CN114835814B

CN114835814B - Pepsinogen II resistant monoclonal antibody, and preparation method and application thereof

Info

Publication number: CN114835814B
Application number: CN202210636189.8A
Authority: CN
Inventors: 卓越; 吴振兴; 马敏娜; 赖思慧; 赵晶磊
Original assignee: Ningbo Saipo Biotechnology Co ltd
Current assignee: Ningbo Saipo Biotechnology Co ltd
Priority date: 2022-06-07
Filing date: 2022-06-07
Publication date: 2023-08-25
Anticipated expiration: 2042-06-07
Also published as: CN114835814A; CN116640221A

Abstract

The invention provides a pepsinogen II-resistant monoclonal antibody, a preparation method and application thereof, wherein the pepsinogen II-resistant monoclonal antibody comprises the following components: antibody 1A7 and/or antibody 3G10; the amino acid sequence of antibody 1A7 is: VH CDR1-3 are respectively shown as SEQ ID NO. 2-4; VL CDRs 1-3 are shown in SEQ ID NOS.5-7, respectively. The amino acid sequence of antibody 3G10 is: VH CDR1-3 are respectively shown as SEQ ID NO. 10-12; VL CDR1-3 is shown in SEQ ID NO. 13-15. As shown. The antibody provided by the invention has high affinity and high specificity, and can be applied to a pepsinogen II resistant kit.

Description

Pepsinogen II resistant monoclonal antibody, and preparation method and application thereof

Technical Field

The invention relates to the field of biotechnology, in particular to the field of antibodies and in vitro detection. In particular, the invention relates to an anti-pepsinogen II antibody, a preparation method and application thereof.

Background

Pepsinogen (pepsinogen) is synthesized by the main cells of acid-secreting glands, and is converted into pepsin in the gastric cavity by the action of hydrochloric acid (HCl) or pepsin (pepsin) with activity, so that protein is decomposed into fat, peptone and a small amount of polypeptide. The optimal pH for the enzyme action is 2, and after entering the small intestine, the enzyme activity is lost.

Pepsinogen is a precursor of pepsin, which is divided into 2 subgroups according to its biochemical properties and immunogenicity, and the 1-5 components have the same immunogenicity, called pepsinogen I (PGI), which is mainly secreted by the main cells of the gastric basal gland and the mucous neck cells; components 6 and 7 are called pepsinogen II (PGII) which is produced by the gastric cardia gland and the mucous neck cells of the pylorus gland of the antrum and the upper duodenal segment in addition to secretion by the main cells of the fundus gland and mucous neck cells.

The pepsinogen levels reflect the morphology and function of gastric mucosa at different sites: PGI is a pointer to detect gastric acid secretion gland cell function, increased gastric acid secretion PGI, decreased secretion or decreased gastric mucosal gland atrophy PGI; PGII has a greater correlation with basal mucosal lesions (relative to antral mucosa), and its elevation is associated with basal gastric tube atrophy, gastric epithelial metaplasia or pseudo-pyloric metaplasia, abnormal proliferation; progressive decrease in PGI/II ratio is associated with progression of gastric mucosal atrophy. Thus, the combined determination of PGI and PGII ratios may serve as a "serological biopsy" of the gastric basal mucosa.

The serum concentrations of PGI and PGII are changed to different degrees in each stomach disease, but in the domestic research on PGII antibodies, the quantity of monoclonal antibodies is small, and the suitability of the antibodies for detecting the concentration of PGII in human serum is poor.

Disclosure of Invention

The object of the present invention is to provide a high affinity and high specificity monoclonal antibody against human pepsinogen II, which is a murine antibody with excellent properties, capable of specifically recognizing and binding to pepsinogen II in human serum.

In one aspect, the invention provides an anti-pepsinogen II monoclonal antibody comprising: antibody 1A7 and/or antibody 3G10;

wherein the amino acid sequence of antibody 1A7 is: VH CDR1 is shown as SEQ ID NO. 2; VH CDR2 is shown in SEQ ID NO. 3; VH CDR3 is shown in SEQ ID NO. 4; VL CDR1 is shown in SEQ ID NO. 5; VL CDR2 is shown in SEQ ID NO. 6; VL CDR3 is shown in SEQ ID NO. 7;

the amino acid sequence of antibody 3G10 is: VH CDR1 is shown in SEQ ID NO. 10; VH CDR2 is shown in SEQ ID NO. 11; VH CDR3 is shown in SEQ ID NO. 12; VL CDR1 is shown in SEQ ID NO. 13; VL CDR2 is shown in SEQ ID NO. 14; VL CDR3 is shown in SEQ ID NO. 15.

Further, the variable region amino acid sequence of antibody 1A7 is: the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 8; the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 9.

Further, the variable region amino acid sequence of antibody 3G10 is: the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 16; the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 17.

In a preferred embodiment, the 3 CDRs contained in the heavy chain variable region and/or the 3 CDRs contained in the light chain variable region are defined by the Chothia numbering system.

Further, the monoclonal antibody is a murine antibody, chimeric antibody, humanized antibody, bispecific antibody or multispecific antibody; the antigen binding fragment is selected from the group consisting of Fab, fab ', (Fab') 2, fv, disulfide-linked Fv, scFv, diabody, or single domain antibody (sdAb).

Further, the monoclonal antibody further comprises: a heavy chain constant region (CH) of a mammalian immunoglobulin or variant 1 thereof, said variant 1 having a substitution, deletion or addition of one or more amino acids compared to the sequence from which it is derived; and, a light chain constant region (CL) of a mammalian immunoglobulin or variant 2 thereof, said variant 2 having a conservative substitution of up to 20 amino acids compared to the sequence from which it is derived.

In the present invention, a monoclonal antibody may include variant 3, which differs from the antibody from which it is derived only by conservative substitutions of one or more amino acid residues; for example, conservative substitutions of up to 20, up to 15, up to 10, or up to 5 amino acids; or at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the antibody from which it is derived, and substantially retains the above-described biological function of the antibody from which it is derived.

Further, the heavy chain constant region of the monoclonal antibody is an IgG heavy chain constant region; the light chain constant region of a monoclonal antibody is a kappa light chain constant region.

Further, the monoclonal antibody is labeled.

The antibody labeling aims to link a label to an antibody, specifically react with an object to be detected to form a multi-component compound, and directly microscopic observation or automatic measurement of test results are carried out by means of a fluorescent microscope, a ray measuring instrument, an enzyme-labeled detector, an electron microscope, a light-emitting immunoassay instrument and other precise instruments. The label in the scheme does not particularly refer to a certain labeling mode, and specifically, the label can be an enzyme label, a biotinylation label, a fluorescent label, a colloidal gold label and the like; and are not limited herein.

In another aspect, the present invention also provides a method for preparing the pepsinogen II-resistant antibody, comprising: culturing the host cell under conditions that allow expression of the monoclonal antibody; monoclonal antibodies are recovered from the cultured host cell culture.

The monoclonal antibodies provided herein can be prepared by various methods known in the art, such as by genetic engineering recombinant techniques. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the invention are obtained by chemical synthesis or PCR amplification. The resulting DNA molecule is inserted into an expression vector and then the host cell is transfected. The transfected host cells are then cultured under specific conditions and express the antibodies of the invention.

The antigen binding fragments of the invention may be obtained by hydrolysis of the intact antibody molecule. Alternatively, these antigen binding fragments may be produced directly by the recombinant host cell. For example, fab' fragments can be obtained directly from the host cell; fab 'fragments can be chemically coupled to form F (ab') 2 fragments. Alternatively, fv, fab or F (ab') 2 fragments may be isolated directly from recombinant host cell culture broth. In general, one of ordinary skill in the art is aware of other techniques for preparing these antigen binding fragments.

In another aspect, the invention provides an isolated nucleic acid molecule for encoding a monoclonal antibody.

In another aspect, the invention also provides a vector comprising the isolated nucleic acid molecule described above.

Further, the vector is a cloning vector or an expression vector. In a preferred embodiment, the vector of the invention is, for example, a plasmid, a cosmid, a phage, or the like. In a preferred embodiment, the vector is capable of expressing the antibody in vivo in a subject, e.g., a mammal.

In another aspect, the invention also provides a host cell comprising an isolated nucleic acid molecule or vector. Such host cells include, but are not limited to: prokaryotic cells such as E.coli cells, eukaryotic cells such as yeast cells, insect cells, plant cells, animal cells such as mammalian cells, and the like. In a preferred embodiment, the host cell of the invention is a mammalian cell, such as CHO, including in particular CHO-K1, CHO-S and CHO DG44.

On the other hand, the invention also provides a kit for detecting pepsinogen II, which comprises the monoclonal antibody.

In one embodiment, the kit comprises a first mab and a second mab; the first monoclonal antibody and the second monoclonal antibody are matched with a detection sample. Wherein the first mab comprises: VH CDR1 shown in SEQ ID No.2, VH CDR2 shown in SEQ ID No.3, VH CDR3 shown in SEQ ID No. 4; and VL CDR1 shown in SEQ ID NO.5, VL CDR2 shown in SEQ ID NO.6, and VL CDR3 shown in SEQ ID NO. 7. The second mab comprises: VH CDR1 shown in SEQ ID No.10, VH CDR2 shown in SEQ ID No.11, VH CDR3 shown in SEQ ID No. 12; and VL CDR1 shown in SEQ ID No.13, VL CDR2 shown in SEQ ID No.14, VL CDR3 shown in SEQ ID No. 15.

In one embodiment, the kit comprises a first mab or a second mab. The primary monoclonal antibody and the secondary monoclonal antibody can also be used independently for detecting pepsinogen I.

In another aspect, the invention also provides a method for using the kit, which comprises the following steps: diluting a tested sample by using a reagent R1, and adding a reagent R2 for reaction; then using a biochemical analyzer to determine the absorbance at 570 nm; and preparing a standard curve by using the pepsinogen I standard substance, and calculating the content of pepsinogen I in the sample to be measured according to the standard curve and the light absorption value.

Wherein, the reagent R1 is a common buffer solution, which can be 0.05mM Tris buffer solution with pH of 7.4, and the preparation method is as follows: taking 6.057g of Tris; adding 800mL of deionized water, and adding concentrated hydrochloric acid to adjust the pH value to 7.4; deionized water was set to a volume of 1L.

The reagent R2 is prepared by mixing a sensitization source 1 formed by coupling the first monoclonal antibody with latex particles and a sensitization source 2 formed by coupling the second monoclonal antibody with the latex particles.

In another aspect, the invention also provides a method for using the kit, which comprises the following steps: diluting a tested sample by using a reagent R1, and adding a reagent R2 for reaction; then using a biochemical analyzer to determine the absorbance at 570 nm; and preparing a standard curve by using the pepsinogen II standard substance, and calculating the content of pepsinogen II in the sample to be measured according to the standard curve and the absorbance value.

Possible principles of the kit of the invention include: when the corresponding antigen exists in the sample, the sensitization source 1 formed by coupling the first monoclonal antibody and the latex particles and the sensitization source 2 formed by coupling the second monoclonal antibody and the latex particles can be combined with the antigen at the same time, and aggregation reaction occurs. The individual sensitizers are within the wavelength of the incident light and light can pass through. When two sensitizers are concentrated, the transmitted light is reduced, which is proportional to the concentration of the sensitizers and also to the amount of antigen. Qualitative and quantitative analysis of pepsinogen II was thus performed.

The first monoclonal antibody and the second monoclonal antibody used for constructing the kit are respectively aimed at different epitopes of human pepsinogen II and have pairing detection effect.

Antibodies of the invention may be derivatized, e.g., linked to another molecule. In general, derivatization of the antibody does not adversely affect its binding to pepsinogen II. Thus, antibodies of the invention also include such derivatized forms. For example, an antibody of the invention may be functionally linked to one or more other molecular groups, such as another antibody, a detection reagent, a pharmaceutical reagent, and/or a protein or polypeptide capable of mediating binding of the antibody or antigen binding fragment to another molecule. Furthermore, the antibodies of the invention may also be derivatized with chemical groups such as polyethylene glycol (PEG), methyl or ethyl, or glycosyl groups. These groups can be used to improve the biological properties of antibodies, such as increasing serum half-life.

Thus, in a preferred embodiment, the antibodies of the invention are conjugated to latex particles. The antibodies of the invention may be conjugated to a detectable label, such as an enzyme, radionuclide, fluorescent dye, luminescent substance, or biotin. The detectable label according to the present invention may be any substance that is detectable by fluorescence, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. Such labels are well known in the art, examples of which include, but are not limited to, enzymes, radionuclides, fluorescent dyes, luminescent substances, magnetic beads, calorimetric labels such as colloidal gold or colored glass or plastic beads, and biotin for binding to the above-mentioned label-modified avidin. Patents teaching the use of such markers include, but are not limited to, U.S. Pat. nos. 3,817,837;3,850,752;3,939,350;3,996,345;4,277,437;4,275,149; and 4,366,241. The detectable labels described above can be detected by methods known in the art. For example, the radiolabel may be detected using a photographic film or scintillation calculator, and the fluorescent label may be detected using a photodetector to detect the emitted light. Enzyme labels are typically detected by providing a substrate to the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate, and calorimetric labels are detected by simply visualizing the coloured label. In certain embodiments, such labels can be suitable for immunological detection, e.g., enzyme-linked immunoassays, radioimmunoassays, fluorescent immunoassays, chemiluminescent immunoassays, and the like. In certain embodiments, a detectable label as described above may be attached to an antibody of the invention through linkers of different lengths to reduce potential steric hindrance.

Definition of terms

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Moreover, the procedures of cell culture, biochemistry, nucleic acid chemistry, immunology laboratories and the like as used herein are all conventional procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

As used herein, the term "antibody" refers to an immunoglobulin molecule that is typically composed of two pairs of polypeptide chains. Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the isotypes of antibodies are defined as IgM, igD, igG, igA and IgE, respectively. Within the light and heavy chains, the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain also comprises a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH 1, CH2 and CH 3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The constant domains are not directly involved in binding of antibodies to antigens, but exhibit a variety of effector functions, such as may mediate binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q). VH and VL regions can also be subdivided into regions of high variability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FR). Each VH and VL is prepared from the following sequence: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 consist of 3 CDRs and 4 FRs arranged from amino-terminus to carboxy-terminus. The variable regions (VH and VL) of each heavy/light chain pair form antigen binding sites, respectively. The assignment of amino acids to regions or domains can be carried out by Kabat, sequences ofProteins of Immunological Interest (National Institutes ofHealth, bethesda, md. (1987 and 1991)), or Chothia & Lesk (1987) J.mol. Biol.196:901-917; chothia et al (1989) Nature 342:878-883.

As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in an antibody variable region that are responsible for antigen binding. Three CDRs, designated CDR1, CDR2 and CDR3, are contained in each of the variable regions of the heavy and light chains. The exact boundaries of these CDRs may be defined according to various numbering systems known in the art, e.g. as in the Kabat numbering system, chothia numbering system or IMGT numbering system. For a given antibody, one skilled in the art will readily identify the CDRs defined by each numbering system. And, correspondence between different numbering systems is well known to those skilled in the art.

In the present invention, the CDRs contained in the antibodies of the present invention can be determined according to various numbering systems known in the art. In certain embodiments, the CDRs contained by the antibodies of the invention are preferably determined by Kabat, chothia or IMGT numbering system. In certain embodiments, the CDRs contained by the antibodies of the invention are preferably determined by the Kabat numbering system.

As used herein, the term "framework region" or "FR" residues refer to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

The term "antibody" is not limited by any particular method of producing an antibody. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibodies may be of different isotypes, for example, igG, igA1, igA2, igD, igE or IgM antibodies.

As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and/or competes with the full-length antibody for specific binding to an antigen, also referred to as an "antigen-binding portion. Non-limiting examples of antigen binding fragments include Fab, fab ', F (ab') 2, fd, fv, complementarity Determining Region (CDR) fragments, scFv, diabodies, monodomain antibodies, chimeric antibodies, linear antibodies, nanobodies, probody, and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen binding ability to the polypeptide.

As used herein, the term "full length antibody" means an antibody consisting of two "full length heavy chains" and two "full length light chains". Wherein "full length heavy chain" refers to a polypeptide chain consisting of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a Hinge Region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain in the N-to C-terminal direction; and, when the full length antibody is an IgE isotype, optionally further comprises a heavy chain constant region CH4 domain. Preferably, a "full length heavy chain" is a polypeptide chain consisting of VH, CH1, HR, CH2 and CH3 in the N-to C-terminal direction. A "full length light chain" is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the N-to C-terminal direction. The two pairs of full length antibody chains are linked together by a disulfide bond between CL and CH1 and a disulfide bond between HR of the two full length heavy chains. The full length antibodies of the invention may be from a single species, e.g., human; chimeric or humanized antibodies are also possible. The full length antibodies of the invention comprise two antigen binding sites formed by VH and VL pairs, respectively, which specifically recognize/bind the same antigen.

As used herein, the term "Fd" means an antibody fragment consisting of VH and CH1 domains; the term "dAb fragment" means an antibody fragment consisting of a VH domain; the term "Fab fragment" means an antibody fragment consisting of VL, VH, CL and CH1 domains; the term "F (ab') 2 fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; the term "Fab 'fragment" means the fragment obtained after reduction of the disulfide bond joining the two heavy chain fragments of the F (ab') 2 fragment, consisting of one complete light and heavy chain Fd fragment (consisting of VH and CH1 domains).

As used herein, the term "Fv" means an antibody fragment consisting of VL and VH domains of a single arm of an antibody. Fv fragments are generally considered to be the smallest antibody fragment that forms the complete antigen binding site. It is believed that the six CDRs confer antigen binding specificity to the antibody. However, even one variable region (e.g., fd fragment, which contains only three CDRs specific for an antigen) is able to recognize and bind antigen, although its affinity may be lower than the complete binding site.

As used herein, the term "Fc" means an antibody fragment formed by disulfide bonding of the second and third constant regions of a first heavy chain of an antibody with the second and third constant regions of a second heavy chain. The Fc fragment of an antibody has a number of different functions, but does not participate in antigen binding.

As used herein, the term "scFv" refers to a single polypeptide chain comprising VL and VH domains, wherein the VL and VH are connected by a linker. Such scFv molecules may have the general structure: NH 2-VL-linker-VH-COOH or NH 2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS) 4 may be used, but variants thereof may also be used and may be used for other linkers of the invention. In some cases, disulfide bonds may also exist between VH and VL of scFv.

As used herein, the term "diabody" means that its VH and VL domains are expressed on a single polypeptide chain, but uses a linker that is too short to allow pairing between two domains of the same chain, forcing the domains to pair with complementary domains of the other chain and creating two antigen binding sites.

As used herein, the term "single domain antibody" has the meaning commonly understood by those skilled in the art and refers to an antibody fragment consisting of a single monomer variable antibody domain that retains the ability to specifically bind to the same antigen to which a full length antibody binds. Single domain antibodies are also known as nanobodies.

Each of the above antibody fragments retains the ability to specifically bind to the same antigen to which the full-length antibody binds and/or competes with the full-length antibody for specific binding to the antigen.

Antigen binding fragments of antibodies can be obtained from a given antibody using conventional techniques known to those skilled in the art, and specifically screened in the same manner as for intact antibodies.

In this context, unless the context clearly indicates otherwise, when referring to the term "antibody" it includes not only whole antibodies, but also antigen-binding fragments of antibodies.

As used herein, the terms "monoclonal antibody," "mAb," and "mAb" have the same meaning and are used interchangeably to refer to an antibody or a fragment of an antibody from a population of highly homologous antibody molecules, i.e., a population of identical antibody molecules except for natural mutations that may occur spontaneously. Monoclonal antibodies have a high specificity for a single epitope on an antigen. Polyclonal antibodies are relative to monoclonal antibodies, which typically comprise at least 2 or more different antibodies, which typically recognize different epitopes on an antigen. Furthermore, the modifier "monoclonal" merely indicates the character of the antibody as being obtained from a population of highly homologous antibodies, and is not to be construed as requiring preparation of the antibody by any particular method.

The monoclonal antibodies of the invention may be prepared by a variety of techniques, such as hybridoma techniques, recombinant DNA techniques, or phage antibody library techniques.

Antibodies can be purified by well-known techniques, such as affinity chromatography using protein a or protein G. Subsequently or alternatively, the specific antigen or epitope thereof may be immobilized on a column and the immunospecific antibody purified by immunoaffinity chromatography.

As used herein, the term "chimeric antibody" refers to an antibody in which a portion of the light chain or/and heavy chain is derived from one antibody and another portion of the light chain or/and heavy chain is derived from another antibody, but which retains binding activity to the antigen of interest in any event. For example, the term "chimeric antibody" may include antibodies (e.g., human murine chimeric antibodies) in which the heavy and light chain variable regions of the antibody are from a first antibody and the heavy and light chain variable regions of the antibody are from a second antibody.

As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence is modified to increase homology with the sequence of a human antibody. Typically, all or part of the CDR regions of a humanized antibody are derived from a non-human antibody and all or part of the non-CDR regions are derived from a human immunoglobulin. Humanized antibodies generally retain the desired properties of the donor antibody, including, but not limited to, antigen specificity, affinity, reactivity, and the like. The donor antibody may be a mouse, rat, rabbit or non-human primate antibody having the desired properties.

The chimeric or humanized antibody of the present invention can be prepared according to the sequence of the murine monoclonal antibody prepared as described above. DNA encoding the heavy and light chains can be obtained from a murine hybridoma of interest and engineered to contain non-murine immunoglobulin sequences using standard molecular biology techniques.

To prepare chimeric antibodies, the murine immunoglobulin variable region can be linked to a human immunoglobulin constant region using methods known in the art. For example, the DNA encoding VH is operably linked to another DNA molecule encoding a heavy chain constant region to obtain a full length heavy chain gene. The sequences of human heavy chain constant region genes are known in the art and DNA fragments comprising these regions can be obtained by standard PCR amplification. The heavy chain constant region may be an IgG1, igG2, igG3, igG4, igA, igE, igM or IgD constant region, but is generally preferably an IgG1 or IgG4 constant region. For example, DNA encoding VL is operably linked to another DNA molecule encoding a light chain constant region CL to obtain a full length light chain gene (as well as Fab light chain gene). The sequences of human light chain constant region genes are known in the art and DNA fragments comprising these regions can be obtained by standard PCR amplification. The light chain constant region may be a kappa or lambda constant region, but is generally preferred.

To prepare humanized antibodies, murine CDR regions can be inserted into a human framework sequence using methods known in the art. Alternatively, transgenic animals can also be utilized that are capable of producing no endogenous immunoglobulins upon immunization and are capable of producing a fully human antibody repertoire. For example, it has been reported that homozygous deletion of the antibody heavy chain Junction (JH) gene in chimeric and germ-line mutant mice can completely suppress endogenous antibody production, and then transferring an array of human germ-line immunoglobulin genes into the germ-line mutant mice will result in the mice producing human antibodies upon antigen stimulation. Non-limiting examples of such transgenic animals include, huMAb mice containing human immunoglobulin gene miniloci encoding unrearranged human heavy chain (μ and γ) and kappa light chain immunoglobulin sequences, plus targeted mutations that inactivate endogenous μ and kappa chain loci; or "KM mouse TM" carrying both human heavy chain transgenes and human light chain transchromosomes. Other methods of antibody humanization include phage display techniques.

As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen against which it is directed. The strength or affinity of a specific binding interaction can be represented by the equilibrium dissociation constant (KD) of the interaction. In the present invention, the term "KD" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and antigen. In certain embodiments, an antibody that specifically binds to (or has specificity for) an antigen means that the antibody binds to or binds to an antigen in an amount of less than about 10 ^-9 M, e.g. less than about 10 ^-9 M、10 ^-10 M、10 ^-11 M or 10 ^-12 M or less affinity (KD) binds the antigen. Specific binding properties between two molecules can be determined using methods well known in the art, for example, using Surface Plasmon Resonance (SPR) in a BIACORE instrument.

As used herein, the term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit, then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matched positions shared by the two sequences divided by the number of positions to be compared x 100. For example, if 6 out of 10 positions of two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions in total are matched). Typically, the comparison is made when two sequences are aligned to produce maximum identity. Such an alignment may be achieved by using, for example, a computer program such as the Align program, conveniently by the method of Needleman et al (1970) J.mol.biol.48:443-453. The percent identity between two amino acid sequences can also be determined using the algorithms of E.Meyers and W.Miller (Comput. Appl biosci.,4:11-17 (1988)) which have been integrated into the ALIGN program (version 2.0), using the PAM120 weight residue table (weight residue table), the gap length penalty of 12 and the gap penalty of 4. Furthermore, percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J mobiol.48:444-453 (1970)) algorithm that has been incorporated into the GAP program of the GCG software package, using the Blossum 62 matrix or PAM250 matrix, and GAP weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6.

As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the desired properties of a protein/polypeptide comprising the amino acid sequence. For example, conservative substitutions may be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions that replace an amino acid residue with an amino acid residue having a similar side chain, e.g., with a residue that is physically or functionally similar to the corresponding amino acid residue. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains, acidic side chains, uncharged polar side chains, nonpolar side chains, beta-branched side chains, and aromatic side chains. Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative substitutions of amino acids are well known in the art.

The twenty conventional amino acids referred to herein are written following conventional usage. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in the present invention, amino acids are generally indicated by single-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

By adopting the technical scheme provided by the invention, the beneficial effects can be achieved: the monoclonal antibody provided by the invention can be specifically combined with PGII protein; the content of PGII protein in human serum can be effectively detected by the double-antibody sandwich immunonephelometry detection method, the sensitivity is high, the minimum is 2 mug/L, the detection linearity is wide, and the requirement of clinical detection can be met.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a linear range plot of PGII of example 2 of the present invention;

FIG. 2 is a graph showing the correlation between PGII detection method of example 2 and a specific brand of alignment reagent/detection method.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The main reagents and instruments of this example are shown in tables 1 and 2, respectively:

table 1: main reagent

Table 2: the main instrument is as follows:

name of the name	Manufacturer' s	Model number
			Liquid transfer device	Eppendorf	/
Constant temperature incubator	Prinsepia and Hiphc	MCO-170AICL-PC
			Biological safety cabinet	JINAN BIOBASE BIOTECH Co.,Ltd.	BSC-1100IIA2-X
Biological microscope	Ningbo Yongxin optical device	N-117M
			Inverted biological microscope	Ningbo Yongxin optical device	XD-202
Desk type centrifugal machine	Lu Xiangyi	TD4
			Plate washer	Shandong Boke	ST-96W
Enzyme label instrument	Primary usTeng (Teng)	ELX800
			Biochemical instrument	HITACHI	7180

Antigen:

recombinant PGII protein from HEK293 cell expression, and the amino acid sequence is shown in SEQ ID NO. 1;

human PGII protein, from human gastric mucosa, available from Lee Biosolutions company under the accession number 440-50.

Experimental animals:

Balb/C mice: balb/C mice, grade SPF (Specific Pathogen Free), were purchased from Shanghai Ling Biotech Inc.

Preparing a buffer solution:

the Phosphate Buffer (PBS) formulation was: naCl,8g; KCl,0.2g; na (Na) ₂ HPO _4· 12H ₂ O，2.9g；KH ₂ PO ₄ 0.2g; H2O constant volume to 1L

The formula of the 10 XPBST lotion is as follows: 80g of NaCl; KCl,2g; na (Na) ₂ HPO ₄ ·12H ₂ O，29g；KH ₂ PO ₄ ，2g；TWEEN-20，5ml；H ₂ Constant volume of O to 1L

The glycine eluent formula at pH 2.7 is: glycine 1.9g, H ₂ O is fixed to 500mL, and the pH value is 2.68-2.72.

The glycine eluent formula at pH 1.9 is: glycine 1.9g, H ₂ O is fixed to 500mL, and the pH value is 1.88-1.92.

Example 1: preparation and screening of PGII monoclonal antibodies

The recombinant PGII protein (1 mg/ml) was mixed with adjuvants CFA and IFA to prepare immunogens. That is, the recombinant protein is first mixed with CFA in a volume ratio of 1:1 to form immunogen A, and then mixed with IFA in a volume ratio of 1:1 to form immunogen B. Immunogen a is primary and immunogen B is secondary, tertiary and fourth booster. Mice were immunized subcutaneously by 2 mice, tail blood was withdrawn on day 14 after the fourth booster immunization, and tail blood antibody titers were assessed using an indirect ELISA method.

Coating an ELISA plate (2 mug/mL) with PGII recombinant protein, adding 100 mug/well, and reacting at 4 ℃ overnight; plates were washed 3 times with PBST solution and blocked with 5% bsa for 2h at 37 ℃; then, after washing the plate 3 times with PBST solution, mouse tail blood diluted with 5% BSA solution was added for reaction at 37℃for 2 hours; then, the plate is washed 3 times by using PBST solution, goat anti-mouse IgG secondary antibody marked by HRP with 1:20000 times dilution is added for reaction for 1h at 37 ℃; washing the plate with PBST solution for 5 times, drying, adding 100 mu L TMB developing solution, and reacting at 37 ℃ in a dark place for 15min; then 50. Mu.L of stop solution (2M H) was added ₂ SO ₄ ) After mixing, the OD450 value was read on an ELISA reader. The results of the indirect ELISA evaluation of mouse tail blood at day 14 after immunization are shown in Table 3.

Table 3: mouse tail blood antibody titer evaluation at day 14 after four PGII immunizations

	No. 1 mouse	No. 2 mouse
			1/10000	2.023	1.954
1/20000	1.707	1.614
			1/40000	1.388	1.323
1/80000	1.104	1.018
			1/160000	0.739	0.66
1/320000	0.494	0.437
			1/640000	0.298	0.268
1/1280000	0.22	0.182
			NC	0.074	0.078

Note that: NC is negative control, PBS.

From the results, the antibody titer of the tail blood of two mice recognizing the PGII recombinant protein exceeds 1:640000 after four times of immunization, the mice are immunized by impact with the PGII recombinant protein, the spleens of the mice are taken on the 3 rd day after the impact immunization, and the spleen cells and myeloma cells SP2/0-Ag14 are separated for cell fusion. More than 3000 hybridoma cell clusters were grown in 96-well plates after fusion, and cell culture supernatants in 96-well plates were evaluated by the indirect ELISA method described previously to screen monoclonal antibodies capable of secreting recognition proteinsFrom the screening results, 48 positive clones were selected for the confirmation experiment of screening, and the experimental procedures were as follows: the ELISA plate (2. Mu.g/mL) was coated with human PGII protein, 100. Mu.L was added to each well and reacted overnight at 4 ℃; plates were washed 3 times with PBST solution and blocked with 5% bsa for 2h at 37 ℃; then, after washing the plate 3 times with PBST solution, adding cell culture supernatant diluted 50 times with PBS solution, and reacting for 2 hours at 37 ℃; then, the plate is washed 3 times by using PBST solution, goat anti-mouse IgG secondary antibody marked by HRP with 1:20000 times dilution is added for reaction for 1h at 37 ℃; washing the plate with PBST solution for 5 times, drying, adding 100 mu L TMB developing solution, and reacting at 37 ℃ in a dark place for 15min; then 50. Mu.L of stop solution (2M H) was added ₂ SO ₄ ) After mixing, the OD450 values were read on an microplate reader and the results are shown in Table 4.

Table 4: confirmation of Positive clones

Clone number	1A7	1H8	2B9	3C8	3G10	4D11	5C2	5D8	5F10	6E6
											OD450	1.699	1.223	1.656	1.335	1.722	1.364	1.542	1.638	0.965	1.128
Clone number	7H11	9A5	9F10	10B4	10D3	10H1	11C5	11E7	11F6	12D9
											OD450	1.356	1.112	1.369	1.568	1.193	1.452	0.996	1.612	1.865	1.036
Clone number	13E11	14B7	14D2	15G9	16H4	17A7	17D6	18C3	18F9	19C2
											OD450	1.551	1.007	1.834	1.683	0.956	1.529	1.658	1.622	1.267	0.989
Clone number	19F10	19H5	20A6	20C9	20F11	21C6	21G5	22B7	22G1	23C1
											OD450	1.869	0.996	1.566	1.339	1.569	1.883	1.681	1.226	1.153	1.598
Clone number	24D9	24H10	26C9	27D3	27G9	29B7	29F9	30E10	NC	PC
											OD450	1.739	1.653	1.381	1.722	0.994	1.146	1.693	1.787	0.064	1.866

Note that: NC is negative control, PBS; PC is a positive control, and the serum of the No. 1 mouse is diluted by 1/10000.

According to the screening result of the positive clones in Table 4, 25 positive clones are selected to continue the sensitivity detection experiment, and a human PGII protein coated ELISA plate (2 mug/mL) is used, 100 mug is added to each well, and the reaction is carried out at 4 ℃ overnight; plates were washed 3 times with PBST solution and blocked with 5% bsa for 2h at 37 ℃; then, after washing the plate 3 times by using a PBST solution, adding cell culture supernatant which is diluted in a gradient manner by using a PBS solution, and reacting for 2 hours at 37 ℃; then, the plate is washed 3 times by using PBST solution, goat anti-mouse IgG secondary antibody marked by HRP with 1:20000 times dilution is added for reaction for 1h at 37 ℃; washing the plate with PBST solution for 5 times, drying, adding 100 mu L TMB developing solution, and reacting at 37 ℃ in a dark place for 15min; then 50. Mu.L of stop solution (2M H) was added ₂ SO ₄ ) After mixing, the OD450 values were read on an microplate reader and the results are shown in Table 5.

Table 5: sensitivity test results

According to the results of Table 5, a total of 15 strains 1A7,3G10,5D8,11E7,11F6,14D2,15G9,17D6,18C3,19F10,21C6,21G5,24D9,24H10,30E10 were selected for purified antibody preparation and screening.

Example 2: preparation of purified antibodies

1) Ascites preparation

Ascites preparation of 1A7,3G10,5D8,11E7,11F6,14D2,15G9,17D6,18C3,19F10,21C6,21G5,24D9,24H10,30E10 antibody was carried out by about 1X 10 ⁶ The cells were injected into the abdominal cavity of 3 Balb/C mice pre-injected with IFA adjuvant, and after about 10 days, ascites produced by each positive clone was extracted, and then centrifuged at 12000rpm for 15min at 4℃to collect the supernatant for further purification of G protein.

2) Purification of mouse monoclonal antibodies

1mL of the column material coupled with the G protein is added into an empty column, after the column material is washed by PBS solution, 2mL of ascites is diluted by 8mL of PBS and then is put into the column, and then the flowing-through liquid is put into the column again; then, elution was performed with glycine eluent of pH2.7, and one tube (100. Mu.L of a neutralization solution comprising 1M Tris-HCl, 10mM EDTA, 1.5M NaCl, pH8.0-8.38 was previously added) was collected per 1mL of the eluent, and 5 tubes were collected in total; following elution with glycine eluent at pH1.9, one tube (300. Mu.L of neutralization solution added in advance) was collected per 1mL of eluent, and 3 tubes were collected in total; then, the OD280 reading is carried out on each tube of eluent respectively, the eluent with the OD280 more than 0.5 is mixed, the OD280 of the mixed liquid is re-measured after mixing, and the antibody concentration is calculated according to the coefficient of 1.4: antibody concentration = OD280/1.4.

Example 3: screening of purified antibodies

Biotin labeling a part of the murine monoclonal antibody after G protein purification, adding-Bio to the labeled antibody, if the 1A7 antibody after biotin labeling is expressed by 1A7-Bio, carrying out antibody pairing screening by using a sandwich ELSIA method, coating an ELISA plate (2 mug/mL) with unlabeled antibody, adding 100 mug of the labeled antibody into each hole, and reacting overnight at 4 ℃; plates were washed 3 times with PBST solution and blocked with 5% bsa for 1.5h at 37 ℃; after washing the plates 3 times with PBST solution, human PGII protein (2. Mu.g/mL) was added and incubated at 37℃for 1.5h; washing the plate 3 times by using PBST solution, adding biotin-labeled antibody diluted in a gradient way by 5% BSA solution, and reacting for 1.5 hours at 37 ℃; plates were washed 3 times with PBST solution, and HRP-labeled streptavidin was added at 1:500 dilution and reacted at 37℃for 1h; washing the plate with PBST solution for 5 times, drying, adding 100 mu L TMB developing solution, and reacting at 37 ℃ in a dark place for 15min; then 50. Mu.L of stop solution (2M H) was added ₂ SO ₄ ) After mixing, the OD450 value was read on an ELISA reader. The results of the evaluation of the purified antibodies are shown in Table 6.

Table 6: sandwich ELISA method paired antibody screening partial result

Note that: NC is negative control, PBS.

According to the results of table 6, antibody 1A7,3G10,19F10 was selected for latex-enhanced immunoturbidimetry paired screening, and antibody 1A7,3G10,19F10 was coupled to latex particles pairwise in different combinations; preparing the coupled latex particles into a detection reagent R2 for the detection of a subsequent calibrator, wherein the detection result is shown in Table 7;

The detection method comprises the following steps:

the detecting instrument is as follows: full-automatic biochemical analyzer 7180

The analysis method comprises the following steps: two-point endpoint method

The reaction direction is as follows: ascending reaction

The calibration mode is as follows: spline

Measurement wavelength: 570nm

Measuring temperature: 37 DEG C

Calibration material: reagent R1: reagent r2=2 μl:120 μl

Table 7: preparation of PGII detection reagent detection calibrator OD570nm readings by different antibody combination modes

According to the results of Table 7, antibodies 1A7 and 3G10 were selected as starting materials for the PGII-content double antibody sandwich-method turbidimetric immunoassay.

The amino acid sequence of the variable region of the murine monoclonal antibody 1A7 was analyzed by sequencing:

the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 8; the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 9.

Further, the CDR sequences of the antibodies were determined using an on-line analysis of Immunolobin BLAST (IG Blast) (http:// www.ncbi.nlm.nih.gov/giblast /), with the amino acid sequences of: VH CDR1 is shown as SEQ ID NO. 2; VH CDR2 is shown in SEQ ID NO. 3; VH CDR3 is shown in SEQ ID NO. 4; VL CDR1 is shown in SEQ ID NO. 5; VL CDR2 is shown in SEQ ID NO. 6; VL CDR3 is shown in SEQ ID NO. 7.

The amino acid sequence of the variable region of the murine monoclonal antibody 3G10 was determined by sequencing analysis:

The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 16; the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 17.

Further, the CDR sequences of the antibodies were determined using an online analysis of Immunolobin BLAST (IGBlast), the amino acid sequences of which were: VH CDR1 is shown in SEQ ID NO. 10; VH CDR2 is shown in SEQ ID NO. 11; VH CDR3 is shown in SEQ ID NO. 12; VL CDR1 is shown in SEQ ID NO. 13; VL CDR2 is shown in SEQ ID NO. 14; VL CDR3 is shown in SEQ ID NO. 15.

Example 4: double-antibody sandwich immunonephelometry method for detecting PGII protein in sample

The pepsinogen II (PGII) content detection method utilizes pepsinogen II detection reagents R1 and R2 and a calibrator to detect pepsinogen II.

The main components of the detection reagent are as follows:

reagent R1: trimethylolaminomethane buffer

Reagent R2: the sensitization source 1 and the sensitization source 2 formed by coupling 1A7 and latex particles and coupling 3G10 and latex particles are mixed in a certain proportion.

Calibration material: human pepsinogen II.

The detection method comprises the following steps:

the detection instrument used was: full-automatic biochemical analyzer 7180

The analysis method comprises the following steps: two-point endpoint method.

The reaction direction is as follows: lifting reaction;

The calibration mode is as follows: a Spline;

measurement wavelength: 570nm;

measuring temperature: 37 ℃;

sample: reagent R1: reagent r2=2 μl:120 μl;

the method comprises the following steps:

step one, taking 2 mu L of a sample and 120 mu L of a reagent R1, adding the sample and the reagent R1 into a reaction cup, uniformly mixing and incubating for 3min;

step two, adding 120 mu L of reagent R2 into the mixed solution, and uniformly mixing;

step three, respectively reading the first reading point transmission absorbance A1 and the second reading point transmission absorbance A2 at 570nm through a full-automatic biochemical analyzer 7180;

and fifthly, calculating delta A=A2-A1 by software and calculating the concentration of pepsinogen II in the tested sample according to a calibration curve.

Linear range determination of pepsinogen II reagent: preparing high-concentration samples by using PGII calibrator, diluting with normal saline according to conventional proportion, repeatedly measuring each sample 3 times by using PGII reagents R1 and R2 in example 2, calculating the mean value, solving the regression equation, calculating the theoretical value by using the regression equation, and obtaining the result shown in figure 1. The result shows that the pepsinogen II reagent has a linear range of 2.0-70 mug/L, high detection sensitivity and wide linearity, and meets the detection requirements of clinical high sensitivity and wide linearity.

The PGII detection method and the reagent for comparing the brand name of China are subjected to a correlation test, 80 serum samples are measured, the measured values are subjected to correlation analysis, and the result is shown in figure 2, wherein the correlation coefficient R of the PGII detection method and the reagent for comparing the brand name of China is the correlation coefficient R of the PGII detection method and the reagent for comparing the brand name of China ² = 0.9959, which indicates that the detection method of the present invention has good correlation with a detection method of a certain known brand comparison reagent in China.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

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Claims

1. A pepsinogen II-resistant monoclonal antibody is characterized in that the monoclonal antibody is

Antibody 1A7, or

A mixture of antibody 1A7 and antibody 3G 10;

wherein the amino acid sequence of the antibody 1A7 is: VH CDR1 is shown as SEQ ID NO. 2; VH CDR2 is shown in SEQ ID NO. 3; VH CDR3 is shown in SEQ ID NO. 4; VL CDR1 is shown in SEQ ID NO. 5; VL CDR2 is shown in SEQ ID NO. 6; VL CDR3 is shown in SEQ ID NO. 7;

The amino acid sequence of the antibody 3G10 is as follows: VH CDR1 is shown in SEQ ID NO. 10; VH CDR2 is shown in SEQ ID NO. 11; VH CDR3 is shown in SEQ ID NO. 12; VL CDR1 is shown in SEQ ID NO. 13; VL CDR2 is shown in SEQ ID NO. 14; VL CDR3 is shown in SEQ ID NO. 15.

2. The monoclonal antibody according to claim 1, wherein the monoclonal antibody,

the amino acid sequence of the variable region of the antibody 1A7 is as follows: the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 8; the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 9.

3. The monoclonal antibody of claim 1, wherein the monoclonal antibody is a mixture of antibody 1A7 and antibody 3G 10;

the amino acid sequence of the variable region of the antibody 3G10 is as follows: the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 16; the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 17.

4. The monoclonal antibody of claim 1, wherein the heavy chain constant region of the monoclonal antibody is an IgG heavy chain constant region; the light chain constant region of the monoclonal antibody is a kappa light chain constant region.

5. The monoclonal antibody of claim 1, wherein the monoclonal antibody is labeled.

6. An isolated nucleic acid molecule for encoding the preparation of a monoclonal antibody according to any one of claims 1 to 5.

7. A vector comprising the isolated nucleic acid molecule of claim 6.

8. The vector of claim 7, wherein the vector is a cloning vector or an expression vector.

9. A method of producing a monoclonal antibody according to any one of claims 1-5, comprising:

culturing a host cell comprising the vector of claim 7 under conditions permitting expression of the monoclonal antibody;

recovering the monoclonal antibody from the cultured host cell culture.

10. Kit for the detection of pepsinogen II, characterized in that it comprises a monoclonal antibody according to any one of claims 1 to 5.