Hybridoma cell strain capable of secreting pigment epithelium-derived factor monoclonal antibody, monoclonal antibody and application of monoclonal antibody

Abstract

The invention relates to the technical field of biology, and particularly discloses a hybridoma cell strain capable of secreting a pigment epithelium-derived factor monoclonal antibody as well as the monoclonal antibody and application of the hybridoma cell strain, and the hybridoma cell strain comprises a hybridoma cell strain JD01 # 1-E6 with the preservation number of GDMCC No: 64528 or a hybridoma cell strain JD01 # 3-B11 with the preservation number of GDMCC No: 64529. Two monoclonal antibodies aiming at different antigen epitopes of the pigment epithelium-derived factor are obtained by obtaining the hybridoma cell strain secreting the monoclonal antibody resisting the pigment epithelium-derived factor, and the specificity, the affinity and the titer of the monoclonal antibodies all meet the detection requirements; the content of the pigment epithelium-derived factor in serum or plasma can be detected through pairing, and the method can be applied to products for detecting change of the content of the pigment epithelium-derived factor or diagnosing diseases characterized by abnormal expression of the pigment epithelium-derived factor.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a hybridoma cell line secreting a monoclonal antibody of a pigment epithelium-derived factor, and its monoclonal antibody and application. Background Art

[0002] Pigment epithelium-derived factor (PEDF) is a secretory glycoprotein composed of 418 amino acids and a molecular weight of 50KD. It was first isolated from the conditioned medium of cultured human fetal retinal pigment epithelial cells. Recent studies have shown that PEDF is a multifunctional, pleiotropic protein with anti-angiogenic, anti-tumor, antioxidant, anti-inflammatory, anti-thrombotic, neurotrophic and neuroprotective properties.

[0003] Pigment epithelium-derived factor participates in maintaining and regulating microvascular homeostasis in the retina, kidney, heart, liver, alveoli, and brain, and its expression level plays different roles in the vascular growth, development, and pathophysiology of organs. Excessive or low expression levels can lead to functional abnormalities in various organs. For example, the lack of pigment epithelium-derived factor in the eyes and kidneys seriously affects the density of capillary plexus under physiological and pathological conditions; the level of pigment epithelium-derived factor in the serum of patients with type IV osteogenesis imperfecta is extremely low or undetectable; the gene deletion of pulmonary pigment epithelium-derived factor leads to delayed development of pulmonary vascular endothelium; the imbalance of the ratio of pigment epithelium-derived factor and vascular endothelial growth factor (VEGF) changes the development and plasticity of early liver blood vessels; the fertility and life expectancy of the pigment epithelium-derived factor gene knockout mice are not affected, but they show pathological characteristics such as pancreatic hyperplasia and prostate hyperplasia; in addition, the changes in the concentration level of pigment epithelium-derived factor in serum not only reflect the changes in the structure and function of the vascular endothelium, but are also closely related to the occurrence and development of diabetic microangiopathy and coronary artery disease. In addition, studies have shown that the level of pigment epithelium-derived factor in diabetic patients is significantly higher than that in non-diabetic patients. The concentration level of pigment epithelium-derived factor is related to insulin resistance in diabetic patients. The concentration level of this factor is closely related to the occurrence and development of diabetic nephropathy and diabetic retinopathy. The research on pigment epithelium-derived factor molecules is increasingly valued by experts in related fields.

[0004] The determination of pigment epithelium-derived factor can be used to diagnose diseases such as the degree of fibrosis in diabetic nephropathy and insulin resistance syndrome. However, in existing studies, the concentration level of pigment epithelium-derived factor is mainly detected by enzyme-linked immunosorbent assay (ELISA), which is not only cumbersome to operate but also has poor reproducibility. In addition, the means of detecting changes in the content of pigment epithelium-derived factor or diagnosing abnormal expression of pigment epithelium-derived factor are not perfect enough.

[0005] Therefore, there is an urgent need for a monoclonal antibody that specifically recognizes pigment epithelium-derived factor in a blood sample and a hybridoma cell line that secretes anti-pigment epithelium-derived factor monoclonal antibody and its application. Summary of the invention

[0006] The purpose of the present application is to overcome the deficiencies of the above-mentioned prior art and provide a hybridoma cell line secreting monoclonal antibodies of pigment epithelium-derived factor and its monoclonal antibodies and applications. The present application obtains hybridoma cell lines (hybridoma cell line JD01#1-E6 and hybridoma cell line JD01#3-B11) that secrete monoclonal antibodies against pigment epithelium-derived factor, and then obtains two monoclonal antibodies against different antigenic epitopes of pigment epithelium-derived factor. The monoclonal antibodies have high specificity, affinity and titer, and can be used to detect the content of pigment epithelium-derived factor in samples through pairing, and are used in products for detecting changes in the content of pigment epithelium-derived factor or diagnosing diseases characterized by pigment epithelium-derived factor.

[0007] To achieve the above purpose, the technical solution adopted by this application is:

[0008] In a first aspect, the present application provides a hybridoma cell strain, wherein the hybridoma cell strain includes a hybridoma cell strain JD01#1-E6 with a deposit number of GDMCC No: 64528 or a hybridoma cell strain JD01#3-B11 with a deposit number of GDMCC No: 64529;

[0009] The hybridoma cell line JD01#1-E6 and hybridoma cell line JD01#3-B11 were both deposited in the Guangdong Provincial Microbiological Culture Collection Center on April 22, 2024, and the storage address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0010] In a second aspect, the present application provides a monoclonal antibody, which is produced by the hybridoma cell line and specifically recognizes pigment epithelium-derived factor.

[0011] The monoclonal antibody secreted by the hybridoma cell line screened in the present application can specifically recognize the pigment epithelium-derived factor in the blood sample. The monoclonal antibody has good specificity and moderate affinity, and can be used to detect the content of the pigment epithelium-derived factor in the blood sample through pairing, and the blood sample for detection does not need to be diluted twice or highly.

[0012] After the monoclonal antibody titer test, the monoclonal antibody titers of hybridoma cell line JD01#1-E6 and hybridoma cell line JD01#3-B11 were 1:128000 and 1:64000, respectively, which were high.

[0013] As a preferred embodiment of the monoclonal antibody described in the present application, the amino acid sequence of the pigment epithelium-derived factor is shown as SEQ ID NO: 1 or SEQ ID NO: 2.

[0014] The monoclonal antibody pairing experiment verified that the monoclonal antibody secreted by the hybridoma cell line JD01#1-E6 had good reactivity with the monoclonal antibody secreted by the hybridoma cell line JD01#3-B11 as a capture antibody, and had little reaction with cross-reactive substances.

[0015] As a preferred embodiment of the monoclonal antibody described in the present application, the monoclonal antibody produced by the hybridoma cell line JD01#1-E6 recognizes the pigment epithelium-derived factor with an amino acid sequence such as SEQ ID NO:1; the monoclonal antibody produced by the hybridoma cell line JD01#3-B11 recognizes the pigment epithelium-derived factor with an amino acid sequence such as SEQ ID NO:2.

[0016] In a third aspect, the present application provides a method for preparing the above-mentioned monoclonal antibody, wherein the enriched hybridoma cell line is inoculated into the peritoneal cavity of mice, and after collecting ascites, the monoclonal antibody is obtained through purification.

[0017] More preferably, the method for preparing the hybridoma cell line comprises the following steps:

[0018] 1) Preparation of pigment epithelium-derived factor fragments: Two pigment epithelium-derived factor fragments were obtained by protein expression, and the amino acid sequences were SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0019] 1) Immunize mice with pigment epithelium-derived factor fragments and detect the titer of mouse serum;

[0020] 2) After the titer of mouse serum reaches 1:10000, the mouse is intraperitoneally boosted with pigment epithelium-derived factor fragments, spleen cells of the immunized mice are fused with mouse myeloma cells, and hybridoma cells with monoclonal antibodies are cloned and screened for expansion, re-culture and preservation.

[0021] More preferably, the mouse myeloma cells are SP2 / 0 mouse myeloma cells.

[0022] More preferably, the method for preparing the above monoclonal antibody comprises the following steps:

[0023] 1) Liquid paraffin was injected into the peritoneal cavity of BALB / c mice, and the hybridoma cell line was inoculated into the peritoneal cavity, and the ascites supernatant was collected for later use;

[0024] 2) Filter the collected ascites solution through a microporous filter membrane, add ammonium sulfate, stir evenly, let stand, collect the precipitate, and redissolve the precipitate with a PBS solution; load the obtained crude pure antibody onto a Protein A-Sepharose affinity chromatography column, wash with 5 column volumes of binding buffer (50 mM PBS, pH 7.0), and then elute the antibody with a glycine-hydrochloric acid solution to obtain a monoclonal antibody.

[0025] In a fourth aspect, the present application provides the use of the above hybridoma cell line or the above monoclonal antibody in detecting pigment epithelium-derived factor.

[0026] In a fifth aspect, the present application provides the use of the above-mentioned monoclonal antibody in the preparation of an in vitro immunoassay reagent or kit for pigment epithelium-derived factor.

[0027] In a sixth aspect, the present application provides an immunodetection reagent, which includes the monoclonal antibody.

[0028] In a seventh aspect, the present application provides a pigment epithelium-derived factor detection kit, wherein the pigment epithelium-derived factor detection kit comprises the monoclonal antibody.

[0029] As a preferred embodiment of the pigment epithelium-derived factor detection kit described in the present application, the pigment epithelium-derived factor detection kit also includes a standard, a detection antibody and a chemiluminescent substrate.

[0030] In an eighth aspect, the present application provides the use of the hybridoma cell line or the monoclonal antibody as described above in the preparation of a product for detecting changes in the content of pigment epithelium-derived factor or diagnosing a disease characterized by pigment epithelium-derived factor.

[0031] Compared with the prior art, this application has the following beneficial effects:

[0032] The present application provides a hybridoma cell line secreting a monoclonal antibody of a pigment epithelium-derived factor, its monoclonal antibody and application. The hybridoma cell line provided in the present application can secrete two monoclonal antibodies targeting different antigenic epitopes of the pigment epithelium-derived factor. The obtained monoclonal antibodies have high specificity, affinity and titer. The content of the pigment epithelium-derived factor in a blood sample can be detected by pairing, and the hybridoma cell line can be used in products for detecting changes in the content of the pigment epithelium-derived factor or diagnosing diseases characterized by abnormal expression of the pigment epithelium-derived factor. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The electrophoresis diagram of 13.0% SDS-PAGE is for the monoclonal antibodies secreted by hybridoma cell lines JD01#1-E2, JD01#1-E6, JD01#3-B5 and JD01#3-B11. DETAILED DESCRIPTION

[0034] In order to better illustrate the purpose, technical solutions and advantages of the present application, the present application will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0035] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified, and the components and raw materials used in each parallel experiment are of the same type.

[0036] Example 1. Preparation of hybridoma cell lines secreting monoclonal antibodies to pigment epithelium-derived factor

[0037] 1) Mouse immunization: BALB / c mice were immunized with recombinant pigment epithelium-derived factor, with 50 μg antigen per mouse per immunization. For the first immunization, recombinant pigment epithelium-derived factor was mixed with Freund's complete adjuvant at a mass ratio of 1:1, and then pigment epithelium-derived factor was mixed with Freund's incomplete adjuvant at a mass ratio of 1:1. Immunization was performed every two weeks for a total of 3 times. Ten days after the third immunization, the tail was cut off and blood was collected to detect the serum titer.

[0038] 2) Hybridoma cell fusion: After the mouse serum titer reaches 1:10000, the mouse is intraperitoneally boosted with pigment epithelium-derived factor (30 μg). Three days later, spleen cells of the immunized mice are aseptically taken and mixed with SP2 / 0 mouse myeloma cells in a 50 mL centrifuge tube. Then, 1 mL of preheated PEG is added for 45 seconds, and then serum-free DMEM medium preheated at 37°C is immediately and slowly added dropwise to 35 mL. The medium is incubated in a 42°C water bath for 15 minutes, and centrifuged at 1000 rpm / min for 10 minutes, the supernatant is discarded, 50 mL of preheated HAT medium is added, the mixture is gently pipetted and mixed, and the mixture is transferred to a 96-well culture plate with feeder cells, 100 μL per well, and cultured in an incubator. The cell supernatant was identified using pigment epithelium-derived factor, and the positive hybridomas were cloned and screened three times, resulting in four hybridoma cell lines secreting specific monoclonal antibodies, which were named hybridoma cell lines JD01#1-E2, JD01#1-E6, JD01#3-B5 and JD01#3-B11, respectively.

[0039] In this embodiment, the preservation number of hybridoma cell line JD01#1-E6 is GDMCC NO: 64528, and the preservation number of hybridoma cell line JD01#3-B11 is GDMCC NO: 64529; hybridoma cell line JD01#1-E6 and hybridoma cell line JD01#3-B11 are deposited in Guangdong Provincial Microbiological Culture Collection Center, and the preservation address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the preservation time is April 22, 2024 respectively.

[0040] Example 2: Preparation, purification and titer detection of monoclonal antibodies against pigment epithelium-derived factor

[0041] 1) Preparation of monoclonal antibody ascites: 10-12 week old BALB / c mice were intraperitoneally injected with liquid paraffin, 0.5 mL / mouse. Seven days later, the above hybridoma cell lines (JD01#1-E2, JD01#1-E6, JD01#3-B5 and JD01#3-B11) were intraperitoneally inoculated with 1×10 6 / mouse, after 7-10 days, when the abdomen of the mouse is swollen, collect the ascites supernatant for later use.

[0042] 2) Antibody purification: The collected ascites was filtered through a 0.45 μm microporous filter, and ammonium sulfate with a final concentration of 50% was added. After stirring evenly at 4°C, the mixture was allowed to stand for 1 hour, and the precipitate was collected at 12,000 rpm. The precipitate was redissolved with a 50 mM PBS solution to obtain crude pure antibody. The crude pure antibody was loaded onto a Protein A-Sepharose affinity chromatography column at a flow rate of 1 mL / min, washed with 5 column volumes of binding buffer (50 mM PBS, pH 7.0), and then eluted with a 0.1 M glycine-hydrochloric acid solution at pH 2.7 (1 M pH 9.0 Tris buffer was added to each collection tube in advance for neutralization) to obtain the target antibody, which was dialyzed 3 times with a 50 mM PBS solution. The dialyzed antibodies were subjected to 13.0% SDS-PAGE electrophoresis. The results showed that the monoclonal antibodies secreted by hybridoma cell lines JD01#1-E2, JD01#1-E6, JD01#3-B5 and JD01#3-B11 had bands at 50kD and 25kD, and the grayscale analysis purity was greater than 95%. Figure 1 shown.

[0043] 3) Monoclonal antibody titer detection: The microplate was coated with 100 μl of 1 μg / ml pigment epithelium-derived factor in carbonate buffer (pH 9.5) at 4°C overnight. The concentration of the antibody after purification in step 2) was adjusted to 1 mg / mL, and each antibody was gradiently diluted (1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, 1:512000), and goat anti-mouse IgG-HRP (1:200000) was added to determine the titer of the purified monoclonal antibody (S / N>2.1). The titers of the monoclonal antibodies secreted by hybridoma cell lines JD01#1-E2, JD01#1-E6, JD01#3-B5 and JD01#3-B11 were 1:256000, 1:128000, 1:128000, and 1:64000, respectively.

[0044] Example 3: Monoclonal Antibody Epitope Identification

[0045] Recombinant truncated pigment epithelium-derived factor construction and expression: According to the amino acid sequence of pigment epithelium-derived factor, the following amino acid sequence (SEQ ID NO: 1-2) was designed, connected to the expression vector pET28a, and loaded into the expression strain BL21 (DE3). In LB medium, IPTG 1mmol / L was used to induce the soluble expression of the truncated recombinant protein of pigment epithelium-derived factor overnight at room temperature. The prokaryotic recombinant expression was affinity purified by polyhistidine tag, and the purified protein was subjected to SDS-PAGE electrophoresis, and the purity was above 90%.

[0046] SEQ ID NO: 1: DPFFKVPVNK LAAAVSNFGY DLYRVRSSTS PTTNVLLSPLSVATALALSALSLGAEQRTESI IHRALYYDLI SSPDIHGT.

[0047] SEQ ID NO:2: LTFPLDYHLN QPFIFVLRDT DTGALLFIGK ILDPRGP.

[0048] ELISA method was used to confirm the recognition epitope of monoclonal antibody: 1 μg / ml pigment epithelium-derived factor fragment was coated with 100 μl carbonate buffer at 4°C overnight, and the monoclonal antibodies secreted by the four hybridoma cell lines were diluted in multiple ratios (800 ng / mL, 400 ng / mL, 200 ng / mL, 100 ng / mL, 50 ng / mL, 255 ng / mL, 12.5 ng / mL and 0 ng / mL) and added to the microwell reaction plate, and then goat anti-mouse IgG-HRP (1:20000) was added. The results are shown in Table 1.

[0049] Table 1 Analysis of antigen epitopes recognized by monoclonal antibodies

[0050]

[0051]

[0052] As shown in Table 1, the results determined that the monoclonal antibodies secreted by hybridoma cell lines JD01#1-E2 and JD01#1-E6 could recognize SEQ ID NO:1, and the monoclonal antibodies secreted by hybridoma cell lines JD01#3-B5 and JD01#3-B11 specifically recognized SEQ ID NO:2.

[0053] Example 4: Antibody ELISA pairing verification

[0054] 1) Monoclonal antibody pairing verification: According to the results of monoclonal antibody recognition of antigen epitopes, the monoclonal antibodies secreted by hybridoma cell lines JD01#1-E2 and hybridoma cell lines JD01#1-E6 both react with the pigment epithelium-derived factor fragment SEQ ID NO:1, but not with the pigment epithelium-derived factor fragment SEQ ID NO:2; while the monoclonal antibodies secreted by hybridoma cell lines JD01#3-B5 and hybridoma cell lines JD01#3-B11 are just the opposite, reacting with the pigment epithelium-derived factor fragment SEQ ID NO:2 but not with the pigment epithelium-derived factor fragment SEQ ID NO:1. It can be considered to pair the monoclonal antibodies secreted by hybridoma cell lines JD01#1-E2 and JD01#1-E6 with the monoclonal antibodies secreted by hybridoma cell lines JD01#3-B5 and JD01#3-B11, respectively.

[0055] 2) ELISA pairing verification: 2 μg / ml of the four monoclonal antibodies were coated on microtiter plates respectively, 25 μl of different concentrations (0-2000 ng / mL) of pigment epithelium-derived factor calibrator were added, and 100 μl of 200 ng / ml of HRP-labeled antibody was added (JD01#1-E2 and JD01#1-E6 coated plates were added with JD01#3-B5 and JD01#3-B11 horseradish peroxidase-labeled antibodies, and JD01#3-B5 and JD01#3-B11 coated plates were added with JD01#1-E2 and JD01#1-E6 horseradish peroxidase-labeled antibodies), incubated at 37°C for 60 min, washed, added 200 μl of substrate, reacted at room temperature in the dark for 15 min, added the stop solution and measured the absorbance with an enzyme reader.

[0056] The results are shown in Table 2. When JD01#1-E6 is used as the coating antibody (JD01#3-B5 or JD01#3-B11 is used as the detection antibody), and when JD01#3-B5 is used as the coating antibody (JD01#1-E2 or JD01#1-E2 is used as the detection antibody), a higher OD value can be obtained. And this group of data shows that when JD01#3-B5 is used as the coating antibody, although there is a higher signal-to-noise ratio, when the concentration of the calibrator is greater than 500ng / mL, different concentrations of hooking effect appear; when JD01#1-E6 is used as the coating antibody, although the signal-to-noise ratio is relatively low, when the calibrator reaches 2000ng / mL, no obvious hooking appears. According to relevant public information, the concentration of pigment epithelium-derived factor in human blood samples is greater than 10μg / mL, so when the linearity after antibody pairing is in the lower part of the entire linear range, in order to achieve accurate detection of blood samples, it is necessary to require the samples to be diluted more times. Therefore, it is more appropriate to use JD01#1-E6 as the coating antibody for the development of the assay reagent than JD01#3-B5; at the same time, when JD01#1-E6 is used as the coating antibody and JD01#3-B11 is used as the detection antibody, the concentration progression is better than JD01#3-B5. In summary, when JD01#1-E6 is used as the coating antibody and JD01#3-B11 is used as the detection antibody, the effect is the best.

[0057] Table 2 Monoclonal antibody pairing results

[0058]

[0059]

[0060] Example 5: Antibody homogeneous reaction system pairing verification and cross-reaction investigation

[0061] Chemiluminescence pairing and cross-reaction verification: Monoclonal antibodies secreted by hybridoma cell lines JD01#1-E6 and JD01#3-B11 were labeled with acridan derivatives (Acridan) and horseradish peroxidase (HRP), respectively.

[0062] Interference sample preparation: Prepare three different interference samples: vascular endothelial growth factor (VEGF) 50 μg / mL, vascular endothelial growth factor receptor protein 1 (VEGF R1) 50 μg / mL, and vascular endothelial growth factor receptor protein 2 (VEGF R2) 50 μg / mL. When detecting interference samples, dilute them 20 times with sample diluent.

[0063] Take 5 μL of different concentrations of pigment epithelium-derived factor calibrators and cross-reactive substances, 60 μL of 3 μg / mL Acridan-labeled antibody, and 60 μL of 250 ng / mL HRP-labeled antibody, incubate at 37°C for 20 min, add luminescent substrate containing hydrogen peroxide, and simultaneously detect the peak area (AUC) of the luminescent signal within 1-3 seconds.

[0064] Table 3 Homogeneous reaction system pairing verification and cross-reaction investigation

[0065]

[0066] The test results are shown in Table 3. The pairing reactivity of Acridan-labeled JD01#1-E6 antibody and HRP-labeled JD01#3-B11 antibody is good, and the cross-reaction rate with VEGF, VEGF R1 and VEGF R2 is low, which will not affect the test results.

[0067] Example 6, Pigment epithelium-derived factor detection kit

[0068] The present embodiment provides a pigment epithelium-derived factor detection kit, which comprises two reagent components, namely, Acridan-labeled monoclonal antibody secreted by hybridoma cell line JD01#1-E6 (Acridan-labeled antibody), HRP-labeled monoclonal antibody secreted by hybridoma cell line JD01#3-B11 (HRP-labeled antibody), sample diluent (composed of phosphate buffer and bovine serum albumin, commercially available), calibrator is liquid, concentration is 0-2000ng / mL, Acridan-labeled antibody, pigment epithelium-derived factor to be tested and HRP-labeled antibody form double antibody sandwich immune complex, after adding substrate, HRP in immune complex catalyzes peroxide in substrate to produce hydroxyl radical, free radical is transferred to Acirdan on sandwich complex, Acridan generates light signal after addition reaction and oxidation reaction, and light signal is positively correlated with concentration of the test object. The detection kit can be used in products for detecting changes in content of pigment epithelium-derived factor or diagnosing diseases characterized by pigment epithelium-derived factor.

[0069] Example 7: Application of Pigment Epithelium-Derived Factor Detection Kit in Diseases

[0070] Diabetic patients and non-diabetic patients (healthy people) were selected from the hospital, 20 cases each. The detection kit in Example 6 was used for detection, and the detection results are shown in Table 4 (unit: μg / mL).

[0071] Table 4 Comparison of the results of pigment epithelium-derived factor detection in the two groups of samples

[0072]

[0073]

[0074] The results of the two groups were analyzed for significance, as shown in Table 4. The results showed that the p values ​​were all less than 0.001, which showed significant differences. Therefore, the content of blood pigment epithelium-derived factor in diabetic patients was significantly different from that in healthy patients. Therefore, the detection of pigment epithelium-derived factor has a certain correlation with diabetes and diabetic metabolic-related diseases.

[0075] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application rather than to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, ordinary technicians in this field should understand that the technical solution of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present application.

Claims

1. A hybridoma cell line, characterized in that The hybridoma cell line includes a hybridoma cell line JD01#1-E6 with a deposit number of GDMCC No: 64528 or a hybridoma cell line JD01#3-B11 with a deposit number of GDMCC No: 64529; The hybridoma cell line JD01#1-E6 and hybridoma cell line JD01#3-B11 were both deposited in the Guangdong Provincial Microbiological Culture Collection Center on April 22, 2024, and the storage address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. A monoclonal antibody, characterized in that: The monoclonal antibody is produced by the hybridoma cell line according to claim 1, and the monoclonal antibody specifically recognizes pigment epithelium-derived factor.

3. The monoclonal antibody according to claim 2, characterized in that The amino acid sequence of the pigment epithelium-derived factor is shown in SEQ ID NO: 1 or SEQ ID NO:

2.

4. The monoclonal antibody according to claim 2, characterized in that The monoclonal antibody produced by the hybridoma cell line JD01#1-E6 recognizes the pigment epithelium-derived factor with an amino acid sequence such as SEQ ID NO:1; the monoclonal antibody produced by the hybridoma cell line JD01#3-B11 recognizes the pigment epithelium-derived factor with an amino acid sequence such as SEQ ID NO:

2.

5. Use of the hybridoma cell line according to claim 1 or the monoclonal antibody according to any one of claims 2 to 4 in detecting pigment epithelium-derived factor.

6. Use of the monoclonal antibody according to any one of claims 2 to 4 in the preparation of an in vitro immunoassay reagent or kit for pigment epithelium-derived factor.

7. An immunoassay reagent, characterized in that: The immunodetection reagent comprises the monoclonal antibody according to any one of claims 2 to 4.

8. A pigment epithelium-derived factor detection kit, characterized in that: The pigment epithelium-derived factor detection kit comprises the monoclonal antibody according to any one of claims 2 to 4.

9. The pigment epithelium-derived factor detection kit according to claim 8, characterized in that: The pigment epithelium-derived factor detection kit also includes a standard substance, a detection antibody and a chemiluminescent substrate.

10. Use of the hybridoma cell line according to claim 1 or the monoclonal antibody according to any one of claims 2 to 4 in the preparation of a product for detecting changes in the content of pigment epithelium-derived factor or diagnosing a disease characterized by pigment epithelium-derived factor.

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