Anti-gastrin antibody as well as hybridoma cell strain and application thereof

Abstract

The invention relates to the technical field of immune globulin, in particular to an anti-gastrin antibody and a hybridoma cell strain and application thereof. The invention provides an anti-gastrin antibody, and also provides a hybridoma cell strain OTI3D11, and the preservation number of the hybridoma cell strain is CGMCC (China General Microbiological Culture Collection Center) No.46359. The invention also provides an anti-gastrin antibody and a hybridoma cell strain OTI3D11. According to the present invention, the hybridoma cell strain OTI3D11 can stably secrete the anti-gastrin monoclonal antibody, and can specifically bind to the Gastrin protein (especially G-17), such that the immunodetection of the hybridoma cell strain OTI3D11 has specificity and sensitivity, and the hybridoma cell strain OTI3D11 can be suitable for the labeling of the Gastrin protein in tissue cells.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of immunoglobulin, in particular to anti-gastrin antibody and its hybridoma cell strain and application. BACKGROUND

[0002] In 1905, a British physiologist Edlkine discovered a kind of gastrointestinal hormone with stimulating gastric acid secretion, and named it gastrin. According to the relative molecular weight, it is divided into different fragments, mainly containing two isomers G-34 (big gastrin) and G-17 (small gastrin), G-34 is mainly secreted by G cells in the duodenum, which is composed of 34 amino acids. 80%~90% of gastrin is G-17, which is mainly secreted by G cells in the antrum, containing 17 amino acids, which is synthesized through four processes of gastrin precursor-glycine type gastrin-amidated gastrin-secretion type, and is one of the sensitive markers that can reflect the function of antral endocrine, which plays a role in promoting gastric acid secretion, stimulating mucosal growth, promoting cell proliferation and apoptosis, stimulating pepsinogen secretion, etc., and can be used for monitoring antral mucosal atrophy and abnormal proliferation.

[0003] The gastrin family mainly has two receptors, one is CCK1 (CCKA) receptor, and the other is CCK2 (CCKB) receptor. The receptor is a cholecystokinin B receptor existing in the stomach, which is not a single receptor subtype. CCK-B receptors show similar affinity to gastrin and cholecystokinin. Gastrin binding to the receptor can induce intracellular calcium ion flow and activate intracellular adenylyl cyclase to increase cAMP, trigger a series of intracellular signal transduction, lead to the expression of cell differentiation early genes such as c-fos and c-jun, and the activation of mitogen-activated protein kinase (MAPK), promote DNA synthesis and cell division. Sand rat experiments have proved that long-term HP infection can cause gastritis, induce hypergastrinemia, and even induce gastric cancer, indicating that gastrin plays an important role in gastritis caused by Helicobacter pylori or cancer induction. Studies have shown that gastrin combined with receptors can inhibit gastric cancer cell apoptosis, stimulate gastric cancer cell infiltration and metastasis. The results of many studies show that the expression of G-17 in cancer tissues is significantly increased, and the expression level of G-17 in advanced gastric cancer tissues is significantly higher than that in early gastric cancer and precancerous lesions.

[0004] G-17 can be an important clue and basis for the diagnosis and pathological staging of gastric cancer. In multiple expert consensus and diagnosis and treatment consensus, PGI, PGII and G-17 detection are used as the diagnostic basis for gastric cancer, gastric mucosa atrophy and other diseases. Whether the detection of G-17 in serum or the detection of G-17 in tissue by immunohistochemical method depends on the high-specificity and high-sensitivity anti-G-17 monoclonal antibody, so developing a monoclonal antibody that can specifically recognize G-17 is crucial to improve the accuracy of diagnosis of atrophic gastritis, gastric cancer and gastrinoma and other diseases. SUMMARY

[0005] Therefore, the present application provides an anti-gastrin antibody, a hybridoma cell strain capable of secreting the anti-gastrin antibody, and applications of the anti-gastrin antibody and the hybridoma cell strain.

[0006] In one aspect, the present application provides an anti-gastrin antibody or an antigen binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising heavy chain complementarity determining regions HCDR1 to HCDR3, and the light chain variable region comprising light chain complementarity determining regions LCDR1 to LCDR3, wherein: the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence shown in SEQ ID NO: 7, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence shown in SEQ ID NO: 3.

[0007] Further, HCDR1 to HCDR3 of the heavy chain variable region and LCDR1 to LCDR3 of the light chain variable region are determined according to Kabat numbering system, AbM numbering system, IMGT numbering system, Chothia numbering system or Contact numbering system.

[0008] Further, the amino acid sequence of HCDR1 is shown in SEQ ID NO: 8, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 9, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 10, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 4, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 5, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 6.

[0009] Further, the light chain variable region comprises an amino acid sequence having 90% or more homology obtained by substitution, deletion and / or addition of one or more amino acids and / or terminal modification of any one or more of the amino acid sequences shown in SEQ ID NO: 3. Preferably, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 3.

[0010] Further, the heavy chain variable region comprises an amino acid sequence having 90% or more homology obtained by substitution, deletion and / or addition of one or more amino acids and / or terminal modification of any one or more of the amino acid sequences shown in SEQ ID NO: 7. Preferably, the VH domain comprises an amino acid sequence as shown in SEQ ID NO: 7.

[0011] Further, the antigen binding fragment is a Fab, Fab', F(ab')2, single chain antibody or disulfide stabilized variable fragment.

[0012] Further, the anti-gastrin antibody or antigen binding fragment thereof comprises a non-CDR region, and the non-CDR region is from human, mouse or rabbit; preferably, the non-CDR region comprises a framework region, a heavy chain constant region and / or a light chain constant region.

[0013] Further, the antibody or antigen binding fragment thereof is produced by the hybridoma cell strain with the accession number CGMCC No. 46359.

[0014] In another aspect, the present application also provides a hybridoma cell strain capable of secreting an anti-gastrin antibody, which is a murine anti-human gastrin monoclonal hybridoma cell strain OTI3D11 with the accession number NO. 46359, deposited in the China General Microbiological Culture Collection Center (CGMCC).

[0015] In another aspect, the present application also provides an application of the anti-gastrin antibody or antigen binding fragment thereof, which is selected from: 1) labeling of gastrin protein in tissues; 2) preparing an immunohistochemical detection kit for gastrin protein.

[0016] Further, the tissues comprise stomach, tonsil, appendix, placenta, lung, prostate, breast, pancreas or thyroid.

[0017] In another aspect, the present application also provides an immunohistochemical detection kit for gastrin protein, which comprises a primary antibody detection reagent containing the anti-gastrin antibody or antigen binding fragment thereof.

[0018] Further, the kit further comprises an antigen repair solution, an endogenous peroxidase blocking agent, a hypersensitive secondary antibody reagent, a DAB substrate buffer, a DAB concentrated color developing solution, a hematoxylin staining solution.

[0019] Compared with the prior art, the application provides a monoclonal antibody capable of specifically binding to human Gastrin protein, wherein the monoclonal antibody against human Gastrin protein is screened by a hybridoma technology. Meanwhile, the application also establishes an immunohistochemical detection method and a detection kit based on the monoclonal antibody against human Gastrin protein. The immunohistochemical detection kit of the application can be used for specifically labeling human Gastrin protein in tissues such as stomach, and can also be used for chemically detecting tumor tissues related to human Gastrin protein expression in immunized tissues. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 A result chart of IHC detection of stomach tissue by the monoclonal antibody secreted by the hybridoma cell strain OTI3D11 of the application;

[0021] Figure 2 A result chart of IHC detection of tonsil, appendix, placenta and lung tissue by the monoclonal antibody secreted by the hybridoma cell strain OTI3D11 of the application;

[0022] Figure 3 A result chart of IHC detection of prostate, breast, thyroid and pancreas tissue by the monoclonal antibody secreted by the hybridoma cell strain OTI3D11 of the application. DETAILED DESCRIPTION

[0023] In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have meanings commonly understood by those skilled in the art. And, the cell culture, molecular genetics, nucleic acid chemistry, immunological laboratory operation steps used herein are conventional steps widely used in the corresponding fields. Meanwhile, in order to better understand the present disclosure, the definitions and explanations of related terms are provided as follows.

[0024] As used herein, the term "antibody" refers to an immunoglobulin molecule that is generally composed of two pairs of polypeptide chains (each pair having one "light" (L) chain and one "heavy" (H) chain). Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define a different antibody class IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is comprised of one domain, CL. The constant regions of antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The VH and VL regions can also be subdivided into regions of hypervariability, termed complementarity determining regions, CDRs, interspersed with regions that are more conserved, termed framework regions, FRs. Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0025] The term "framework" or "FR" refers to those variable region residues that are not within a CDR. FR residues are those variable domain residues other than hypervariable region residues or CDR residues.

[0026] The terms "hypervariable region," "complementarity determining region," and "CDR" are used interchangeably. "CDR" refers to one of three hypervariable regions (H1, H2, or H3) within the non- framework region of an immunoglobulin (Ig or antibody) VH beta-sheet framework, or one of three hypervariable regions (L1, L2, or L3) within the non-framework region of an antibody VL beta-sheet framework. The location of CDRs and FRs can be determined using various definition methods well known in the art, e.g., the Kabat numbering system, the AbM numbering system, the IMGT numbering system, the Chothia numbering system, or the Contact numbering system.Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (see, e.g., Chothia, C. et al., Nature, 342, 877-883 (1989); and Al-Lazikani, B. et al., J. Mol. Biol., 273, 927-948 (1997)), Kabat based on the variability of antibody sequences (see, e.g., Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242), AbM (Martin, A.C.R. and J. Allen (2007) “Bioinformatics tools for antibody engineering,” in S. Dübel (ed.), Handbook of Therapeutic Antibodies. Weinheim: Wiley-VCH Verlag, pp. 95-118), Contact (MacCallum, R. M. et al., (1996) J. Mol. Biol. 262:732-745), IMGT (Lefranc, M.-P., 2011 (6), IMGT, the International ImMunoGeneTics Information System Cold Spring Harb Protoc.; and Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003)), and North CDR definitions based on affinity propagation clustering with a large number of crystal structures. It will be understood by those skilled in the art that, unless otherwise specified, the terms “CDR” and “complementarity determining region” of a given antibody or region thereof (e.g., variable region) are understood to encompass complementarity determining regions as defined by any of the above known schemes described by the present invention. It is noted that CDR regions can also be determined by a combination of various numbering systems, e.g., a combination of Kabat and Chothia numbering systems, a combination of Kabat and AbM numbering systems, or a combination of Kabat and IMGT numbering systems.Thus, for example, the term "CDR1 as set forth in a particular VH or VL" includes any CDR1 as defined by the exemplary CDR numbering systems described above, but is not limited thereto. Once the amino acid sequence of a variable region (e.g., VH or VL) is given, one of skill in the art will appreciate that the CDRs within that region can be defined by different numbering systems or combinations thereof. In this document, CDRs are determined according to the amino acid sequence and position in the light or heavy chain. Since the "position" of a CDR within an immunoglobulin variable region domain is conserved between species, it is easy to identify CDR and framework region amino acid residues by aligning the variable region domain amino acid sequence with the structural features divided by using the known numbering systems.

[0027] The term "antibody" is not limited by any particular method of producing the antibody. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody can be an antibody of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0028] As used herein, the terms "monoclonal" and "monoclonal antibody" refer to one antibody or one fragment of an antibody from a population of highly homogenous antibody molecules, i.e., a population of identical antibody molecules except for natural mutations that can spontaneously occur. Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies are in contrast to monoclonal antibodies and generally comprise at least 2 or more different antibodies that generally recognize different epitopes on an antigen. Monoclonal antibodies can generally be obtained using the hybridoma technology first reported by Kohler et al., but can also be obtained using recombinant DNA technology.

[0029] As used herein, the term "immunohistochemistry" or "immunohistochemical" refers to immunochemical studies performed at the tissue level. "Immunohistochemical staining" is a kind of immunostaining, which uses a modified antibody labeled with a fluorescent or colorable chemical substance to selectively recognize antigens in tissue sections or smear cells by the immunological principle of specific binding between the modified antibody and antigens in biological tissues, so as to detect the presence of target antigens in cells or tissues; this method can not only be used to detect the expression amount of antigens, but also can be used to observe the location of antigen expression, and can improve the sensitivity and specificity of pathological diagnosis. In immunohistochemical staining, the specific antibody modified by human beings is labeled with a color developing agent, such as an enzyme, a metal ion, an isotope, etc.; and the target antigen of the cell or tissue to be detected can be any substance that can be bound by the antibody, that is, an antigenic substance, including proteins, polypeptides, nucleic acids, polysaccharides, pathogens, etc. Through the antigen-antibody reaction of immunology and the color developing reaction of histochemistry, combined with the morphological analysis of tissues and cells, the localization, qualification and quantification of antigens in tissues or cells are performed.

[0030] The present application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not used to limit the scope of the present application. The experimental methods in the following examples, if not otherwise specified, are generally carried out according to the conventional conditions, the conditions described in the laboratory manual or the conditions suggested by the manufacturer.

[0031] Example 1 Design of immunogen

[0032] The Gastrin gene NM_000805 was selected from Genebank, and the antigenic epitope analysis of the amino acid sequence of Gastrin protein was carried out, mainly to evaluate the biological characteristics, antigenicity, hydrophilicity / hydrophobicity, amino acid category and distribution, homology of human sequence and immunized animals, and to compare the amino acid sequences of G-17 and G-34. The polypeptide sequence of N-terminal of G-17 was designed, and glutamine (Gln) was cyclized to pyroglutamic acid (pGlu). The G-17 polypeptide was chemically synthesized according to the sequence, and the specific sequence is shown in SEQ ID NO: 1. KLH was coupled at the C-terminal as an immunogen (G-17-KLH). The first glutamine (Gln) of G-34 was cyclized to pyroglutamic acid (pGlu), and the G-34 polypeptide was chemically synthesized according to the sequence, which was used as a cross-screening detection material, and the specific sequence is shown in SEQ ID NO: 2.

[0033] Example 2 Animal immunization and preparation of hybridoma cell lines

[0034] The BALB / c mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were immunized with the immunogen (G-17-KLH) according to the standard method. The specific method is as follows:

[0035] 1. Animal immunization: The G-17 polypeptide antigen (G-17-KLH) was emulsified with complete Freund's adjuvant, and 6-8 week old BALB / c mice were immunized by intraperitoneal injection, with a dose of 400 μg per mouse. The next round of immunization was carried out every two weeks, with a dose of 300 μg per mouse, emulsified with incomplete Freund's adjuvant. After three times of immunization, the tail blood was taken to determine the serum titer by ELISA method with gradient dilution. According to the results, it was determined whether the immune response of the immunized animal was induced, and the mouse with antibody titer meeting the requirements was selected to take its spleen for cell fusion.

[0036] 2. Cell fusion: the hybridoma technique was applied to prepare the fusion cells, and the myeloma cells were sp2 / 0 cells in the logarithmic growth phase; the spleen of a mouse with a satisfactory immune response was taken to prepare a lymphocyte single cell suspension; the mouse spleen lymphocytes and myeloma cells were mixed at a ratio of 1:7, 1 mL of 37°C preheated 50% PEG (pH 8.0) was added dropwise, and the reaction was static for 1 min, then 10 mL of incomplete medium was slowly added to terminate the reaction, the supernatant was discarded after centrifugation, and then HAT medium was added for suspension and mixing, the MC medium containing methyl cellulose was added to 50 mL, and then the medium was divided into 3.5 cm culture dishes, which were placed in a wet box and cultured in a 37°C, 5% CO2 constant temperature incubator. The growth state of the clone group was observed every day, and the clones were selected under a dissecting microscope and cultured in a 96-well plate after about 8 days.

[0037] Example 3 Screening of G-17 hybridoma cell strains with specific recognition and preparation of antibodies

[0038] The specific monoclonal cell strains recognizing G-17 were screened by comprehensively using ELISA and IHC detection. The specific hybridoma cell strains screened by ELISA detection were subjected to IHC detection of the corresponding supernatant.

[0039] 1. ELISA detection method for screening specific hybridoma cell strains

[0040] The process is as follows: (1) Detection of the original coating: according to the number of clones of the hybridoma supernatant to be detected, the synthesized G-17 and G-34 polypeptides were diluted to 4 μg / mL with coating buffer, and 100 μL / well was added to the 96-well plate, which was incubated at 4°C overnight; (2) Blocking: 5% skimmed milk powder was added at a volume of 100 μL / well, and the plate was blocked at 37°C for 1 h, then the supernatant was discarded, and the plate was washed with deionized water and dried for standby; (3) Incubation of primary antibody: the hybridoma cell culture supernatant was used as the primary antibody, 100 μL / well was added to the G-17 and G-34 polypeptide coated plates, and the plates were incubated at 37°C for 1 h, then the supernatant was discarded, and the plates were washed with deionized water for 3 times; (4) Incubation of secondary antibody: HRP-labeled goat anti-mouse IgG was added at a volume of 100 μL / well, and the plates were incubated at 37°C for 1 h, then the supernatant was discarded, and the plates were washed with deionized water for 3 times; (5) Substrate color development: TMB color developing solution was added at a volume of 100 μL / well, and the plates were incubated at 37°C for 15 min; (6) Reaction termination: 50 uL / well of 2M sulfuric acid was added to terminate the reaction; (7) Data analysis: the wavelength of 450 nm was set on the enzyme label instrument, and the data was read; (8) Data interpretation: the clone showing positive results on G-17 polypeptide coated plate and negative results on G-34 polypeptide coated plate is judged to specifically recognize G-17, and the supernatant thereof is subjected to IHC detection.

[0041] 2. Screening of specific hybridoma cell strain by IHC detection method

[0042] The procedure is as follows: (1) Take formalin-fixed gastric tissue blocks for paraffin embedding, set the thickness of Leica tissue sectioning machine to 4 μm, and perform sectioning, spreading, fishing, and drying; (2) De-paraffinization and hydration: the above-processed slices are sequentially subjected to 10 min x 3 times of analytical pure xylene, 1 min x 3 times of anhydrous ethanol, 1 min of 95% ethanol, 1 min of 85% ethanol, 1 min of 75% ethanol, and 2 min x 3 times of deionized water immersion, to achieve the purpose of de-paraffinization and hydration; (3) Antigen repair: high-temperature and high-pressure are used for antigen repair treatment, and antigen repair solution (EDTA antigen repair solution, pH 8.0) is added into the pressure cooker for heat repair for 2.5 min, and when the temperature of the pressure cooker decreases to about 90℃, the pressure cooker is opened, the slices are taken out, and then naturally cooled to room temperature, and deionized water is immersed for 2 min x 3 times; (4) Inactivation: 3% hydrogen peroxide is used to inactivate endogenous peroxidase in the tissue, and the solution is placed at room temperature in the dark for 15 min, and deionized water is immersed for 2 min x 3 times; (5) Incubation of primary antibody: an immunohistochemical pen is used to frame the tissue periphery, and a reagent addition area is set, and the tissue is washed with 0.1% PBST for 2 min x 1 time. The hybridoma cell culture supernatant is used as the primary antibody, 200 μl of the supernatant is added to the reaction area, the slices are placed in a wet box, and incubated at 37℃ for 60 min. The slices are washed with 0.1% PBST for 2 min x 3 times; (6) Incubation of secondary antibody: 100 μl of hypersensitive HRP enzyme-labeled goat anti-mouse / rabbit IgG polymer is added to the reaction area, and incubated at 37℃ for 30 min. The slices are washed with 0.1% PBST for 2 min x 3 times; (7) DAB color development: 120 μl of freshly prepared DAB color developing solution is added, and the reaction is terminated after 5 min of reaction at room temperature, and the slices are washed for 3 times; (8) Hematoxylin re-staining, differentiation, and blue returning: the slices are placed in hematoxylin solution for 12 s, washed for 3 times to terminate the color development, differentiated in 1% hydrochloric acid ethanol solution, washed with deionized water for 3 times to terminate the reaction, placed in boiled Tris-EDTA disodium solution with pH 8.0 for blue returning, and then placed in room temperature Tris-EDTA disodium solution with pH 8.0 for a few seconds, and washed with tap water for 3 times; (9) Dehydration and clearing: After blueing, the slides were treated with 75% ethanol for 1 min, 85% ethanol for 1 min, 95% ethanol for 1 min, 100% ethanol for 1 min × 3 times, and xylene for 1 min × 3 times, and then mounted with neutral resin. (10) Microscopic examination: Observe the staining results under a microscope.

[0043] The supernatant, which stains on G cells in gastric tissue and appears negative in other cells, can be considered positive. After screening, the antibody secreted by the hybridoma cell line with clone number OTI3D11 met the specificity requirements. Specific preservation information is as follows: Biological material: OTI3D11; Classification and nomenclature: Mouse anti-human Gastrino-17 monoclonal hybridoma cell line; Preservation number: CGMCC No. 46359; Preservation date: April 26, 2025; Preservation institution: China General Microbiological Culture Collection Center (CGMCC); Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0044] 3. Expression and purification of monoclonal antibodies

[0045] The hybridoma cell line OTI3D11 was cultured in DMEM containing 15% serum in 10 cm culture dishes and expanded to approximately 4 × 10⁶ cells / year. 7 Centrifuge at 800 rpm for 5 min, discard the supernatant and transfer the cells to a 2 L roller flask. Add serum-free culture medium to bring the cell density to approximately 3 × 10⁶ cells / min. 5 Cells / ml. After culturing for another 1.5 weeks, when the cell death rate reached 60%, the cell suspension was collected and centrifuged at 6000 rpm for 20 min. The supernatant was collected and purified using a Protein G column (antibody subtype: IgG1). The concentration of the purified monoclonal antibody was determined, lyophilized, and aliquoted (100 μg / tube), and finally stored at -20℃.

[0046] Example 4: Amino acid sequence analysis of the variable region of anti-human Gastrin protein monoclonal antibody

[0047] Purchase SMARTer from Takara Bio USA ® The RACE 5' / 3' kit uses 5'RACE (Rapid Amplification of cDNA Ends) technology to amplify the variable region light and heavy chain gene sequences of hybridoma cell functional antibodies. For detailed experimental procedures, please refer to the SMARTer kit from Takara Bio USA. ® RACE 5' / 3' Kit User Manual.

[0048] Based on the fact that the antibody is an IgG1 subtype, specific gene primers pRace-H-GSP and pRace-K-GSP targeting the 3' end of its Ig and Kappa constant regions were designed. The primer sequences are as follows: pRace-H-GSP: 5'-CATCDGTCTATCCACTGGCCCCTG-3' (SEQ ID NO:11); pRace-K-GSP: 5'-CTTCCCACCATCCAGTGAGCAGTT-3' (SEQ ID NO:12).

[0049] mRNA was extracted from hybridoma cells OTI3D11 and reverse transcribed into cDNA. DNA fragments of the antibody heavy and light chains were amplified using RACE. The amplified light and heavy chains were ligated into the cloning vector PUC119 by enzyme digestion. Positive clones were selected using blue-white screening, and the positive plasmids were purified and sequenced using an ABI 3730 sequencer with universal primers M13F and M13R.

[0050] Using the IMGT / V-QUEST analysis software at http: / / www.imgt.org, the sequencing results of the light and heavy chains of the anti-human Gastrino monoclonal antibody were analyzed. The amino acid sequence of the VL domain of the anti-human Gastrino monoclonal antibody is shown in SEQ ID NO.3, and the amino acid sequence of the VH domain is shown in SEQ ID NO.7. The VL domain is 104 amino acids in length. The number of amino acids in the four domains of FR are 26, 17, 36, and 12, respectively, and the number of amino acids in the three domains of LCDR are 6, 3, and 5, respectively. The regions of LCDR1, LCDR2, and LCDR3 are 27-32 aa, 50-52 aa, and 89-93 aa, respectively. Their amino acid sequences are: QDINKF (SEQ ID NO.4), RTS (SEQ ID NO.5), and LQYDD (SEQ ID NO.6). The VH domain is 111 amino acids long. The four domains of its FR have 24, 17, 38 and 11 amino acids, respectively. The three domains of HCDR have 25-32 amino acids, 50-57 amino acids and 96-100 amino acids, respectively. HCDR1, HCDR2 and HCDR3 have 8, 8 and 5 amino acids, respectively. Their amino acid sequences are: GYTFTSYG (SEQ ID NO.8), INPYNGES (SEQ ID NO.9) and ARWGP (SEQ ID NO.10).

[0051] Example 5: Immunohistochemical detection kit containing monoclonal antibody against human Gastriin protein

[0052] An immunohistochemical assay kit containing an anti-human Gastrin protein monoclonal antibody includes antigen retrieval solution [1 mM EDTA, 10 mM Tris buffer (pH 8.0)], endogenous peroxidase inhibitor (hydrogen peroxide), anti-human Gastrin protein monoclonal antibody prepared in Example 3, ultrasensitive enzyme-labeled goat anti-mouse / rabbit IgG polymer, DAB chromogenic solution, and hematoxylin staining solution.

[0053] Example 6 Immunohistochemical detection of Gastrinoin protein in different human tissues

[0054] The expression of Gastrino protein in gastric, tonsil, appendix, placenta, lung, prostate, breast, pancreas, and thyroid tissues was detected using the immunohistochemical detection kit prepared in Example 5. The immunohistochemical detection procedure was the same as the IHC detection procedure in Example 3, wherein the tissues involved in step (1) included the above-mentioned tissues, and the reagents in steps (3)-(8) were from the immunohistochemical detection kit prepared in Example 5. The primary antibody in step (5) was the anti-human Gastrino protein monoclonal antibody prepared in Example 3, and the corresponding concentration of the anti-G-17 monoclonal antibody was 47.5 ng / mL. The results were examined under a microscope after detection.

[0055] The stomach contains various hormone-secreting cells in varying numbers, generally fewer than 20 cells per gland, with most glands containing fewer than 10 cells. The pyloric mucosa has slightly more endocrine cells, with approximately 20-50 per crypt. In the pyloric mucosa, endocrine cells are most commonly found in the gland neck, and about 50% of the entire endocrine cell population are G cells. G cells are open-type endocrine cells containing large, round granules; their main function is to secrete gastrin, regulating gastric acid secretion and gastrointestinal function.

[0056] Figure 1 The images show the results of gastric tissue analysis. Figure a on the left shows the results under low magnification, with positive staining mainly concentrated at the base and a few scattered staining areas in the neck. Figure b on the right is a magnified view of the boxed area in Figure a. The number of positive cells varies in different glands. As an example, the G cells indicated by the arrow show positive staining, while parietal cells, chief cells, and lymphocytes in the interstitium all show negative staining. The staining results are consistent with the expression and distribution characteristics of Gastrinoin protein in gastric tissue.

[0057] Figure 2 The four tissues in the figure are: Figure a tonsils, including germinal centers, mantle zones and squamous epithelial cells; Figure b lungs, including alveolar epithelium, vascular endothelial cells and inflammatory cells; Figure c appendix, including glandular epithelial cells and lymphocytes; and Figure d placental trophoblast cells. All types of cells did not show brown staining and were judged as negative, that is, there was no expression of Gastrinoin protein.

[0058] Figure 3 The four tissues in the study were: Figure a, the prostate gland, including acinar epithelial cells, basal cells, and stromal cells; Figure b, the thyroid gland, including follicular epithelium and parafollicular cells; Figure c, the mammary gland, including ductal epithelial cells, acinar epithelial cells, myoepithelial cells, adipocytes, and smooth muscle cells; and Figure d, the pancreas, including acinar epithelium and islet cells. All cell types showed no brown staining, indicating a negative result, meaning no Gastrino protein expression. This aligns with the expression characteristics of Gastrino protein in these tissues. Therefore, the monoclonal antibody secreted by the OTI3D11 hybridoma cell line demonstrated excellent specificity in immunohistochemical staining. All results were obtained under an antibody concentration of 47.5 ng / mL, exhibiting extremely high sensitivity.

[0059] The above results indicate that the anti-human Gastrino monoclonal antibody secreted by the hybridoma cell OTI3D11 described in this application has high specificity and sensitivity, is expressed only in secretory G cells, and exhibits excellent staining effect at a concentration of 47.5 ng / mL. Therefore, the Gastrino monoclonal antibody secreted by OTI3D11 can be used for immunohistochemical detection and for the study of Gastrino protein.

[0060] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. An antigastrin antibody or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises heavy chain complementarity-determining regions HCDR1 to HCDR3, and the light chain variable region comprises light chain complementarity-determining regions LCDR1 to LCDR3, characterized in that, The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the amino acid sequence shown in SEQ ID NO:7, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the amino acid sequence shown in SEQ ID NO:

3.

2. The antigastrin antibody or its antigen-binding fragment according to claim 1, characterized in that, The heavy chain variable regions HCDR1 to HCDR3 and the light chain variable regions LCDR1 to LCDR3 are determined according to the Kabat numbering system, AbM numbering system, IMGT numbering system, Chothia numbering system or Contact numbering system.

3. The antigastrin antibody or its antigen-binding fragment according to claim 2, characterized in that, The amino acid sequence of HCDR1 is shown in SEQ ID NO:8, the amino acid sequence of HCDR2 is shown in SEQ ID NO:9, the amino acid sequence of HCDR3 is shown in SEQ ID NO:10, the amino acid sequence of LCDR1 is shown in SEQ ID NO:4, the amino acid sequence of LCDR2 is shown in SEQ ID NO:5, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:

6.

4. The antigastrin antibody or its antigen-binding fragment according to any one of claims 1 to 3, characterized in that, The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:3, or an amino acid sequence with more than 90% homology obtained by substituting, deleting and / or adding one or more amino acids and / or terminal modification of any one or more amino acids in the amino acid sequence shown in SEQ ID NO:

3.

5. The antigastrin antibody or its antigen-binding fragment according to any one of claims 1 to 3, characterized in that, The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:7, or an amino acid sequence with more than 90% homology obtained by substituting, deleting and / or adding one or more amino acids and / or terminal modification of any one or more amino acids in the amino acid sequence shown in SEQ ID NO:

7.

6. The antigastrin antibody or its antigen-binding fragment according to any one of claims 1 to 5, characterized in that, The antigen-binding fragment is Fab, Fab', F(ab')2, a single-chain antibody, or a disulfide-bonded stable variable fragment.

7. The antigastrin antibody or its antigen-binding fragment according to any one of claims 1 to 6, characterized in that, The antibody or its antigen-binding fragment was produced by a hybridoma cell line with accession number CGMCC No. 46359.

8. A hybridoma cell line capable of secreting antigastrin antibodies, characterized in that, The hybridoma cell line is the mouse anti-human gastrin monoclonal hybridoma cell line OTI3D11, with accession number CGMCC No. 46359, deposited at the China General Microbiological Culture Collection Center (CGMCC).

9. The use of the antigastrin antibody or its antigen-binding fragment as described in any one of claims 1 to 7, or the hybridoma cell line as described in claim 8, in immunohistochemical detection or preparation of a detection kit.

10. An immunohistochemical detection kit, characterized in that, The immunohistochemical detection kit includes a primary antibody detection reagent, which contains the antigastrin antibody or its antigen-binding fragment as described in any one of claims 1-7.

11. The reagent kit according to claim 10, characterized in that, The kit also includes antigen retrieval solution, endogenous peroxidase inhibitor, hypersensitive secondary antibody reagent, DAB substrate buffer, DAB concentrated chromogenic solution, and hematoxylin staining solution.

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