Anti-pepsinogen II antibodies, reagents and kits for detecting pepsinogen II

Abstract

The present invention discloses an anti-pepsinogen II antibody or a reagent and kit for detecting pepsinogen II, relating to the field of antibodies. The anti-pepsinogen II antibody disclosed in the present invention comprises a heavy chain complementary determining region and a light chain complementary determining region. The antibody provides an important raw material source for the detection of pepsinogen II and has good affinity or activity.

Description

Technical Field

[0001] The present invention relates to the technical field of antibodies, in particular to an anti-pepsinogen II antibody, a reagent and a kit for detecting pepsinogen II. Background Art

[0002] Pepsinogen (PG) is the precursor of pepsin in gastric juice. The protein molecule is approximately 43 kDa and consists of 300 amino acids. Based on their biochemical and immunological activity, it can be divided into two major subgroups: PGI (also known as PGA) and PGII (also known as PGC). Pepsinogen A (PGA) originates from the chief cells and mucous neck cells of the fundic glands, while pepsinogen C (PGC) originates from all gastric glands (e.g., ventral, fundic, and pyloric glands of the antrum) and the Brunner glands of the distal duodenum. Small amounts of PGC are also produced by the prostate and pancreas. Gastric mucosal synthesis accounts for approximately 25% of the total PGC. Most of the synthesized PG enters the gastric lumen and is activated into pepsin by acidic gastric juice. Only a small amount (approximately 1%) enters the blood circulation through the gastric mucosal capillaries. The amount of PG entering is very stable, so serum PG concentration reflects the secretion level of gastric mucosal glands and indirectly reflects the secretory function of different parts of the gastric mucosa. When pathological changes occur in the gastric mucosa, the serum PG content also changes, which is called a "serological biopsy" of the gastric mucosa.

[0003] The normal blood PG I level is 60.6±13.9 ng / ml; the PG II level is 11.9±4.2 ng / ml, with a PG I / PG II ratio of 5.52±1.86. Clinical studies both domestically and internationally have shown a strong correlation between PG II and gastric fundic mucosal lesions. Elevated PG II levels are associated with fundic glandular duct atrophy, intestinal metaplasia or pseudopyloric metaplasia, and dysplasia. Measuring PG II levels can help detect other gastrointestinal diseases, such as duodenal ulcers, atrophic gastritis, and gastric cancer, and is therefore useful for clinical testing and physical screening. While it is unrealistic to perform a gastroscopy on every patient in population-based gastric disease screening, non-invasive serum PG testing can identify high-risk individuals for superficial gastritis, erosive gastritis, gastric ulcers, duodenal ulcers, and gastric cancer, followed by a gastroscopy. This is a feasible approach. The PG I / PG II ratio is a key indicator for gastric cancer screening. As a non-invasive method, PG II testing reduces patient pain, is simple, and cost-effective, making it valuable for general screening.

[0004] The detection of pepsinogen II adopts serological detection methods, which mainly include double antibody sandwich ELISA, immune complex lysis detection, ultrasensitive EIA, enzyme-linked immunofluorescence assay, etc. The double antibody sandwich method is currently widely used to detect pepsinogen II antigen. However, the above methods all require monoclonal antibodies against pepsinogen II. Therefore, technicians in this field have a strong demand for anti-pepsinogen II antibodies with good activity and affinity. Summary of the Invention

[0005] The present application provides an antibody or a functional fragment thereof, which provides an important source of raw materials for the detection of pepsinogen II and has good activity and affinity.

[0006] To achieve the above-mentioned object, according to one aspect of the present invention, an antibody or a functional fragment thereof is provided, wherein the antibody or the functional fragment thereof comprises HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, wherein the HCDR1, HCDR2, HCDR3 have amino acid sequences consistent with the HCDR1, HCDR2, HCDR3 of the heavy chain variable region shown in any one of SEQ ID NOs: 17, 18, and 19; and the LCDR1, LCDR2, LCDR3 have amino acid sequences consistent with the LCDR1, LCDR2, LCDR3 of the light chain variable region shown in any one of SEQ ID NOs: 20, 21, and 22.

[0007] To achieve the above-mentioned object, according to the second aspect of the present invention, an antibody or a functional fragment thereof is provided, comprising a heavy chain variable region and / or a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID NOs: 17, 18, and 19; and the amino acid sequence of the light chain variable region is shown in any one of SEQ ID NOs: 20, 21, and 22.

[0008] In order to achieve the above-mentioned object, according to the third aspect of the present invention, an antibody or a functional fragment thereof is provided, comprising a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is shown in any one of SEQ ID NOs: 23, 24, and 25; and the amino acid sequence of the light chain is shown in any one of SEQ ID NOs: 26, 27, and 28.

[0009] In order to achieve the above object, according to the fourth aspect of the present invention, an antibody conjugate is provided, wherein the antibody conjugate comprises the above antibody or a functional fragment thereof.

[0010] In order to achieve the above object, according to the fifth aspect of the present invention, a reagent or kit is provided, wherein the reagent or kit comprises the above-mentioned antibody or its functional fragment or the above-mentioned antibody conjugate.

[0011] In order to achieve the above-mentioned object, according to the sixth aspect of the present invention, a method for detecting pepsinogen II is provided, comprising: a) contacting the above-mentioned antibody or its functional fragment, antibody conjugate, or reagent or kit with the pepsinogen II antigen in the sample to be detected under conditions sufficient for an antibody / antigen binding reaction to occur to form an immune complex; and b) detecting the presence of the immune complex, wherein the presence of the complex indicates the presence of the antigen in the test sample.

[0012] In order to achieve the above object, according to the seventh aspect of the present invention, a nucleic acid is provided, which encodes the above antibody or a functional fragment thereof.

[0013] In order to achieve the above object, according to the eighth aspect of the present invention, a vector is provided, wherein the vector comprises the above nucleic acid.

[0014] To achieve the above object, according to a ninth aspect of the present invention, a cell is provided, wherein the cell comprises the above nucleic acid, vector or expresses the above antibody or a functional fragment thereof.

[0015] In order to achieve the above object, according to the tenth aspect of the present invention, a method for preparing the above antibody or a functional fragment thereof is provided, which comprises culturing the above cell.

[0016] In order to achieve the above-mentioned object, according to the eleventh aspect of the present invention, there is provided a use of the above-mentioned antibody or functional fragment thereof, antibody conjugate, reagent or kit in detecting or preparing a product for detecting pepsinogen II. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0018] Figure 1 The results of reducing SDS-PAGE of Anti-HPG 4E3Rmb1 are shown. DETAILED DESCRIPTION

[0019] In a first aspect, an embodiment of the present invention provides an antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof comprises HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, wherein the HCDR1, HCDR2, HCDR3 are amino acid sequences consistent with the HCDR1, HCDR2, HCDR3 of the heavy chain variable region shown in any one of SEQ ID NOs: 17, 18, and 19; and the LCDR1, LCDR2, LCDR3 are amino acid sequences consistent with the LCDR1, LCDR2, LCDR3 of the light chain variable region shown in any one of SEQ ID NOs: 20, 21, and 22.

[0020] It should be noted that HCDR1, HCDR2 and HCDR3 are amino acid sequences consistent with HCDR1, HCDR2 and HCDR3 of the same heavy chain variable region defined in the antibody or its functional fragment described in the first aspect, and LCDR1, LCDR2 and LCDR3 are amino acid sequences consistent with LCDR1, LCDR2 and LCDR3 of the same light chain variable region defined in the antibody or its functional fragment described in the first aspect.

[0021] For example, the HCDR1, HCDR2, and HCDR3 have amino acid sequences consistent with those of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region shown in SEQ ID NO: 17; and the LCDR1, LCDR2, and LCDR3 have amino acid sequences consistent with those of LCDR1, LCDR2, and LCDR3 in the light chain variable region shown in SEQ ID NO: 20.

[0022] Under the Kabat definition, the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region SEQ ID NO:17 are shown as SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively. Then, the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the antibody or its functional fragment are also shown as SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively.

[0023] Under the Kabat definition, the amino acid sequences of LCDR1, LCDR2, and LCDR3 of the light chain variable region SEQ ID NO:20 are shown as SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively. Then, the amino acid sequences of LCDR1, LCDR2, and LCDR3 of the antibody or its functional fragment are also shown as SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

[0024] In the present invention, the term "antibody" is used in the broadest sense and may include full-length monoclonal antibodies, bispecific or multispecific antibodies, and chimeric antibodies, so long as they exhibit the desired biological activity.

[0025] As used herein, the terms "complementarity determining region," "CDR," or "CDRs" refer to the hypervariable regions of the heavy and light chains of immunoglobulins, and include one or more, or even all, of the amino acid residues that contribute substantially to the binding of an antibody or antigen-binding fragment to its recognized antigen or epitope. In specific embodiments of the present invention, CDRs refer to the hypervariable regions of the heavy and light chains of the antibody.

[0026] In the present invention, the heavy chain complementarity determining region is represented by HCDR, which includes HCDR1, HCDR2 and HCDR3; the light chain complementarity determining region is represented by LCDR, which includes LCDR1, LCDR2 and LCDR3.

[0027] Methods for defining CDRs are well known in the art, and include the Kabat definition, the Chothia definition, the IMGT definition, the Contact definition, and the AbM definition. As used herein, the "Kabat definition" refers to the definition system described by Kabat et al., U.S. Patent No. 200,255,854,554, "Sequence of Proteins of Immunological Interest" (1983). The "Chothia definition" refers to the definition system described by Chothia et al., J Mol Biol 196:901-917 (1987). Other CDR definition methods may not strictly follow one of the above schemes, but may still overlap with at least a portion of the Kabat-defined CDR region, although they may be shortened or lengthened based on predictions or experimental results of specific residues or residue groups. Exemplary defined CDRs are listed in Table 1 below. Given the variable region amino acid sequence of an antibody, a person skilled in the art can routinely determine which residues comprise a specific CDR. It should be noted that CDRs defined by other methods other than those in Table 1 also fall within the scope of protection of the present disclosure.

[0028] Table 1: CDR Definition 1

[0029] CDR Kabat <![CDATA[AbM 2 ]]> IMGT Chothia HCDR1 <![CDATA[H31~H35 3 ]]> <![CDATA[H26~H35 3 ]]> <![CDATA[H26~H33..35 5 ]]> H26~H32..344 HCDR2 H50~H65 H50~H58 H51~H57 H52~H56 HCDR3 H95~H102 H95~H102 H93~H102 H95~H102 LCDR1 L24~L34 L24~L34 L27~L32 L24~L34 LCDR2 L50~L56 L50~L56 L50~L51 L50~L56 LCDR3 L89~L97 L89~L97 L89~L97 L89~L97

[0030] 1The numbering of all CDR definitions in Table 1 is based on the Kabat numbering system (see below), with amino acid numbers on the heavy chain represented by "H+number" and amino acid numbers on the light chain represented by "L+number." One of ordinary skill in the art can unambiguously assign this Kabat numbering system to any variable region sequence without relying on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described in Kabat et al., U.S. Pat. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0031] 2 "AbM" as used in Table 1 with a lowercase "b" refers to CDRs defined by Oxford Molecular's "AbM" antibody modeling software.

[0032] 3 If both H35A and H35B are absent, CDR-H1 ends at position 35; if only H35A is present, CDR-H1 ends at position 35A; if both H35A and H35B are present, CDR-H1 ends at position 35B.

[0033] 4 If both H35A and H35B are absent, CDR-H1 ends at position 32; if only H35A is present, CDR-H1 ends at position 33; if both H35A and H35B are present, CDR-H1 ends at position 34.

[0034] 5 If both H35A and H35B are absent, CDR-H1 ends at position 33; if only H35A is present, CDR-H1 ends at position 34; if both H35A and H35B are present, CDR-H1 ends at position 35.

[0035] According to an embodiment of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one of the Kabat, Chothia, IMGT, AbM or Contact systems or a combination of multiple systems.

[0036] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat system.

[0037] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Chothia system.

[0038] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by an IMGT system.

[0039] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the AbM system.

[0040] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a Contact system.

[0041] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a combination of Kabat, Chothia, IMGT, AbM or Contact systems.

[0042] According to an embodiment of the present invention, the Kabat numbering positions corresponding to the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 defined by the Kabat, Chothia, AbM or IMGT systems are as follows:

[0043] CDR Kabat AbM IMGT Chothia HCDR1 H31~H35 H26~H35 H26~H33 H26~H32 HCDR2 H50~H65 H50~H58 H51~H57 H52~H56 HCDR3 H95~H102 H95~H102 H93~H102 H95~H102 LCDR1 L24~L34 L24~L34 L27~L32 L24~L34 LCDR2 L50~L56 L50~L56 L50~L51 L50~L56 LCDR3 L89~L97 L89~L97 L89~L97 L89~L97

[0044] According to an embodiment of the present invention, the HCDRs and LCDRs are defined by the Kabat system.

[0045] In an optional embodiment, an embodiment of the present invention provides an antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof comprises the following complementarity determining regions:

[0046] HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 1.

[0047] HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 2.

[0048] HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 3.

[0049] LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 4.

[0050] LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 5.

[0051] LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 6.

[0052] In the present invention, the "framework region" or "FR" region includes the heavy chain framework region and the light chain framework region, and refers to the region of the antibody heavy chain variable region and the light chain variable region excluding CDR; wherein the heavy chain framework region can be further subdivided into adjacent regions separated by CDR, including HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework region can be further subdivided into adjacent regions separated by CDR, including LFR1, LFR2, LFR3 and LFR4 framework regions.

[0053] In the present invention, the heavy chain variable region is obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0054] In an optional embodiment, the antibody or functional fragment thereof further has at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4;

[0055] The HFR1 comprises / is SEQ ID NO: 7 or an amino acid sequence having at least 80% identity thereto;

[0056] The HFR2 comprises / is as SEQ ID NO: 8 or an amino acid sequence having at least 80% identity thereto;

[0057] The HFR3 comprises / is SEQ ID NO: 9 or an amino acid sequence having at least 80% identity thereto;

[0058] The HFR4 comprises / is as SEQ ID NO: 10 or an amino acid sequence having at least 80% identity thereto;

[0059] The LFR1 comprises / is SEQ ID NO: 11 or an amino acid sequence having at least 80% identity thereto;

[0060] The LFR2 comprises / is SEQ ID NO: 12 or an amino acid sequence having at least 80% identity thereto;

[0061] The LFR3 comprises / is SEQ ID NO: 13 or an amino acid sequence having at least 80% identity thereto;

[0062] The LFR4 comprises / is SEQ ID NO: 14 or an amino acid sequence having at least 80% identity thereto;

[0063] It should be noted that, in other embodiments, the amino acid sequences of the framework regions of the antibodies or functional fragments thereof provided by the present invention may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the corresponding framework regions (SEQ ID NO: 7, 8, 9, 10, 11, 12, 13 or 14).

[0064] In an optional embodiment, the HFR3 comprises / is represented by the amino acid sequence of SEQ ID NO: 9, 29 or 30.

[0065] In an optional embodiment, the LFR1 includes / is represented by the amino acid sequence of SEQ ID NO: 11 or 31.

[0066] In an optional embodiment, the LFR3 comprises / is represented by the amino acid sequence of SEQ ID NO: 13 or 32.

[0067] In an optional embodiment, the antibody or its functional fragment has a KD of less than 8.65×10 -10 M binds pepsinogen II with high affinity.

[0068] In an optional embodiment, the antibody or its functional fragment has a KD≤10 -10 M or KD≤10 -11 M binds pepsinogen II with high affinity.

[0069] In an optional embodiment, the antibody or its functional fragment has a KD ≤ 2.11×10 -11 M binds pepsinogen II with high affinity.

[0070] There are many methods for determining antibody affinity (KD). Based on the detection principle, they can be divided into thermodynamic detection methods, kinetic detection methods, and dynamic equilibrium detection methods. Among them, thermodynamic detection methods such as isothermal titration calorimetry (ITC) are common; kinetic detection methods such as surface plasmon resonance (SPR) and biofilm interferometry (BLI) are common; and dynamic equilibrium detection methods such as enzyme-linked immunosorbent assay (ELISA) are common.

[0071] In alternative embodiments, KD is determined using a kinetic assay; alternatively, biomembrane interferometry, for example by using a biosensor system such as the AMC system.

[0072] In a second aspect, an embodiment of the present invention provides an antibody or a functional fragment thereof, comprising a heavy chain variable region and / or a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID NOs: 17, 18, and 19, and the amino acid sequence of the light chain variable region is shown in any one of SEQ ID NOs: 20, 21, and 22.

[0073] In an optional embodiment, the antibody or functional fragment thereof described in the first aspect or the second aspect comprises any combination of the following heavy chain variable regions and light chain variable regions:

[0074]

[0075] In an alternative embodiment, the antibody or functional fragment thereof further comprises a constant region.

[0076] In alternative embodiments, the constant region comprises a heavy chain constant region and / or a light chain constant region.

[0077] In an alternative embodiment, the heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD, and the light chain constant region is selected from the κ type or λ type light chain constant region.

[0078] In an alternative embodiment, the species origin of the constant region is cow, horse, dairy cow, pig, sheep, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, fighting cock or human.

[0079] In an alternative embodiment, the species origin of the constant region is mouse.

[0080] In an optional embodiment, the heavy chain constant region sequence (CH) is shown as SEQ ID NO:15, and the light chain constant region (CL) sequence is shown as SEQ ID NO:16.

[0081] It should be noted that, in other embodiments, the constant region sequence may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the above constant region (SEQ ID NO: 15 or 16).

[0082] In an optional embodiment, the functional fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

[0083] The functional fragments of these antibodies generally possess the same binding specificity as the antibodies from which they are derived. Based on the disclosure herein, those skilled in the art will readily appreciate that these functional fragments can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or chemical reduction to cleave disulfide bonds. Based on the structure of the intact antibody disclosed herein, those skilled in the art can readily obtain these functional fragments.

[0084] Functional fragments of the above antibodies can also be synthesized by recombinant genetic techniques known to those skilled in the art or by, for example, an automatic peptide synthesizer, such as those sold by Applied BioSystems.

[0085] In a third aspect, the present invention provides an antibody or a functional fragment thereof, comprising a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is shown in any one of SEQ ID NOs: 23, 24, and 25, and the amino acid sequence of the light chain is shown in any one of SEQ ID NOs: 26, 27, and 28.

[0086] combination H L 1 SEQ ID NO:23 SEQ ID NO:26 2 SEQ ID NO:24 SEQ ID NO:26 3 SEQ ID NO:25 SEQ ID NO:26 4 SEQ ID NO:23 SEQ ID NO:27 5 SEQ ID NO:23 SEQ ID NO:28 .

[0087] In a fourth aspect, the present invention provides an antibody conjugate comprising the above-mentioned antibody or a functional fragment thereof.

[0088] In an optional embodiment, the above-mentioned antibody conjugate further comprises biotin or a biotin derivative coupled to the antibody or a functional fragment thereof.

[0089] In an optional embodiment, the antibody conjugate further comprises a label coupled to the antibody or a functional fragment thereof.

[0090] In an optional embodiment, the above-mentioned marker refers to a class of substances with properties that can be directly observed by the naked eye or detected or detected by an instrument, such as luminescence, color development, radioactivity, etc., through which qualitative or quantitative detection of the corresponding target can be achieved.

[0091] In an alternative embodiment, the label includes but is not limited to fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents and nanoparticle labels.

[0092] In actual use, those skilled in the art can select a suitable marker according to the detection conditions or actual needs. No matter which marker is used, it falls within the scope of protection of the present invention.

[0093] In an optional embodiment, the fluorescent dyes include but are not limited to fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, C y5, Cy5.5, Cy3, etc. or their analogs), Alexa series dyes and their derivatives (for example, including but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs) and protein dyes and their derivatives (for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (preCP), etc.).

[0094] In alternative embodiments, the enzyme includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxidase.

[0095] In an optional embodiment, the radioactive isotopes include but are not limited to 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 47Sc, 110mIn, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu and 18F.

[0096] In an optional embodiment, the chemiluminescent reagent includes but is not limited to luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, and peroxalates and their derivatives.

[0097] In an optional embodiment, the nanoparticle markers include but are not limited to nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles and rare earth complex nanoparticles.

[0098] In alternative embodiments, the colloid includes, but is not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.

[0099] In an alternative embodiment, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0100] In an optional embodiment, the colloidal metal is colloidal gold.

[0101] In an optional embodiment, the above-mentioned antibody conjugate further includes a solid phase carrier coupled to the antibody or its functional fragment.

[0102] In an alternative embodiment, the solid support is selected from microspheres, plates, and membranes.

[0103] In an optional embodiment, the solid phase carrier includes but is not limited to magnetic microspheres, plastic microspheres, plastic particles, microplates, glass, capillaries, nylon and nitrocellulose membranes.

[0104] In a fifth aspect, the present invention provides a reagent or a kit, which comprises the above-mentioned antibody or a functional fragment thereof or the above-mentioned antibody conjugate.

[0105] As previously mentioned, the antibodies or functional fragments thereof in some embodiments or examples of the present invention can effectively bind to the pepsinogen II antigen. Therefore, the reagents or kits comprising the pepsinogen II antibodies or functional fragments thereof can effectively perform qualitative or quantitative detection of pepsinogen II. The reagents or kits provided by the present invention can be used, for example, in immunoblotting, immunoprecipitation, and other assays that utilize the specific binding properties of pepsinogen II and its antibodies. As previously mentioned, the antibodies or functional fragments thereof in some embodiments or examples of the present invention have higher binding activity or affinity for pepsinogen II. Therefore, the reagents or kits comprising the antibodies or functional fragments thereof have higher detection sensitivity or specificity.

[0106] In a sixth aspect, the present invention provides a method for detecting pepsinogen II, comprising: a) contacting the above-mentioned antibody or functional fragment thereof, antibody conjugate, reagent or kit with a pepsinogen II antigen in a sample to be tested under conditions sufficient for an antibody / antigen binding reaction to occur, to form an immune complex; and b) detecting the presence of the immune complex, wherein the presence of the complex indicates the presence of the antigen in the test sample;

[0107] In an optional embodiment, the immune complex further comprises a second antibody, which binds to the antibody or a functional fragment thereof.

[0108] In an alternative embodiment, the immune complex further comprises a second antibody that binds to the pepsinogen II antigen.

[0109] In a seventh aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned antibody or a functional fragment thereof.

[0110] In an eighth aspect, the present invention provides a vector comprising the above-mentioned nucleic acid molecule.

[0111] In a ninth aspect, the present invention provides cells containing the above-mentioned vector.

[0112] In a tenth aspect, the present invention provides a method for preparing an antibody or a functional fragment thereof, comprising: culturing the cell as described above.

[0113] In an eleventh aspect, the present invention provides use of the above-mentioned antibody or functional fragment thereof, antibody conjugate, or the above-mentioned reagent or kit in detecting pepsinogen II or preparing a product for detecting pepsinogen II.

[0114] Based on the amino acid sequence of the antibody or its functional fragment disclosed in the present invention, those skilled in the art can easily conceive of using genetic engineering technology or other technologies (chemical synthesis, recombinant expression) to prepare the antibody or its functional fragment. For example, the antibody or its functional fragment can be isolated and purified from the culture product of a recombinant cell that can recombinantly express the antibody or its functional fragment as described in any of the above items. This is easy to achieve for those skilled in the art. Based on this, no matter what technology is used to prepare the antibody or its functional fragment of the present invention, it falls within the scope of protection of the present invention.

[0115] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0116] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the preparations or unit doses herein, some methods and materials are now described. Unless otherwise indicated, the techniques employed or contemplated herein are standard methods. Materials, methods, and examples are illustrative and non-limiting only.

[0117] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a person skilled in the art. The technique is fully explained in the literature, for example, Molecular Cloning: A Laboratory Manual, 2nd ed. (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); and PCR: The Polymerase Chain Reaction. Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (J.E. Colligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0118] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0119] Example 1 Preparation of Anti-HPG-4E3 Monoclonal Antibody

[0120] In this example, restriction endonucleases and Prime Star DNA polymerase were purchased from Takara. MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART TMThe RACE cDNA Amplification Kit was purchased from Takara. The pMD-18T vector was also purchased from Takara. The plasmid extraction kit was purchased from Tiangen. Primer synthesis and gene sequencing were performed by Invitrogen. The hybridoma cell line secreting the anti-HPG-4E3 monoclonal antibody was a pre-existing hybridoma cell line that was revived and used for future reference.

[0121] (1) Antibody gene preparation

[0122] mRNA was extracted from a hybridoma cell line secreting the Anti-HPG-4E3 monoclonal antibody. DNA products were obtained by RT-PCR. The products were then inserted into the pMD-18T vector after A addition reaction using rTaq DNA polymerase and transformed into DH5α competent cells. After colonies grew, the Heavy Chain and Light Chain gene clones were obtained, and four clones each were sent to a gene sequencing company for sequencing.

[0123] (2) Sequence analysis of the variable region gene of the Anti-HPG-4E3 antibody

[0124] The gene sequences obtained by the above sequencing were placed in the Kabat antibody database for analysis, and VNTI11.5 software was used for analysis to determine that the genes amplified by the heavy chain and light chain primer pairs were correct. Among them, in the gene fragment amplified by the Light Chain, the VL gene sequence was 315 bp, preceded by a 57 bp leader peptide sequence; in the gene fragment amplified by the Heavy Chain primer pair, the VH gene sequence was 375 bp, belonging to the VH1 gene family, and preceded by a 57 bp leader peptide sequence.

[0125] (3) Construction of recombinant antibody expression plasmid

[0126] pcDNA TM 3.4 The vector is a recombinant antibody eukaryotic expression vector constructed by using the pMD-18T vector. Multiple cloning restriction sites such as HindIII, BamHI, and EcoRI have been introduced into the vector, and the vector is named pcDNA3.4A expression vector, hereinafter referred to as 3.4A expression vector. Based on the sequencing results of the antibody variable region genes in the pMD-18T vector, specific primers for the VL and VH genes of the antibody were designed, with HindIII and EcoRI restriction sites and protective bases at both ends, respectively. PCR amplification was used to amplify a 0.71kb Light Chain gene fragment and a 1.43kb Heavy Chain gene fragment.

[0127] The Heavy Chain and Light Chain gene fragments were digested with HindIII / EcoRI, and the 3.4A vector was digested with HindIII / EcoRI. After the fragments and vectors were purified and recovered, the Heavy Chain gene and Light Chain gene were respectively connected to the 3.4A expression vector to obtain the recombinant expression plasmids of Heavy Chain and Light Chain, respectively.

[0128] 2. Recombinant Antibody Production

[0129] Resuscitate HEK293 cells in advance and subculture them into 200 ml system to make the cell density reach 3-5×10 6 cells / ml cell density reaches the selected antibody concentration and cells, cell viability>95%; centrifuge and wash the cells, re-dissolve with culture medium, and adjust the cell density to 2.9×10 6 Cells were washed at 100 μg / ml and re-dissolved in culture medium, which also served as a cell dilution. Plasmid DNA and transfection reagent dilutions were prepared separately using culture medium. The transfection reagent dilution was added to the plasmid DNA dilution, mixed, and allowed to stand at room temperature for 15 minutes. This mixture was slowly added to the cell dilution over 1 minute. After mixing, samples were collected and counted. Cell viability after transfection was recorded and observed. Cells were incubated in a 35°C incubator at 120 rpm and 8% CO2 for 13 days. Samples were collected by centrifugation. The supernatant was affinity purified using a protein A affinity column. A 6 μg portion of the purified antibody was subjected to reducing SDS-PAGE. The electropherogram is shown in the figure. Reducing SDS-PAGE revealed two bands: one with an Mr of 50 kD (heavy chain) and the other with an Mr of 28 kD (light chain).

[0130] The obtained antibody was named Anti-HPG 4E3Rmb1. Anti-HPG 4E3Rmb1 was mutated to obtain a mutant antibody. The sequences of the heavy chain (H) and light chain (L) of the above antibody are shown in the following table: Table 2 Antibody Sequence

[0131] Antibody name Heavy chain (H) Light chain (L) Anti-HPG 4E3Rmb1 SEQ ID NO:23 SEQ ID NO:26 Anti-HPG 4E3Rmb1 SEQ ID NO:24 SEQ ID NO:26 Anti-HPG 4E3Rmb2 SEQ ID NO:25 SEQ ID NO:26 Anti-HPG 4E3Rmb3 SEQ ID NO:23 SEQ ID NO:27 Anti-HPG 4E3Rmb4 SEQ ID NO:23 SEQ ID NO:28

[0132] Example 2 Performance testing of antibodies

[0133] 1. Affinity Analysis

[0134] Using the AMC sensor, the purified antibody was diluted to 10 μg / ml with PBST, and PGII (purchased from FITZGERALD, product number 30R-AP039) was serially diluted with PBST.

[0135] Runtime: Equilibrate in buffer 1 (PBST) for 60 seconds, immobilize the antibody in the antibody solution for 300 seconds, incubate in buffer 2 (PBST) for 180 seconds, bind in the antigen solution for 420 seconds, dissociate in buffer 2 for 1200 seconds, regenerate the sensor with 10 mM GLY solution, pH 1.69, and buffer 3 (PBST), and output data. (KD represents the equilibrium dissociation constant (affinity); kon represents the association rate; kdis represents the dissociation rate. PBST consists primarily of Na₂HPO₄, NaCl, and TW-20).

[0136] Table 3 Affinity data

[0137]

[0138] 2. Activity Identification

[0139] Coat the microplate with 1ug / ml goat anti-mouse IgG diluted in coating solution (main component NaHCO3), 100uL per well, overnight at 4°C; the next day, wash twice with washing solution (main component Na2HPO4+Nacl), pat dry; add blocking solution (20% BSA+80% PBS), 120uL per well, incubate at 37°C for 1 hour, pat dry; add diluted purified antibody, 100uL / well, incubate at 37°C for 60 minutes; shake off the liquid in the plate, pat dry, add 20% mouse negative blood for blocking, 120ul per well, incubate at 37°C for 1 hour; shake off the liquid in the plate, pat dry, and add diluted PG II (purchased from FITZGERALD, product number 30R-AP039), 100uL per well, 37℃, 40min; wash with detergent 5 times and pat dry; add HRP-labeled anti-PGII monoclonal antibody (paired with purified antibody), 100uL per well, 37℃, 30min; add colorimetric solution A (50uL / well), add colorimetric solution B (50uL / well), 10min; add stop solution, 50uL / well; read the OD value at 450nm (reference 630nm) on a microplate reader.

[0140] Note: Solution A (main ingredients: citric acid + sodium acetate + acetanilide + carbamide peroxide); Solution B (main ingredients: citric acid + EDTA·2Na + TMB + concentrated HCL); Stop solution (EDTA·2Na + concentrated H2SO4)

[0141] Table 4 Activity data

[0142] Concentration (ng / ml) 15.63 7.81 3.91 1.95 0.98 0 comparison 1.632 1.241 0.752 0.323 0.127 0.007 Anti-HPG 4E3Rmb1 2.157 1.839 1.210 0.737 0.358 0.057 Anti-HPG 4E3Rmb1 2.018 1.808 1.178 0.714 0.317 0.048 Anti-HPG 4E3Rmb2 2.112 1.826 1.201 0.727 0.342 0.051 Anti-HPG 4E3Rmb3 2.218 1.871 1.245 0.741 0.361 0.055 Anti-HPG 4E3Rmb4 2.109 1.793 1.209 0.727 0.359 0.043

[0143] 3. Stability assessment

[0144] The above-mentioned antibody was placed at 4°C (refrigerator), -80°C (freezer), and 37°C (incubator) for 21 days. Samples were collected at 7, 14, and 21 days for observation, and the 21-day sample was assayed for activity. The results showed no significant changes in protein status under all three assay conditions, and the activity did not decrease with increasing assay temperature, indicating that the antibody is stable. Table 5 below shows the OD results of the enzyme immunoassay for Anti-hPG-4E3Rmb1 after 21 days of assay.

[0145] Table 5 Stability data

[0146] Sample concentration (ng / ml) 7.81 3.91 0 4℃, 21-day samples 1.845 1.228 0.022 -80℃, 21-day sample 1.844 1.231 0.021 37℃, 21-day samples 1.851 1.227 0.021

[0147] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

[0148] Some of the amino acid sequences involved in this application are as follows:

[0149]

Claims

1. A pepsinogen II antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof comprises HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, characterized in that: The HCDR1, HCDR2, and HCDR3 are amino acid sequences consistent with the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in any one of SEQ ID NOs: 17, 18, and 19; the LCDR1, LCDR2, and LCDR3 are amino acid sequences consistent with the LCDR1, LCDR2, and LCDR3 of the light chain variable region shown in any one of SEQ ID NOs: 20, 21, and 22, and the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the variable region are defined by any one of the systems of Kabat, Chothia, IMGT, AbM, or Contact.

2. An anti-pepsinogen II antibody or a functional fragment thereof, characterized in that: The antibody or its functional fragment comprises the following complementarity determining regions: HCDR1, the amino acid sequence of which is shown in SEQ ID NO: 1; HCDR2, the amino acid sequence of which is shown in SEQ ID NO: 2; HCDR3, the amino acid sequence of which is shown in SEQ ID NO: 3; LCDR1, the amino acid sequence of which is shown in SEQ ID NO:4; LCDR2, the amino acid sequence of which is shown in SEQ ID NO:5; LCDR3, the amino acid sequence of which is shown in SEQ ID NO:

6.

3. The antibody or functional fragment thereof according to claim 1 or 2, characterized in that The antibody or a functional fragment thereof further comprises HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4.

4. The antibody or functional fragment thereof according to claim 3, characterized in that The HFR1 comprises SEQ ID NO: 7 or an amino acid sequence having at least 80% identity thereto; The HFR2 comprises SEQ ID NO: 8 or an amino acid sequence having at least 80% identity thereto; The HFR3 comprises SEQ ID NO: 9 or an amino acid sequence having at least 80% identity thereto; The HFR4 comprises SEQ ID NO: 10 or an amino acid sequence having at least 80% identity thereto; The LFR1 comprises SEQ ID NO: 11 or an amino acid sequence having at least 80% identity thereto; The LFR2 comprises SEQ ID NO: 12 or an amino acid sequence having at least 80% identity thereto; The LFR3 comprises SEQ ID NO: 13 or an amino acid sequence having at least 80% identity thereto; The LFR4 comprises SEQ ID NO: 14 or an amino acid sequence having at least 80% identity thereto.

5. The antibody or functional fragment thereof according to any one of claims 1 to 2 and 4, characterized in that: The antibody or its functional fragment has a KD of less than 8.65×10 -10 M binds pepsinogen II with high affinity.

6. An anti-pepsinogen II antibody or a functional fragment thereof, comprising a heavy chain variable region and a light chain variable region, characterized in that: The amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID NOs: 17, 18, and 19; the amino acid sequence of the light chain variable region is shown in any one of SEQ ID NOs: 20, 21, and 22.

7. The antibody or functional fragment thereof according to claim 6, wherein the heavy chain variable region and the light chain variable region are selected from any one of the following groups:

8. The antibody or functional fragment thereof according to any one of claims 1 to 2, 4, 6 to 7, wherein: The antibody or functional fragment thereof further comprises a constant region.

9. The antibody or functional fragment thereof according to claim 8, characterized in that The constant region includes a heavy chain constant region and a light chain constant region.

10. The antibody or functional fragment thereof according to claim 9, characterized in that The heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light chain constant region is selected from the κ type or λ type light chain constant region.

11. The antibody or functional fragment thereof according to claim 8, characterized in that The species origin of the constant region is cow, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human.

12. The antibody or functional fragment thereof according to claim 8, characterized in that The species origin of the constant region is mouse.

13. The antibody or functional fragment thereof according to claim 9, characterized in that: The heavy chain constant region sequence is shown in SEQ ID NO: 15, and the light chain constant region sequence is shown in SEQ ID NO: 16 or has at least 80% identity thereto.

14. The antibody or functional fragment thereof according to any one of claims 1 to 2, 4, 6 to 7, wherein: The functional fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

15. An anti-pepsinogen II antibody comprising a heavy chain and a light chain, characterized in that: The amino acid sequence of the heavy chain is shown in any one of SEQ ID NOs: 23, 24, and 25; the amino acid sequence of the light chain is shown in any one of SEQ ID NOs: 26, 27, and 28.

16. An antibody conjugate, characterized in that The antibody conjugate comprises the antibody or functional fragment thereof according to any one of claims 1 to 14 or the antibody according to claim 15 and biotin, a label or a solid phase carrier coupled to the antibody or functional fragment thereof.

17. The antibody conjugate according to claim 16, characterized in that The label is selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents and nanoparticle labels.

18. The antibody conjugate according to claim 16, characterized in that The solid support is selected from microspheres, plates and membranes.

19. A reagent or kit, characterized in that: The reagent or kit comprises the antibody or functional fragment thereof according to any one of claims 1 to 14, the antibody according to claim 15, or the antibody conjugate according to any one of claims 16 to 18.

20. Use of the antibody or functional fragment thereof according to any one of claims 1 to 14, the antibody according to claim 15, or the antibody conjugate according to any one of claims 16 to 18 in the preparation of a product for detecting pepsinogen II, characterized in that: include: a) contacting the antibody or functional fragment thereof according to any one of claims 1 to 14, the antibody according to claim 15, or the antibody conjugate according to any one of claims 16 to 18 with a pepsinogen II antigen in a sample to be tested under conditions sufficient for an antibody / antigen binding reaction to form an immune complex; and b) detecting the presence of the immune complex, wherein the presence of the complex indicates the presence of the antigen in the test sample.

21. The use according to claim 20, characterized in that The immune complex further comprises a second antibody that binds to the antibody or a functional fragment thereof.

22. The use according to claim 20, characterized in that The immune complex also includes a second antibody that binds to the pepsinogen II antigen.

23. A nucleic acid, characterized in that It encodes the antibody or functional fragment thereof according to any one of claims 1 to 14 or the antibody according to claim 15.

24. A carrier, characterized in that It contains the nucleic acid according to claim 23.

25. A cell, characterized in that It contains the nucleic acid according to claim 23 or the vector according to claim 24.

26. A method for preparing the antibody or functional fragment thereof according to any one of claims 1 to 14 or the antibody according to claim 15, characterized in that: It includes: Cultivate the cell of claim 25.

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