Gel sol protein monoclonal antibody and application thereof

Abstract

According to the monoclonal antibody capable of being specifically combined with the gelsolin and the kit containing the monoclonal antibody, the monoclonal antibody is high in affinity, and the kit prepared from the monoclonal antibody is good in precision and can meet the requirement for clinical in-vitro detection of the gelsolin level.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a gel sol protein monoclonal antibody and its application. Background Technology

[0002] Gelsolin (GSN) is a highly conserved actin-binding protein widely distributed in the cytoplasm and body fluids. It participates in key physiological processes such as cell migration, inflammatory responses, and tissue repair by regulating the dynamic assembly and disassembly of actin filaments. Its diagnostic value has been extensively studied, and results show that Gelsolin exhibits significant expression changes under various pathological conditions, including:

[0003] Acute myocardial infarction: GSN is one of the main factors independently predicting major adverse cardiovascular events within 3 months. Changes in GSN levels can reflect myocardial perfusion levels after emergency PCI and have certain predictive value for short-term prognosis.

[0004] Sepsis: Serum coagulant levels are closely related to disease severity and prognosis, and an early decrease in these levels can serve as a warning marker.

[0005] Tumors: In solid tumors such as lung cancer and breast cancer, abnormal expression of gelsin is associated with metastatic potential;

[0006] Neurodegenerative diseases: Fragments of gelling proteins in cerebrospinal fluid may be involved in the pathological process of Alzheimer's disease.

[0007] It is evident that diseases closely related to changes in gelspore protein levels are often severe and have a high incidence rate, posing a significant health threat to patients' lives and impacting families and society. Developing GSN into a diagnostic reagent for the early diagnosis, classification, and monitoring of treatment efficacy in these diseases would undoubtedly have significant clinical and social implications.

[0008] However, there are currently no approved gel protein detection reagents based on immunoassay worldwide. The main reason for this is that the performance of existing antibodies is insufficient, and the affinity of monoclonal antibodies is low, which affects the sensitivity of the detection and makes it impossible to capture trace protein changes in pathological states, making it difficult to prepare detection reagent products that meet the requirements.

[0009] Therefore, developing next-generation monoclonal antibodies against gel proteins and improving their affinity and detection sensitivity has become an urgent need for in vitro diagnostics of gel proteins. Summary of the Invention

[0010] This invention addresses the aforementioned technical problems by combining hybridoma technology with antibody engineering to obtain a monoclonal antibody targeting a unique conformational epitope of a gelling protein, exhibiting excellent affinity. This monoclonal antibody specifically binding to a gelling protein, or its antigen-binding fragment, includes a heavy chain variable region and a light chain variable region, wherein...

[0011] The heavy chain variable region includes the heavy chain CDR1 sequence shown in SEQ ID NO.2, the heavy chain CDR2 sequence shown in SEQ ID NO.3, and the heavy chain CDR3 sequence shown in SEQ ID NO.4;

[0012] The light chain variable region includes the light chain CDR1 sequence shown in SEQ ID NO.6, the light chain CDR2 sequence LVS, and the light chain CDR3 sequence shown in SEQ ID NO.7.

[0013] Furthermore, the monoclonal antibody or its antigen-binding fragment that specifically binds to colloid proteins described in this invention has the heavy chain variable region amino acid sequence shown in SEQ ID NO.1 and the light chain variable region amino acid sequence shown in SEQ ID NO.5.

[0014] Furthermore, the monoclonal antibody or its antigen-binding fragment that specifically binds to the gelling protein is a Fab fragment, Fab' fragment, F(ab')2 fragment, single-chain antibody, or humanized antibody. Because the antigen-binding fragment retains the variable regions of both the light and heavy chains, it can also recognize and bind to the gelling protein epitope.

[0015] The present invention also relates to a nucleic acid molecule comprising encoding the aforementioned specific gelling protein-binding monoclonal antibody or its antigen-binding fragment, and an expression vector comprising the aforementioned nucleic acid molecule, said expression vector being capable of expressing the aforementioned specific gelling protein-binding monoclonal antibody or its antigen-binding fragment. Furthermore, the present invention also relates to a recombinant comprising the aforementioned nucleic acid molecule or the aforementioned expression vector, which can produce the aforementioned specific gelling protein-binding monoclonal antibody or its antigen-binding fragment.

[0016] The present invention also relates to the application of the above-mentioned monoclonal antibody or antigen-binding fragment thereof that specifically binds to colloid proteins in the preparation of detection products for colloid proteins, such as the GSN monoclonal antibody or antigen-binding fragment thereof of the present invention being prepared into detection reagents / kits by different methods such as chemiluminescence, immunofluorescence, enzyme-linked immunosorbent assay or colloidal gold method, for the detection of colloid protein levels in body fluids (such as blood samples).

[0017] Furthermore, this invention provides a detection kit for sol-gel proteins, and more specifically, an immunoassay kit employing a double-antibody sandwich method. This kit contains a monoclonal antibody or its antigen-binding fragment that specifically binds to sol-gel proteins as the detection antibody; the capture antibody uses other monoclonal or polyclonal antibodies against sol-gel proteins, preferably the rabbit polyclonal antibody from Example 1 of this invention. The detection antibody and the capture antibody are interchangeable; that is, a monoclonal antibody that specifically binds to sol-gel proteins or its antigen-binding fragment can be used as the capture antibody. The kit can be manufactured using different methods such as chemiluminescence, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), or colloidal gold assay. The raw materials and excipients required for preparation, except for the detection antibody and the capture antibody, can all be selected from techniques known in the art.

[0018] In a preferred embodiment, a fluorescent immunochromatographic assay kit containing the monoclonal antibody of this invention was prepared using a double-antibody sandwich immunofluorescence method. The kit demonstrated good precision, with intra-assay repeatability coefficient of variation within 5% and inter-assay precision coefficient of variation within 7%. This precision is considered high among similar methodologies. Furthermore, the kit is unaffected by interference from substances commonly found in clinical settings, such as lipemia, hemolysis, jaundice, and / or anticoagulants, which may affect the test results. The invention also tested the kit's effectiveness in diagnosing specific clinical conditions with previously reported correlations. Results showed that the kit can be used to determine GSN levels in clinical samples, aiding in the diagnosis of acute myocardial infarction. Using 97.2 ng / ml as a diagnostic reference value, the sensitivity and specificity were both 82%, significantly higher than the previously reported sensitivity of 54.8% (specificity 80.6%), with an area under the diagnostic curve (AUC) of 0.89. Different methodologies, such as immunofluorescence, chemiluminescence, enzyme-linked immunosorbent assay (ELISA), and colloidal gold assays, each have their own advantages and disadvantages. Compared to fluorescence methods, chemiluminescence methods offer higher sensitivity and specificity. Depending on different clinical needs, the monoclonal antibody of this invention can be formulated into various immunoassay kits. These kits can be used to detect sol-gel protein levels in various populations with related diseases.

[0019] Instructions for the Preservation of Biological Materials

[0020] The monoclonal antibody hybridoma cell line that specifically binds to colloid proteins, as described in this invention, is named mouse hybridoma cell line 3472. It has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46340, on April 2, 2025. The address of the China General Microbiological Culture Collection Center is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China.

[0021] The monoclonal antibody of this invention has high affinity and sensitivity, and is not affected by high-abundance proteins in blood samples such as hemoglobin. The fluorescence immunochromatographic assay kit prepared with this monoclonal antibody has good intra- and inter-batch precision, which can fully meet the clinical requirements for in vitro detection of gel sol protein levels. Attached Figure Description

[0022] Figure 1 This is a ROC curve showing the diagnostic efficacy of the gel sol protein kit of the present invention in acute myocardial infarction and angina pectoris. Detailed Implementation

[0023] This invention utilizes hybridoma technology to prepare a gel sol protein monoclonal antibody, and the monoclonal antibody was sequenced. Its heavy chain variable region amino acid sequence consists of 118 amino acids, specifically EVQLVESGGGLVQPK GSLKLSCAAS. GFTFNTNA MNWVRQAPGKGLEWVAR IRTKSNNYAP FYADSVKDRFTI SRDDSQSMLYLQMNNLKTEDTAMYYC VGSGYAMDF WGQGTTVTVSS (SEQ ID NO.1), wherein the underlined sequences are CDR1, CDR2, and CDR3 in sequence; its light chain variable region amino acid sequence is 109 amino acids, and its specific sequence is DIVLTQSPASLAVSLGQRATISYRAS KSVSTSDYSY MHW NQQKPGQPPRLLIY LVS NLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYC QHIRELT R SEGAPSWKT (SEQ ID NO.5), where the underlined sequences are CDR1, CDR2, and CDR3, respectively. This monoclonal antibody has been verified to have high affinity for colloid proteins and can be used to prepare reagents / kits for detecting GSN.

[0024] Various antibody fragments, i.e., antigen-binding fragments, capable of binding to sol proteins, can also be prepared using existing techniques in the art based on the monoclonal antibody that specifically binds to sol proteins according to this invention. Examples include, but are not limited to, Fab, Fab', and F(ab')2. The Fab fragment is the antigen-binding region in the antibody structure, consisting of a complete light chain and a variable region VH and a constant region CH1 domain (Fd segment) of the heavy chain. Both the light and heavy chains have a constant region and a variable region, and disulfide bonds link the light and heavy chains. These antigen-binding fragments can be used to detect sol proteins or to prepare products for detecting sol proteins.

[0025] To illustrate the technical content, objectives, and effects of the present invention in detail, the following description is provided in conjunction with specific embodiments.

[0026] Example 1

[0027] Rabbit polyclonal antibody preparation

[0028] Healthy male white rabbits were selected. 1 mg of recombinant human soluble coagulant protein (purchased from AVISCERABIOSCIENCE) and 1 ml of Freund's complete adjuvant were mixed thoroughly with a stirrer until a milky white, viscous water-in-oil emulsion was formed. Multiple subcutaneous injections of at least 0.1 ml were performed along both sides of the rabbit's spine. Four weeks later, 1 mg of recombinant human soluble coagulant protein and 1 ml of Freund's incomplete adjuvant were mixed thoroughly with a stirrer and administered as a second immunization at different points on the same site. Two weeks later, blood was collected from the ear vein. An ELISA plate coated with recombinant human soluble coagulant protein was used, and the antibody titer was measured using an indirect ELISA method. A titer of 1:512000 was acceptable; a third booster immunization was performed if the titer was high. One week later, rabbit blood was collected via cardiac blood collection. The collected blood was quickly injected into a 30 ml sterile centrifuge tube and incubated at room temperature for 4 hours. After the blood clots and contracted, the tube was centrifuged at 5000 rpm for 15 minutes, and the serum was transferred to another sterile centrifuge tube. The polyclonal antibody was purified according to the instructions of the "Montage antibodypurification prosep-G kit". The content of the purified antibody was measured by UV spectrophotometer OD280. After aliquoting, the purified antibody was stored in a -20℃ freezer for later use.

[0029] Example 2

[0030] Preparation of mouse monoclonal antibodies

[0031] 1. Animal immunization and potency determination

[0032] Healthy 6-8 week old female Balb / c mice were selected. Recombinant human soluble gel sol protein, 250 μl of complete Freund's adjuvant, and 150 μl of water for injection were thoroughly emulsified in a mixer per mouse. Mice were immunized subcutaneously and intraperitoneally. Four weeks later, a second immunization was performed (50 μg antigen, 250 μl incomplete Freund's adjuvant, and 200 μl water for injection, 500 μl / mouse). After thorough emulsification, the mixture was immunized subcutaneously and intraperitoneally. Three days before fusion, a booster immunization was performed intraperitoneally (50 μg antigen, 450 μl water for injection, 500 μl / mouse, without adjuvant). Seven to ten days after the third immunization, blood was collected from the tail vein of the mice. Recombinant human soluble gel sol protein was coated onto an ELISA plate, and the serum titer of the immunized mice was detected using indirect ELISA. Mice with the highest antibody titers were selected for cell fusion.

[0033] 2. Preparation of monoclonal antibody hybridoma cell lines

[0034] Frozen SP2 / 0 cells were removed from liquid nitrogen and quickly thawed in a 37°C water bath. They were then cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator. The cells were passaged once the day before fusion to achieve a seeding concentration of 4–5 × 10⁻⁵ cells / day. 5 / ml. Mice were euthanized by cervical dislocation and disinfected by immersing in 75% alcohol for 3-5 minutes; the abdominal skin was carefully cut open and the spleen was removed. A spleen cell suspension was prepared and injected into a 50ml centrifuge tube, and washed once with 20ml of serum-free culture medium. SP2 / 0 myeloma cells and spleen cells were mixed at a ratio of 1:9 and centrifuged in serum-free RPMI 1640 medium at 1200 rpm for 8 min in a 50 ml plastic centrifuge tube. The cells were washed once, the supernatant was discarded, and any residual liquid was aspirated with a dropper. The bottom of the centrifuge tube was gently tapped to loosen the cell pellet slightly. The cells were then fused in a 37°C water bath. 1 ml of preheated 40% PEG was added within 60 seconds, and the centrifuge tube was gently rotated or tapped with a finger from time to time. Preheated serum-free RPMI 1640 medium was added to stop the effect of PEG. The cells were centrifuged at 800 rpm for 6 min and the supernatant was discarded. 2 ml of HAT-RPMI 1640 medium containing 20% ​​fetal bovine serum was added and the cells were gently resuspended to prevent the fused cells from dispersing. The fused cell suspension was added to 96-well plates containing feeder cells at a density of 100 μl / well and incubated at 37°C in a 5% CO2 incubator. Hybrid cell colonies appeared after 2–3 weeks. When the colonies reached 1 / 3 of the well, antibody titers were detected using indirect ELISA. Using a limiting dilution method, monoclonal antibody hybridoma cells with high positive values ​​were selected for cloning and subcloning until the positive rate reached 100%, ultimately obtaining 5 monoclonal antibody cell lines, numbered 3471, 3472, 3473, 3474, and 3475.

[0035] 3. Preparation and purification of monoclonal antibody ascites fluid

[0036] Eight-week-old male BALB / c mice were injected intraperitoneally with 0.5 ml of paraffin oil; 10 days later, they were injected intraperitoneally with 1×10⁻⁶ g of paraffin oil thoroughly washed with physiological saline. 6 Five monoclonal antibody cell lines were injected separately. Ten days after cell inoculation, when significant ascites production was observed in mice, the mice were euthanized by cervical retraction, and the ascites was collected. The cells were centrifuged at 2000 rpm and 4°C for 20 min, and the supernatant was collected. The monoclonal antibody was purified according to the "Montage antibody purification prosep-G kit" instructions. The concentration of purified antibody was measured using a UV spectrophotometer at OD280. The purified antibody was aliquoted and stored at -20°C for later use (each monoclonal antibody was distinguished by the cell line number that secreted the antibody).

[0037] Example 3

[0038] Screening of hybridoma cell lines

[0039] 1. Sensitivity evaluation of GSN monoclonal antibody detection

[0040] The purified GSN monoclonal antibody (as a capture antibody) was coated onto a 96-well microplate, blocked with BSA, and then serially diluted recombinant human soluble gel sol protein was added, followed by HRP-labeled rabbit polyclonal antibody from Example 1. The plate was then developed with TMB chromogenic buffer for 10 min, and the reaction was terminated with stop solution. Data were read using a microplate reader (as shown in Table 1).

[0041] Table 1 Results of monoclonal antibody sensitivity testing The test results showed that the sensitivity of gel sol monoclonal antibodies 3472 and 3473 was significantly higher than that of the other three groups, and also higher than the level of several hundred pg / ml of existing GSN monoclonal antibodies.

[0042] 2. Evaluation of GSN monoclonal antibody affinity

[0043] Three highly sensitive monoclonal antibodies were selected for Biocore binding assay to analyze antibody affinity. Different antibody strains were diluted 1:100 with PBS. After sensor pretreatment, antibodies were introduced at a rate of 5 μl / min for 1.5 min for binding. Subsequently, 1 mg / ml recombinant human soluble gel sol protein was introduced at a rate of 30 μl / min for 8 min, with the dissociation time being the same as the binding time. Chip regeneration was performed by washing with 10 mM glycine-HCl buffer (pH = 1.7) at a rate of 30 μl / min for 30 s. The antibody-antigen interaction was detected using the Biocore binding assay, and the results are detailed in Table 2. Ka represents the binding rate constant, the rate of binding between reacting molecules; kd represents the dissociation rate constant, the rate of dissociation of the reacting molecular complex; KD represents the dissociation equilibrium constant (KD = kd / ka). A higher Ka value indicates a faster antibody-antigen binding reaction; a lower KD value indicates a slower antibody-antigen dissociation rate; and a lower KD value indicates a higher antibody affinity for the antigen. See Table 2 for detailed results.

[0044] Table 2 Results of monoclonal antibody affinity assay

[0045] Monoclonal antibodies <![CDATA[Ka(10 4 1 / Ms)]]> <![CDATA[Kd(10 -4 1 / s)]]> <![CDATA[KD(10 -9 M)]]> 3472 25.63±2.59 5.46±1.14 2.13±0.46 3473 12.99±3.26 7.50±1.65 5.77±0.67 3474 15.28±1.77 6.48±1.41 4.24±0.49

[0046] The test results showed that among the three gel sol monoclonal antibodies, 3472 had the smallest dissociation equilibrium constant, indicating that this monoclonal antibody had the highest affinity for the antigen.

[0047] Based on the above performance evaluation results, the monoclonal antibody secreted by mouse hybridoma cell line 3472 exhibited the best sensitivity and potency. The inventors have deposited this cell line with the China General Microbiological Culture Collection Center (CGMCC), with registration number CGMCC No. 46340, on April 2, 2025. The address of the China General Microbiological Culture Collection Center is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China.

[0048] Example 4

[0049] Monoclonal antibody sequencing

[0050] Mouse hybridoma cell line 3472 containing monoclonal antibodies was used. Total RNA was extracted from the hybridoma cells using the Trizol method, and cDNA was obtained by reverse transcription. Primers for amplifying the heavy and light chain genes of the mouse monoclonal antibody were designed according to Chapter 3, "Genetic Engineering Antibody Technology," of the second edition of *Modern Immunology Experimental Techniques*. PCR amplification was performed, with a preheating temperature of 95℃ for 3 min, followed by 32 cycles of 95℃ for 30 seconds, 58℃ for 30 seconds, and 72℃ for 30 seconds, and a final extension at 72℃ for 7 min. The resulting cells were ligated into the pMD18-T vector and transformed into *E. coli* HB101. Positive clones were selected for sequencing. The hybridoma sequencing results were analyzed using the IgBLAST analysis module in the NCBI website to identify the variable regions of the antibody's heavy and light chains.

[0051] Sequence analysis revealed that the heavy chain variable region of the monoclonal antibody secreted by mouse hybridoma cell line 3472 contains 118 amino acids, specifically: EVQLVESGGGLVQPKGSLKLSCAAS GF TFNTNA MNWVRQAPGKGLEWVAR IRTKSNNYAP FYADSVKDRFTISRDDSQSMLYLQ MNNLKTEDTAMYYC VGSGYAMDF WGQGTTVTVSS (SEQ ID NO.1), wherein: CDR1 is located at 26-33 aa, with the amino acid sequence GFTFNTNA (SEQ ID NO.2); CDR2 is located at 51-60 aa, with the amino acid sequence IRTKSNNYAP (SEQ ID NO.3); CDR3 is located at 99-107 aa, with the amino acid sequence VGSGYA MDF (SEQ ID NO.4). The light chain variable region amino acid sequence of the monoclonal antibody secreted by mouse hybridoma cell line 3472 is 109 amino acids, and its specific sequence is DIVLTQSPASLAVSLGQRATISYRAS. KS VSTSDYSY MHWNQQKPGQPPRLLIY LVS NLESGVPARFSGSGSGTDFTLNIHPVEEEDA ATYYC QHIRELTR SEGAPSWKT (SEQ ID NO.5), wherein CDR1 is located at 27-36 aa and has the amino acid sequence KSVSTSDYSY (SEQ ID NO.6); CDR2 is located at 54-56 aa and has the amino acid sequence LVS; and CDR3 is located at 93-100 aa and has the amino acid sequence QHIRELTR (SEQ ID NO.7).

[0052] Example 5

[0053] Monoclonal antibody performance analysis — The effect of the gel sol monoclonal antibody of this invention on the detection of GSN content in human blood samples (fluorescence method)

[0054] Based on the double-antibody sandwich principle, a fluorescent pad (approximately 0.1 μg / person) is prepared using mouse anti-sol protein monoclonal antibody 3472 labeled with fluorescent microspheres. A rabbit anti-sol protein polyclonal antibody prepared according to this invention is coated onto the T-line of a nitrocellulose membrane (detection pad) (approximately 0.5 μg / person), and goat anti-mouse IgG polyclonal antibody is coated onto the C-line of the nitrocellulose membrane (detection pad) (approximately 0.3 μg / person). This process is used to prepare a sol protein fluorescence immunochromatographic detection kit. The specific preparation method is well-known in the art and will not be elaborated further.

[0055] (1) Precision test

[0056] Three batches (20240715, 20240716, and 20240717) of gel sol protein fluorescence immunochromatographic assay kits were prepared according to the above method. Three different instrument models were used to test three samples (serum samples at high, medium, and low concentrations) within one day. Each sample was tested 10 times, resulting in 30 results per sample per instrument model. Data analysis was performed. The study showed that the intra-batch repeatability coefficient of variation (CV) for each concentration of samples measured by the three batches of gel sol protein assay kits was less than 5%, and the inter-batch precision coefficient of variation (CV) was less than 7%. Compared with similar methodologies, this precision result is excellent.

[0057] (2) Analysis of anti-interference capability

[0058] The Glycosol Protein Fluorescent Immunochromatographic Detection Kit is mainly used to detect the content of GSN in human blood. However, blood samples often contain substances that may affect the test results, such as lipemia, hemolysis, jaundice, and / or anticoagulants. Therefore, the interference screening experiment is used to identify whether triglycerides, hemoglobin, bilirubin, and heparin sodium are interferences detected by the kit of this invention.

[0059] Preparation of basic samples: Fresh serum samples were collected from healthy individuals who had never taken any medication. Pure analytes of gel sol protein were added to each sample, and the samples were mixed to prepare two basic samples with concentrations of approximately 50.00 mg / L and 250.00 mg / L, respectively.

[0060] Preparation of interferon stock solutions: Prepare a triglyceride stock solution with a concentration of 148 mmol / L using anhydrous ethanol; prepare a hemoglobin stock solution with a concentration of 20 g / L using sample diluent; prepare a bilirubin stock solution with a concentration of 1.36 mol / L using sample diluent; prepare a heparin sodium stock solution with a concentration of 20 mg / mL using sample diluent.

[0061] Preparation: Take 4 portions of the basic sample, each 9.5 mL, and add 0.5 mL of the stock solution of each of the different interfering substances.

[0062] Preparation of control samples: Take 4 basic samples, 9.5 mL each, and add 0.5 mL of different interfering solvents to each sample.

[0063] The experimental and control samples were tested alternately, repeated 7 times. For example, each experimental sample (T) containing an interfering agent and each control sample (C) were aliquoted into 7 portions and labeled as T1, T2, T3, T4, T5, T6, T7, C1, C2, C3, C4, C5, C6, and C7. The concentration of gelling protein in the samples was detected using this kit, and the sample determination order was C1, T1, C2, T2, C3, T3, C4, T4, C5, T5, C6, T6, C7, and T7.

[0064] Analysis of the test results revealed that 7.4 mmol / L triglycerides, 1 g / L hemoglobin, 68 μmol / L bilirubin, and 1 mg / mL heparin sodium did not interfere with the detection results of GSN in human blood by the kit of this invention.

[0065] (3) Performance analysis for diagnostics

[0066] Blood samples to be tested: 50 serum samples from each of the acute myocardial infarction group, angina pectoris group, and health check-up group.

[0067] The level of GSN in each sample was detected using a batch of gel sol-gel fluorescence immunochromatographic assay kits used for precision studies. Statistical analysis was performed using GraphpadPrism 9.0 and SPSS Statistics 27.0 software to compare the diagnostic efficacy of GSN in acute myocardial infarction and angina pectoris.

[0068] The results showed that GSN expression differed significantly between the acute myocardial infarction group and other groups. The median expression was 122.2 mg / L in the healthy group, 80.36 mg / L in the acute myocardial infarction group, and 112.8 mg / L in the angina pectoris group. Compared with the healthy group, the GSN level in the acute myocardial infarction group was significantly lower (p < 0.001), while the GSN level in the angina pectoris group was slightly lower, but the difference was not statistically significant (p > 0.05). (See Table 3). ROC curve analysis... Figure 1 The area under the diagnostic curve (AUC) of GSN in acute myocardial infarction reached 0.89. Using 97.2 ng / ml as the diagnostic reference value, the sensitivity and specificity for diagnosis were both 82%, significantly higher than previously reported. These results suggest that the monoclonal antibody and double-antibody sandwich immunofluorescence method of this invention can be used to determine the GSN content in clinical samples, aiding in the diagnosis of acute myocardial infarction.

[0069] Table 3. Levels of GSN in blood samples from each group (ng / mL)

[0070] Healthy group (n=50) Acute myocardial infarction group (n=50) Angina group (n=50) gender Male / Female(23 / 27) Male / Female(29 / 21) Male / Female(26 / 24) GSN <![CDATA[122.2(71.5,172.5) *** ]]> <![CDATA[80.4(35.6,134.7) *** ]]> <![CDATA[112.8(54.7,185.1) ns ]]>

[0071] ***p<0.001, *p<0.05, ns p>0.05

[0072] The present invention has been described in detail above, with the aim of enabling those skilled in the art to understand and implement the invention. However, this description should not be construed as limiting the scope of protection of the invention. All equivalent changes or modifications made in accordance with the spirit and essence of the invention should be included within the scope of protection of the invention.

Claims

1. A monoclonal antibody or antigen-binding fragment thereof that specifically binds to a gelling protein, comprising a heavy chain variable region and a light chain variable region, wherein, The heavy chain variable region includes the heavy chain CDR1 sequence shown in SEQ ID NO.2, the heavy chain CDR2 sequence shown in SEQ ID NO.3, and the heavy chain CDR3 sequence shown in SEQ ID NO.4; The light chain variable region includes the light chain CDR1 sequence shown in SEQ ID NO.6, the light chain CDR2 sequence LVS, and the light chain CDR3 sequence shown in SEQ ID NO.

7.

2. The monoclonal antibody or its antigen-binding fragment as described in claim 1, characterized in that, The heavy chain variable region amino acid is the sequence shown in SEQ ID NO.1, and the light chain variable region amino acid is the sequence shown in SEQ ID NO.

5.

3. The monoclonal antibody or its antigen-binding fragment as described in claim 1 or 2, characterized in that, The antibody or antigen-binding fragment is a Fab fragment, Fab' fragment, F(ab')2 fragment, single-chain antibody, or humanized antibody.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule contains a nucleic acid encoding the antibody or its antigen-binding fragment as described in any one of claims 1-3.

5. An expression carrier, characterized in that, The expression vector contains the nucleic acid molecule as described in claim 4.

6. A recombinant, characterized in that, The recombinant comprises the nucleic acid molecule of claim 4 or the expression vector of claim 5.

7. The use of the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-3 in the preparation of a detection product for gel sol protein.

8. A detection kit for gel proteins, characterized in that, The kit contains the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-3.

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