阅读说明：本技术 一种抗fgf19抗体及其应用 (anti-FGF 19 antibody and application thereof ) 是由 肖彦羚 陈才伟 张亚飞 戴平 姜威 于 2021-03-22 设计创作，主要内容包括：本发明提供了一种抗FGF19抗体及其应用,所述抗FGF19抗体由杂交瘤细胞系产生,所述杂交瘤细胞系命名为鼠抗人FGF19单克隆抗体杂交瘤细胞系MJ19-7,保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC),保藏编号为CGMCC No.21492,保藏日期为2021年1月20日。本发明的抗FGF19单克隆抗体MJ19-7与人源FGF19蛋白具有特异性高亲和力,构建的FGF19检测试剂盒灵敏度高、特异性好,应用前景广阔。(The invention provides an anti-FGF 19 antibody and application thereof, wherein the anti-FGF 19 antibody is generated by a hybridoma cell line, the hybridoma cell line is named as a mouse anti-human FGF19 monoclonal antibody hybridoma cell line MJ19-7 and is preserved in the China general microbiological culture Collection center (CGMCC), the preservation number is CGMCC No.21492, and the preservation date is 2021 month and 20 days in 2021 year. The anti-FGF 19 monoclonal antibody MJ19-7 has high specificity affinity with human FGF19 protein, and the constructed FGF19 detection kit has high sensitivity, good specificity and wide application prospect.)

1. The anti-FGF 19 antibody is produced by a hybridoma cell line, wherein the hybridoma cell line is named as a mouse anti-human FGF19 monoclonal antibody hybridoma cell line MJ19-7 and is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No.21492 and the preservation date of 2021 year, 1 month and 20 days.

2. The anti-FGF 19 antibody according to claim 1, characterized in that the antigen of the anti-FGF 19 antibody is a human FGF19 protein;

the human FGF19 protein comprises an amino acid sequence shown in SEQ ID NO. 1.

3. The anti-FGF 19 antibody according to claim 1 or 2, characterized in that the anti-FGF 19 antibody is a heavy chain IgG1, a light chain kappa-type murine monoclonal antibody;

the anti-FGF 19 antibody is modified with a conjugate;

the conjugate comprises any one of horseradish peroxidase, alkaline phosphatase, biotin or a fluorescent group.

4. A method of producing the anti-FGF 19 antibody of any one of claims 1-3, comprising the steps of:

(1) taking spleen cells of mice immunized by the human FGF19 protein, and fusing the spleen cells with myeloma cells to prepare hybridoma cells;

(2) mice were immunized with the prepared hybridoma cells, and anti-FGF 19 antibodies were obtained from the mice.

5. An FGF19 immunohistochemical detection kit, wherein the FGF19 immunohistochemical detection kit comprises the anti-FGF 19 antibody of any one of claims 1-3;

the FGF19 immunohistochemical detection kit further comprises any one or combination of at least two of immunohistochemical antigen repair buffer solution, confining liquid, enzyme-labeled secondary antibody, color developing agent or hematoxylin complex staining solution.

6. An FGF19 immunoblot detection kit, characterized in that the FGF19 immunoblot detection kit comprises the anti-FGF 19 antibody of any one of claims 1-3;

the FGF19 immunoblotting detection kit further comprises any one or combination of at least two of sodium dodecyl sulfate-polyacrylamide gel, nitrocellulose membrane, confining liquid or enzyme-labeled secondary antibody.

7. An FGF19 ELISA kit, comprising the ELISA plate coated with the anti-FGF 19 antibody of any one of claims 1-3;

the enzyme-linked immunosorbent assay kit also comprises any one or the combination of at least two of confining liquid, enzyme-labeled secondary antibody, chromogenic solution or stop solution.

8. An FGF19 immunofluorescence assay kit, wherein the FGF19 immunofluorescence assay kit comprises the anti-FGF 19 antibody of any one of claims 1-3;

the anti-FGF 19 antibody is modified with a fluorophore, and the fluorophore is any one of FITC, Cy3 or Cy 5.

9. A FGF19 flow cytometry detection kit, wherein the FGF19 flow cytometry detection kit comprises the anti-FGF 19 antibody of any one of claims 1-3.

10. Use of the anti-FGF 19 antibody according to any one of claims 1 to 3, the FGF19 immunohistochemical detection kit according to claim 5, the FGF19 immunoblot detection kit according to claim 6, the FGF19 enzyme-linked immunosorbent assay kit according to claim 7, the FGF19 immunofluorescence assay kit according to claim 8, or the FGF19 flow cytometry assay kit according to claim 9 for the preparation of a product for detecting expression of FGF19 protein.

Technical Field

The invention belongs to the technical field of immunochemistry, and relates to an anti-FGF 19 antibody and application thereof.

Background

The human Fibroblast Growth Factor (FGF) signaling pathway plays an important role in a variety of cellular processes, such as cell proliferation, differentiation, and angiogenesis. Human fibroblast growth factor 19 (FGF 19) is a member of the FGF protein family, has a gene at 11q13.1, a molecular weight of about 21 kDa, and is a secreted protein. FGF19 is expressed in fetal brain, cartilage, retina, adult gall bladder, and tumor cells.

FGFR4 was shown to be the only specific receptor for FGF 19. The combination of FGF19 and FGFR4 can inhibit apoptosis and NF-kappa beta signals and up-regulate related genes of cell proliferation, thereby promoting the proliferation of tumor cells. The amplification and overexpression of FGF19 are closely related to the occurrence and development of malignant tumors, such as hepatocellular carcinoma, pancreatic cancer, bladder cancer, colon cancer, breast cancer, prostate cancer, cervical cancer, myeloma and other tumor tissues, and the expression of FGF19 is obviously higher than that of normal tissues. Further studies have shown that FGF19 in human hepatocellular carcinoma cells causes hepatocyte proliferation and hepatocellular carcinoma formation by activating FGFR4 on the hepatocyte surface. FGF19 is therefore a potential therapeutic target for hepatocellular carcinoma (HCC).

The BLU-554 is a novel targeting drug in clinical trial stage, and the action target is FGFR 4. Phase I experimental data for advanced hepatocellular carcinoma with aberrant FGF19-FGFR4 signaling pathways showed a total remission rate of 17% for BLU-554 in 66 FGF19 immunohistochemically positive (TC ≧ 1%, IHC +) patients and 0% for 32 FGF19 immunohistochemically negative (TC <1%, IHC-) patients, indicating that the expression level of FGF19 may direct the use of FGFR4 inhibitors. Therefore, the preparation of an anti-FGF 19 antibody with high specificity and sensitivity is necessary for clinical diagnosis.

At present, the number of antibodies capable of specifically and highly sensitively detecting FGF19 on the market is very small, and especially, an anti-FGF 19 monoclonal antibody capable of being applied to Immunohistochemistry (IHC) is available. Through searching and screening of commercial reagents, among monoclonal antibodies against human FGF19 on the market, antibodies with better specificity and sensitivity include clone D1N3R (cat No. 83348) of CST, clone H-12 (cat No. sc-390621) of Santa Cruz and clone #032 (cat No. 12226-R032) of Chinesia.

Therefore, the preparation of the anti-FGF 19 monoclonal antibody with strong specificity, high sensitivity and wide application range has important practical significance and application value.

Disclosure of Invention

Aiming at the defects and practical requirements of the prior art, the invention provides an anti-FGF 19 antibody and application thereof, wherein the anti-FGF 19 antibody has high specificity and affinity to FGF19, and has important application prospects in preparation of an FGF19 detection kit.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the invention provides an anti-FGF 19 antibody, wherein the anti-FGF 19 antibody is generated by a hybridoma cell line, the hybridoma cell line is named as a mouse anti-human FGF19 monoclonal antibody hybridoma cell line MJ19-7, which is preserved in China general microbiological culture Collection center (CGMCC), the address is No. 3 of Xilu No. 1 of Beijing Kogyang district, the postal code is 100101, the preservation number is CGMCC number 21492, and the preservation date is 2021 year, 1 month and 20 days.

In the invention, the humanized FGF19 recombinant protein shown in SEQ ID NO. 1 is used as immunogen to immunize a mouse, spleen cells of the immunized mouse are taken to be fused with SP2/0 cells to form hybridoma cells, and the hybridoma cell line clone MJ19-7 with the strongest sensitivity and specificity is screened out to be used for preparing the anti-FGF 19 antibody.

Preferably, the antigen of the anti-FGF 19 antibody is a human FGF19 protein.

Preferably, the human FGF19 protein comprises the amino acid sequence shown in SEQ ID NO. 1.

Preferably, the anti-FGF 19 antibody is a heavy chain IgG1, light chain kappa-type murine monoclonal antibody.

Preferably, when the anti-FGF 19 antibody is applied to the construction of a FGF19 detection kit, the anti-FGF 19 antibody may be modified with a conjugate, wherein the conjugate includes horseradish peroxidase (HRP), Alkaline Phosphatase (AP), Biotin (Biotin), Fluorescein Isothiocyanate (FITC), Cy3, Cy5, and the like, and the coupling mode includes chemical bond coupling, electrostatic adsorption, or hydrophilic-hydrophobic adsorption.

In a second aspect, the present invention provides a method for preparing the anti-FGF 19 antibody of the first aspect, the method comprising the steps of:

(1) taking spleen cells of mice immunized by the human FGF19 protein, and fusing the spleen cells with myeloma cells to prepare hybridoma cells;

(2) mice were immunized with the prepared hybridoma cells, and anti-FGF 19 antibodies were obtained from the mice.

In a third aspect, the present invention provides an FGF19 immunohistochemical detection kit, wherein the FGF19 immunohistochemical detection kit comprises the anti-FGF 19 antibody of the first aspect.

Preferably, the FGF19 immunohistochemical detection kit further comprises any one or a combination of at least two of immunohistochemical antigen retrieval buffer, blocking solution, enzyme-labeled secondary antibody, color developing agent or hematoxylin complex staining solution.

In a fourth aspect, the present invention provides an FGF19 immunoblotting detection kit, wherein the FGF19 immunoblotting detection kit comprises the anti-FGF 19 antibody of the first aspect.

Preferably, the FGF19 immunoblotting detection kit further comprises any one of or a combination of at least two of sodium dodecyl sulfate-polyacrylamide gel, nitrocellulose membrane, a blocking solution or an enzyme-labeled secondary antibody.

In a fifth aspect, the invention provides an enzyme-linked immunosorbent assay kit for FGF19, which comprises the enzyme-linked immunosorbent assay plate coated with the anti-FGF 19 antibody of the first aspect.

Preferably, the enzyme-linked immunosorbent assay kit further comprises any one or a combination of at least two of a blocking solution, an enzyme-labeled secondary antibody, a developing solution or a stop solution.

In a sixth aspect, the present invention provides an FGF19 immunofluorescence assay kit, wherein the FGF19 immunofluorescence assay kit comprises the anti-FGF 19 antibody of the first aspect.

Preferably, the anti-FGF 19 antibody is modified with a fluorophore, preferably any one of FITC, Cy3, and Cy 5.

In a seventh aspect, the present invention provides a FGF19 flow cytometry detection kit, wherein the FGF19 flow cytometry detection kit comprises the anti-FGF 19 antibody of the first aspect.

In an eighth aspect, the invention provides an anti-FGF 19 antibody of the first aspect, an FGF19 immunohistochemical detection kit of the third aspect, an FGF19 immunoblot detection kit of the fourth aspect, an FGF19 enzyme-linked immunosorbent assay kit of the fifth aspect, an FGF19 immunofluorescence detection kit of the sixth aspect, or an FGF19 flow cytometry detection kit of the seventh aspect, for use in preparing an FGF19 protein expression detection product.

Compared with the prior art, the invention has the following beneficial effects:

(1) according to the invention, a mouse spleen cell immunized by human FGF19 recombinant protein is fused with SP2/0 cell to form a hybridoma cell, and the prepared anti-FGF 19 monoclonal antibody has good specificity and strong affinity with human FGF 19;

(2) the human FGF19 detection kit based on the anti-FGF 19 monoclonal antibody is high in sensitivity, can accurately detect the expression levels of FGF19 proteins in different clinical samples compared with commercially available products, is superior to commercially available products in specificity and sensitivity, and has important application prospects.

Drawings

FIG. 1 is a Western Blot analysis of recombinant human FGF19 protein, wherein lane 1 is a positive control, lanes 2 and 3 are negative controls, lane 4 is recombinant human FGF19 protein, and M is a protein marker;

FIG. 2 is a SDS-PAGE electrophoresis of purified human FGF19 recombinant protein, wherein lane 1 is BSA, lane 2 is purified human FGF19 recombinant protein, and lane M is protein marker;

FIG. 3 is a diagram showing the results of indirect ELISA assay of hybridoma cell line clones MJ19-1 to MJ 19-32;

FIG. 4 shows SDS-PAGE of purified monoclonal antibody MJ19-7, wherein lane 1 is BSA, lane 2 is purified monoclonal antibody, and lane M is protein marker;

FIG. 5 is a diagram showing the results of ELISA for specific verification of the purified clone MJ19-7, wherein the commercial monoclonal antibodies are clone D1N3R (cat 83348) of CST, clone H-12 (cat sc-390621) of Santa Cruz, and clone #032 (cat 12226-R032) of Chinesia;

FIG. 6 is a graph showing the results of immunohistochemical detection of human gallbladder tissue, normal liver tissue and hepatocellular carcinoma tissue at different FGF19 expression levels using purified clone MJ19-7 monoclonal antibody, wherein the commercial monoclonal antibodies are clone D1N3R of CST (cat No. 83348), clone H-12 of Santa Cruz (cat No. sc-390621), and clone #032 of Chinesota (cat No. 12226-R032);

FIG. 7 is a diagram showing the results of immunoblotting experiments performed on purified clone MJ19-7 monoclonal antibody to detect FGF19 proteins from different expression sources, wherein lane 1 is prokaryotic FGF19 protein, lane 2 is eukaryotic FGF19 protein, lane 3 is eukaryotic expression positive control, and lane M is protein marker.

Detailed Description

To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.

Example 1 preparation of human FGF19 antigen

The amino acid sequence of the human FGF19 protein (O95750) is shown in SEQ ID NO. 1, in the embodiment, the FGF19 gene is connected with pcDNA3.4 by a homologous recombination method to construct a recombinant vector pcDNA3.4-FGF 19;

SEQ ID NO: 1：

LAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK；

pcDNA3.4-FGF19 was transiently transfected into 293F cells, cultured for 120 h, and cell cultures harvested for Western Blot validation.

As can be seen from FIG. 1, the human FGF19 protein was successfully expressed, and the recombinant protein had a His tag at the C-terminus and was about 22.3 kD in size. And (3) adopting an AKTA instrument to carry out affinity purification on the recombinant protein, carrying out SDS-PAGE gel electrophoresis, and checking the size and purity of the protein. The results are shown in FIG. 2, the FGF19 protein band after purification is correctly positioned and is >95% pure.

EXAMPLE 2 preparation of FGF19 resistant hybridomas

1) Immunization

Carrying out abdominal subcutaneous immunization on a 4-6-week-old female Balb/c mouse by using the purified human FGF19 recombinant protein (20 muL serum is taken from an orbit before immunization as a negative control); diluting FGF19 recombinant protein with the primary immunization dose of 50 mug to 200 mug by using physiological saline for each mouse, mixing the diluted protein with an equal volume of Freund's complete adjuvant, and injecting the mixture after full emulsification; subcutaneous boosting immunization is carried out once every 14 days, FGF19 recombinant protein with the dose of 25 mug is diluted to 200 mug L by normal saline, and the diluted FGF19 recombinant protein is mixed with equal volume of Freund incomplete adjuvant, and is injected after full emulsification; and (3) taking blood from the orbit 7 days after the 3 rd boosting immunization, detecting the serum antibody titer by ELISA, carrying out impact immunization on the mouse meeting the requirement, diluting the FGF19 recombinant protein with the immunization dose of 50 mug to 100 mug by using normal saline, carrying out intraperitoneal injection, and fusing after 3 days.

2) Fusion

Placing the freshly picked mouse spleen on a cell sieve, grinding, filtering, mixing with SP2/0 cells according to the ratio of 3:1, centrifuging at 1000 rpm at room temperature for 5 min, and removing the culture medium; the centrifuge tube containing the cell pellet was placed in a 37 ℃ water bath and 1 mL of polyethylene glycol solution (Sigma, P7181-5X5 ML) was added slowly with stirring; standing in water bath for 1 min, slowly adding 10 mL serum-free DMEM medium (Hyclone, SH30243.01B), centrifuging at 1000 rpm for 5 min, and removing supernatant; adding a certain amount of serum-containing DMEM medium according to the total number of the cells,and the cells were seeded into 96-well cell culture plates to a cell density of 1X 10⁵Each well is 150 muL, and the well is placed at 37 ℃ and 5% CO₂Culturing in an incubator; after 24 hours, 50 mu L of a culture medium containing 4 XHAT is added, and then liquid change is carried out every 3-5 days.

3) Screening

When the cell amount is about 1/2 of the bottom area of the culture dish, taking the cell culture supernatant to carry out indirect ELISA detection, screening hybridoma cell strains secreting anti-FGF 19 antibodies, and carrying out subcloning on positive clone strains by adopting a limiting dilution method.

The indirect ELISA method was performed as follows: coating an enzyme label plate with 100 mu L of antigen containing 0.5 mu g/mL human FGF19 recombinant protein, taking serum of a 1:5000 immune mouse as a positive control, taking supernatant of a culture medium without clone growth as a negative control, adding 100 mu L of 1:5000 HRP-goat anti-mouse IgG into each well, measuring an OD value of 450 nm, judging that the OD450 value is greater than 0.5, and judging that the positive clone can be subcloned.

4) Establishment of hybridoma cell lines

32 hybridoma cell lines which aim at FGF19 and stably secrete anti-FGF 19 monoclonal antibodies are obtained by co-screening through 3 times of subcloning and indirect ELISA screening, the clone numbers are MJ19-1 to MJ19-32, and the indirect ELISA result is shown in figure 3. Combining the IHC detection result, selecting a hybridoma cell line clone with the strongest sensitivity and specificity, namely a mouse anti-human FGF19 monoclonal antibody hybridoma cell line MJ19-7, which is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and has the address of No. 3 of Xilu No. 1 of Beijing Kogyo-sunward-Yang district, the postal code 100101, the preservation number CGMCC number 21492 and the preservation date of 2021 year, 1 month and 20 days.

Example 3 preparation of anti-FGF 19 monoclonal antibody MJ19-7 based on hybridoma cell line MJ19-7

1) Preparation of ascites

MJ19-7 hybridoma cells in logarithmic growth phase were washed with serum-free medium and resuspended at a cell density of 5X 10⁵Per mL; intraperitoneal injecting the suspended cells into mice sensitized by paraffin oil in advance, and collecting ascites after 7 days; the ascites fluid taken out is centrifuged at 4000 rpm for 10 min at 4 ℃,the ascites fluid in the middle was carefully aspirated and collected in a centrifuge tube and stored at 4 ℃.

2) Monoclonal antibody subtype identification

Diluting goat anti-mouse IgG (SL 200101, Solai Bo Ou Biotechnology Co., Ltd. in Hangzhou) with 100 mM PBS (pH7.4) to 1 μ g/mL, adding 100 μ L per well into an enzyme label plate, and coating overnight at 4 ℃; emptying the liquid, washing the liquid for 5 times by using PBST containing 0.05% Tween-20, adding 300 mu L of sealing liquid into each hole, and sealing the hole for 1 h at 37 ℃; emptying the liquid, washing with PBST for 5 times, adding 100 mu L MJ19-7 hybridoma cell supernatant into each well, and incubating for 1 h at 37 ℃; emptying the liquid, washing with PBST for 5 times, adding 100 mu L of HRP-labeled goat anti-mouse (kappa, lambda) antibody diluted with blocking solution 1:5000 or HRP-labeled goat anti-mouse (IgM, IgA, IgG1, IgG2a, IgG2b, IgG2c, IgG 3) antibody diluted with blocking solution 1:5000 to each well, and incubating for 1 h at 37 ℃; the liquid was emptied, washed 5 times with PBST, 100 μ L of substrate solution was added to each well, developed for 5 min at room temperature, and the OD value was measured at a wavelength of 450 nm. The results show that the clone MJ19-7 monoclonal antibody is a heavy chain IgG1, light chain kappa type murine monoclonal antibody.

3) Purification of monoclonal antibodies

Monoclonal antibodies were purified from ascites fluid using HiTrap protein G FF (GE Healthcare, 17061802) affinity chromatography packing and AKTA system, and purity and Nanodrop concentration determinations were performed using SDS-PAGE gels. The SDS-PAGE results are shown in FIG. 4, and the purified anti-FGF 19 monoclonal antibody MJ19-7 band is correctly positioned and is >95% pure.

Example 4 specific detection of anti-FGF 19 monoclonal antibody MJ19-7

The human FGF19 has higher homology with murine FGF15 (mFGF 15), human FGF21 and human FGF23, and the specificity of the clone MJ19-7 monoclonal antibody is detected by an indirect ELISA method in the embodiment, which comprises the following steps:

respectively coating an enzyme label plate with 100 mu L of antigen containing 0.5 mu g/mL eukaryotic expression human FGF19 recombinant protein, prokaryotic expression human FGF19 recombinant protein, mouse FGF15 (mFGF 15), human FGF21 and human FGF 23; adding 100 muL of purified clone MJ19-7 antibody with the concentration of 1 mug/mL, taking an antibody diluent as a negative control, taking clone D1N3R (good 83348) of CST, clone H-12 (good sc-390621) of Santa Cruz and clone #032 (good 12226-R032) of Hovenia as a positive control, and adding the amount of the antibody diluent to the clone MJ 19-7; adding 100 mu L1: 5000 HRP-goat anti-mouse IgG into each hole, and finally measuring the OD value of 450 nm.

As a result, as shown in fig. 5, the clone MJ19-7 monoclonal antibody was able to specifically recognize the eukaryotic FGF19 protein and the prokaryotic FGF19 protein, but did not recognize the mouse FGF15 (mFGF 15), the human FGF21, and the human FGF 23; clone #032 monoclonal antibody from Chinesia angustifolia has cross recognition on human FGF23 and poor specificity. Under the same antibody dosage, clone MJ19-7 recognized the FGF19 protein with the highest OD compared to the commercial 3 antibodies, indicating that the sensitivity is higher than that of the commercial 3 antibodies.

Example 5 immunohistochemical detection based on anti-FGF 19 monoclonal antibody MJ19-7

The clinical samples adopted in this example, human gallbladder tissue, normal liver tissue and hepatocellular carcinoma tissue, were from the tissue sample bank of meijie transformed medical research (suzhou) ltd, and the samples were formalin-fixed and paraffin-embedded, and were pathologically confirmed to have informed consent of the patients; wherein, human gallbladder tissue is FGF19 expression positive tissue as positive control, normal liver tissue is FGF19 expression negative tissue as negative control, hepatocellular carcinoma samples 1, 2 and 3 are FGF19 expression strong positive, medium positive and weak positive tissues respectively as negative control;

after the sample was sliced, Immunohistochemical (IHC) detection was performed on a Leica BOND-MAX (national mechanical appliance 20140277) full-automatic histochemical system, and the specific procedures are shown in Table 1, wherein the primary antibody is the optimal using concentration of the antibody, and the commercial monoclonal antibodies are clone D1N3R (cat No. 83348) of CST, clone H-12 (cat No. sc-390621) of Santa Cruz, and clone #032 (cat No. 12226-R032) of Yi Qiao Shen.

TABLE 1

The immunohistochemical result is shown in figure 6, the expression of human normal gallbladder tissue FGF19 is detected to be strong positive by adopting a clone MJ19-7 monoclonal antibody, the expression of normal liver tissue FGF19 is negative, the expression of hepatocellular carcinoma tissue FGF19 from three different sources is respectively strong positive, medium positive and weak positive, and the result accords with the early verification result; among the 3 commercial antibodies, H-12 has obvious staining on normal liver tissues, which indicates that the specificity is poor; D1N3R failed to detect FGF19 expression on medium-positive and weak-positive hepatocellular carcinoma tissues, indicating that the sensitivity is poor; #032 had a clear staining on normal liver tissue and a darker background, indicating poor specificity. Therefore, the clone MJ19-7 monoclonal antibody can specifically detect the expression of FGF19 in hepatocellular carcinoma tissues, has high sensitivity and strong specificity, and can effectively detect the expression conditions of FGF19 in different tissue samples.

Example 6 immunoblot assay based on anti-FGF 19 monoclonal antibody MJ19-7

Respectively loading 0.5 mu g of eukaryotic expression FGF19 protein and prokaryotic expression FGF19 protein on 15% SDS-PAGE gel, and taking eukaryotic commercial proteins with the same loading amount as positive controls; after electrophoresis is finished, soaking SDS-PAGE gel in 1 × transfer solution for 10 min, and treating a PVDF membrane with methanol and then transferring the PVDF membrane at 100V for 60 min; sealing the membrane in PBST sealing solution containing 5% skimmed milk powder at 37 deg.C for 1 h; adding 2 mug/mL of purified anti-FGF 19 monoclonal antibody MJ19-7, and incubating for 60 min at room temperature; adding HRP-goat anti-mouse IgG 1:5000 diluent, incubating at room temperature for 60 min, and exposing with ChemiDoc ™ MP imaging system.

As shown in FIG. 7, the clone MJ19-7 monoclonal antibody can be used in immunoblot experiments to detect FGF19 proteins from different expression sources.

In conclusion, the hybridoma cell line constructed by the invention is used for preparing the anti-FGF 19 monoclonal antibody, and the obtained anti-FGF 19 monoclonal antibody has good specificity, high sensitivity and strong affinity with human FGF19, and has potential application value in the field of FGF19 protein detection.

The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

Sequence listing

<110> Mejie transformation medical research (Suzhou) Co., Ltd

<120> anti-FGF 19 antibody and application thereof

<130> 20210209

<160> 1

<170> PatentIn version 3.3

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<211> 192

<212> PRT

<213> Artificial sequence

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Leu Ala Phe Ser Asp Ala Gly Pro His Val His Tyr Gly Trp Gly Asp

1 5 10 15

Pro Ile Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His Gly Leu Ser

20 25 30

Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp Cys Ala Arg

35 40 45

Gly Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala Leu Arg

50 55 60

Thr Val Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys Met Gly

65 70 75 80

Ala Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys

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Ala Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg Ser

100 105 110

Glu Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln Arg Gln

115 120 125

Leu Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe Leu Pro Met

130 135 140

Leu Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly His Leu Glu

145 150 155 160

Ser Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met Asp Pro Phe

165 170 175

Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser Phe Glu Lys

180 185 190