupling reaction and storage of conjugate: After the reaction is completed, take out the magnetic bead-antibody conjugate solution, wash it with TBST buffer for 3 times, add an appropriate amount of CE510 blocking agent and block it 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).

[0025] Example 3 Preparation of AE-antibody labeled complex The preparation process is briefly described as follows.

[0026] Antibody labeling reaction: The secondary antibody was washed with 0.2 M NaHCO 3 The solution was diluted to a concentration of 1 mg / mL (pH 9.0). 5 μL of 5 mmol / L AE solution (Helison (Xiamen) Biotechnology Co., Ltd., HS-13016011) was added to 100 μL of the secondary antibody solution, and the mixture was centrifuged slowly for 1 h (20°C, 500 rpm), and then 25 μL of the stop solution containing 10% lysine was added and the mixture was shaken for 1 h.

[0027] Purification and storage of AE-antibody labeled complex: AE-antibody labeled complex was eluted by chromatography column using PBS as eluent, and the collected AE-antibody labeled complex was stored in PBS, adjusted to a concentration of 10 μg / mL, and stored at 2-8°C away from light.

[0028] Example 4 Standard Curve Drawing 4.1 Use PBS to prepare MYBPC3 antigen standards with concentrations of 10pg / mL, 25pg / mL, 50pg / mL, 100pg / mL, 250pg / mL, and 1000pg / mL, respectively. The test instrument is the Cosma SMART 6500 chemiluminescence detection platform. The loading method is a one-step method. Add 50μL of the magnetic bead-antibody conjugate prepared in Example 2, 50 μL of the MYBPC3 antigen standard, and 50μL of the AE-antibody labeled complex prepared in Example 3 in sequence, incubate at 37°C for 30 min, wash with PBS, and add 100 μL of acidic excitation solution (nitric acid, hydrogen peroxide) and 100 μL of alkaline excitation solution (NaOH) in sequence, record the luminescence value results at 470nm, and draw a standard curve. The results are as follows: Figure 1 As shown in Figure 4, within a fairly large concentration range, there is a good linear relationship between the antigen concentration and the detected luminescence value (RLU).

[0029] 4.2 In order to determine whether there is a linear relationship between the antigen concentration and the luminescence value detected in the low concentration range (20 pg / mL - 100 pg / mL), we prepared MYBPC3 antigen standards at 20 pg / mL, 40 pg / mL, 60 pg / mL, 80 pg / mL, and 100 pg / mL, tested them according to the above method, and drew a standard curve. The results are shown in the figure. Figure 2 In the case of extremely low antigen concentration, the double antibody sandwich method of the present invention combined with acridinium ester chemiluminescence can still give quantitative detection results.

[0030] Example 5 Serum sample detection Preparation of serum samples: 200 pg / mL MYBPC3 antigen standard was diluted 2-fold, 5-fold and 10-fold with healthy human serum to obtain 2-fold, 5-fold and 10-fold serum dilution samples. These serum dilution samples were tested according to the method described in Example 4, and based on Figure 2 The detection results obtained with the standard curve are shown in Table 1.

[0031] Table 1 Test results of serum dilution samples The results in Table 1 indicate 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 to detect the 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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