CP-IgM antibody, preparation method and application thereof, nucleic acid molecule, carrier, cell, quality control product, kit and detection method of CP-IgM antibody

Abstract

The invention relates to the technical field of biological detection, and relates to a CP-IgM antibody, a preparation method and application thereof, a nucleic acid molecule, a carrier, a cell, a quality control product, a kit and a detection method of the CP-IgM antibody. The CP-IgM antibody has CDR-L1 to CDR-L3 of which the amino acid sequences are as shown in SEQ ID NO.2 to SEQ ID NO.4, and CDR-H1 to CDR-H3 of which the amino acid sequences are as shown in SEQ ID NO.6 to SEQ ID NO.8. As a quality control antibody, the CP-IgM can overcome the problems of low signal, too strong matrix effect and the like of the traditional CP-IgM.

Description

Technical Field

[0001] This application relates to the field of biodetection technology, specifically to CP-IgM antibodies and their preparation methods and applications, nucleic acid molecules, vectors, cells, quality control materials, reagent kits, and detection methods for CP-IgM antibodies. Background Technology

[0002] Chlamydia pneumoniae (CP) is an important pathogen causing respiratory diseases in humans, primarily resulting in atypical pneumonia, bronchitis, sinusitis, and other respiratory illnesses. It is transmitted through droplets or respiratory secretions and can spread between families and in crowded places. Due to its unique parasitic and antigenic characteristics, Chlamydia pneumoniae often causes direct or indirect harm to the human body.

[0003] After a person is infected with Chlamydia pneumoniae, specific laboratory tests are often required to detect it. The main detection methods include the detection of Chlamydia pneumoniae pathogen and the detection of antibodies against Chlamydia pneumoniae in human serum or body fluids.

[0004] Pathogen detection methods include pathogen isolation and culture, pathogen-specific antigen detection, and gene detection. Isolation and culture is the "gold standard" for laboratory diagnosis of Chlamydia pneumoniae; however, the culture conditions for Chlamydia pneumoniae are demanding and time-consuming, resulting in low detection sensitivity and limited clinical applicability. Pathogen-specific antigen detection suffers from difficulties in collection and limited sample sizes. While gene detection technologies, such as polymerase chain reaction (PCR), have high specificity and sensitivity, their accuracy is easily affected by experimental conditions and the operator's skill level.

[0005] To quickly diagnose Chlamydia pneumoniae infection and provide doctors with a basis for treatment, clinical practice uses reagent kits to detect the presence of IgM antibodies against Chlamydia pneumoniae in serum for early diagnosis. During the IgM antibody detection process, quality control materials are used to confirm the precision and accuracy of the reagents and instruments to ensure the reliability of the test results.

[0006] Commonly used quality control materials include selectively using appropriately diluted clinical positive serum samples. However, CP-IgM clinical positive samples are scarce and difficult to collect in large quantities, resulting in limited sources and collection volumes, making positive clinical samples expensive. Furthermore, the significant variability between individual serum samples poses a risk of batch-to-batch instability. More importantly, serum samples present potential for biotransfection and are prohibitively expensive.

[0007] Compared to using clinical positive serum samples as quality control, screening suitable CP-IgM antibodies as quality control can solve problems such as source, batch-to-batch variation and cost. However, traditional CP-IgM has problems such as low signal and excessive matrix effect. Summary of the Invention

[0008] Based on this, one or more embodiments of this application provide CP-IgM antibodies and their preparation methods and applications, nucleic acid molecules, vectors, cells, quality control materials, reagent kits, and detection methods for CP-IgM antibodies. These include the following technical solutions:

[0009] One or more embodiments of this application provide a CP-IgM antibody having amino acid sequences as shown in SEQ ID NO.2 to 4, namely CDR-L1 to CDR-L3, and amino acid sequences as shown in SEQ ID NO.7 to 9, namely CDR-H1 to CDR-H3.

[0010] In some embodiments of this application, the species origin of the heavy chain variable region and the light chain variable region backbone region of the CP-IgM antibody is independently mouse, human, cattle, horse, pig, sheep, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck or goose.

[0011] In some embodiments of this application, the amino acid sequence of the light chain variable region of the CP-IgM antibody is as shown in SEQ ID NO.5 or / and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.10.

[0012] In some embodiments of this application, the CP-IgM antibody includes a chimeric antibody;

[0013] Optionally, the species source of the light chain constant region and heavy chain constant region of the CP-IgM antibody includes humans.

[0014] In some embodiments of this application, the amino acid sequence of the light chain of the CP-IgM antibody is as shown in SEQ ID NO. 6 or / and the amino acid sequence of the heavy chain is as shown in SEQ ID NO. 11.

[0015] One or more embodiments of this application provide a CP-IgM antibody, wherein the amino acid sequence of the heavy chain constant region of the CP-IgM antibody is shown as positions 118 to 540 of SEQ ID NO.11.

[0016] One or more embodiments of this application provide a nucleic acid molecule that encodes the CP-IgM antibody.

[0017] One or more embodiments of this application provide a vector comprising the nucleic acid molecule described above.

[0018] One or more embodiments of this application provide a cell that expresses the CP-IgM antibody, the nucleic acid molecule, or the vector.

[0019] One or more embodiments of this application provide a method for constructing the cell described above, the method comprising the step of introducing the nucleic acid molecule or the vector into a target cell.

[0020] One or more embodiments of this application provide a method for preparing the CP-IgM antibody, the method comprising the steps of culturing the cells and isolating the CP-IgM antibody from the resulting culture.

[0021] One or more embodiments of this application provide a quality control product, the quality control product comprising the CP-IgM antibody described above.

[0022] One or more embodiments of this application provide a reagent kit, the reagent kit including the aforementioned quality control material.

[0023] One or more embodiments of this application provide a method for detecting CP-IgM antibodies, wherein the detection method uses the CP-IgM antibody as a quality control antibody, or uses the quality control material, or uses the kit described above during the detection of CP-IgM antibodies in the sample to be tested.

[0024] Compared with traditional technologies, this application has the following advantages: This application provides a CP-IgM antibody, which, as a quality control antibody, can overcome the problems of low signal and excessive matrix effect of traditional CP-IgM. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 The results are for CP antigen SDS-PAGE.

[0027] Figure 2 This is a diagram of the antibody light chain expression vector.

[0028] Figure 3 This is a diagram of the antibody heavy chain expression vector.

[0029] Figure 4 SDS-PAGE of CP-8-IgM-1 (Lane1) and CP-8-IgM-2 (Lane2) antibodies.

[0030] Figure 5For the specific detection of CP-IgM antibody. Detailed Implementation

[0031] The present application will be further described in detail below with reference to the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for descriptive purposes only and is not intended to be limiting of the application.

[0033] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0034] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0035] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0036] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0037] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0038] In this document, terms such as “preferred,” “better,” “more suitable,” and “ideal” are merely used to describe implementation methods or examples that achieve better results, and should be understood not to limit the scope of protection of this application.

[0039] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0040] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0041] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0042] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0043] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0044] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0045] In this application, %(w / w) and wt% both represent weight percentage, %(v / v) refers to volume percentage, and %(w / v) refers to mass-volume percentage.

[0046] All references to this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the purpose and / or technical solution of this application, all cited references are incorporated herein by reference in their entirety and for all purposes. When references are cited in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. Examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0047] Chlamydia pneumoniae (CP) primarily causes atypical pneumonia in humans, but can also lead to bronchitis, pharyngitis, sinusitis, otitis media, iritis, and other diseases. CP-IgM specific antibody testing is one of the more reliable indicators of clinical CP infection. Chlamydia pn. IgM generally begins to rise within 1-2 weeks after infection, peaks at 3-4 weeks, and lasts for 4 months. Therefore, a positive CP-IgM result indicates the acute phase of infection and is a specific indicator for early diagnosis. Furthermore, clinical IgM and IgG combined testing can differentiate between acute and past infections. This application develops a chimeric antibody against CP-IgM. After obtaining the sequence of the murine variable region, it was cloned together with the human IgM constant region into an expression vector. The conventional human IgM constant region has four constant regions: CH1, CH2, CH3, and CH4. The IgM constant region of this application is a tandem combination of human IgM CH1 and three CH3 regions. Because the binding site of the anti-human IgM secondary antibody is located in the CH3 region, the signal of the obtained CP-IgM chimeric antibody is much higher than that of the traditional IgM chimeric antibody. At the same time, it solves the problem of low signal and low sensitivity of the IgM chimeric antibody used as a quality control product in this project.

[0048] In a first aspect, this application provides a CP-IgM antibody having amino acid sequences as shown in SEQ ID NO. 2 to 4, namely CDR-L1 to CDR-L3, and amino acid sequences as shown in SEQ ID NO. 7 to 9, namely CDR-H1 to CDR-H3.

[0049] The antibody of this application may have the aforementioned CDRs, or a derivative fragment having the aforementioned CDRs. The derivative fragment is formed by replacing amino acids at no more than six sites relative to its corresponding CDR (“conservative modification” or “conservative substitution”), retaining the biological activity consistent with its corresponding complementarity-determining region. For example, the derivative fragment may replace one amino acid with another, or one amino acid with multiple amino acids (e.g., two), at sites 1, 2, 3, 4, 5, or 6 of its corresponding complementarity-determining region.

[0050] In the CDRs provided in this application, the derived fragments (conserved variants) refer to polypeptides formed by replacing one, two, or three amino acids with amino acids of similar or related properties compared to the amino acid sequence of the antibody in this application. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0051] Table A

[0052]

[0053] "Conservative modification" or "conservative substitution" refers to the replacement of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation, and rigidity), allowing for frequent alterations without changing the protein's biological activity. Those skilled in the art will recognize that, in general, the substitution of a single amino acid in a non-essential region of a polypeptide does not substantially alter its biological activity (see, for example, Watson et al. (1987), Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th edition)). Furthermore, substitutions of structurally or functionally similar amino acids are unlikely to disrupt biological activity.

[0054] The three-letter and single-letter codes for amino acids used in this application are as described in J.biol.chem, 243, p3558 (1968).

[0055] The "antibody" mentioned in this application refers to immunoglobulins. A complete antibody is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the heavy chain of immunoglobulins differ, thus their antigenicity also differs. Based on this, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, based on differences in the amino acid composition of its hinge region and the number and position of disulfide bonds in its heavy chain, it can be further divided into different subclasses; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chains are classified as κ chains or λ chains based on differences in their constant regions. Each of the five classes of Ig can have either a κ chain or a λ chain.

[0056] The sequence of approximately 110 amino acids near the N-terminus of both the antibody heavy and light chains varies considerably and is known as the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are called the constant region. The variable region includes three hypervariable regions (HVR) and four relatively conserved backbone regions (FR). The three hypervariable regions determine the antibody's specificity and are also called complementarity-determining regions (CDR). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDR regions and four FR regions, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

[0057] The antibody in this application may be a murine antibody, a chimeric antibody, or a humanized antibody.

[0058] In this application, the term "mouse antibody" refers to a CP-IgM monoclonal antibody prepared in accordance with the knowledge and skills in the art. Preparation involves using CP or its epitopes as the antigen injection target, followed by isolation of hybridomas expressing antibodies with the desired sequence or functional characteristics. In some examples, the CPCP-IgM antibody or its antigen-binding fragment may further comprise a light chain constant region of a mouse κ, λ chain or its variants, or further comprise a heavy chain constant region of mouse IgG1, IgG2, IgG3 or its variants.

[0059] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody. It can reduce the immune response induced by murine antibodies. To create a chimeric antibody, a hybridoma that secretes murine-specific monoclonal antibodies must first be established. Then, the variable region gene is cloned from the murine hybridoma cells. Next, the constant region gene of the human antibody is cloned as needed. The murine variable region gene and the human constant region gene are then linked to form a chimeric gene, which is inserted into an expression vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic system.

[0060] The term "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody produced by grafting a non-human species' CDR sequence into the variable region framework of a human antibody, i.e., a human antibody framework sequence of different types. This overcomes the heterologous response induced by chimeric antibodies carrying a large number of heterologous protein components. Such framework sequences can be obtained from public DNA databases containing germline antibody gene sequences or from publicly available references. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available at www.mrccpe.com.ac.uk / vbase) and in Kabat, E.A. et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition. To avoid a decrease in activity along with a decrease in immunogenicity, minimal reverse or reversion mutations can be performed on the aforementioned human antibody variable region framework sequence to maintain activity. The humanized antibodies in this application also include humanized antibodies further derived from yeast exhibiting affinity maturation mutations of CDR.

[0061] In this application, the antibody or its antigen-binding fragment may further include a light chain constant region of human or mouse κ, λ chains or variants thereof, or further include a heavy chain constant region of human or mouse IgG1, IgG2, IgG3, IgG4 or variants thereof; preferably, it includes a heavy chain constant region of human IgG1, IgG2 or IgG4, or uses an IgG1, IgG2 or IgG4 variant with an amino acid mutation (e.g., L234A and / or L235A mutation, and / or S228P mutation).

[0062] The "variants" of the heavy chain constant region and light chain constant region of human antibodies described in this application refer to prior art variants of the heavy chain constant region or light chain constant region derived from humans that do not alter the structure and function of the antibody variable region. Exemplary variants include IgG1, IgG2, IgG3, or IgG4 heavy chain constant region variants that involve site-specific modifications and amino acid substitutions in the heavy chain constant region; specific substitutions include prior art known YTE mutations, L234A and / or L235A mutations, S228P mutations, and / or mutations that obtain a knock-in-hole structure (giving the antibody heavy chain a knock-Fc and hole-Fc combination), which have been shown to give antibodies new properties without altering the function of the antibody variable region.

[0063] The terms "human antibody," "human-derived antibody," "fully human antibody," and "completely human antibody" are used interchangeably. An antibody can be derived from a human being or obtained from a transgenic organism "modified" to produce specific human antibodies in response to antigenic stimulation, and can be produced by any method known in the art. In some techniques, human heavy and light chain locus elements are introduced into cell lines derived from embryonic stem cell lines, where endogenous heavy and light chain loci are targeted and disrupted. The transgenic organism can synthesize human antibodies specific to human antigens, and can be used to produce hybridomas that secrete human antibodies. A human antibody can also be an antibody in which the heavy and light chains are encoded by nucleotide sequences derived from one or more human DNA sources. Completely human antibodies can also be constructed using gene or chromosome transfection methods and phage display technology, or from in vitro activated B cells, all of which are known in the art.

[0064] The terms “full-length antibody,” “intact antibody,” “complete antibody,” and “all antibody” are used interchangeably herein to refer to an antibody in substantially its complete form, as distinguished from the antigen-binding fragment as defined below. This term specifically refers to antibodies containing constant regions in both the light and heavy chains. The term “antibody” in this application includes “full-length antibodies” and their antigen-binding fragments.

[0065] The full-length antibody of this application includes the full-length antibody formed by linking the light chain variable region with the light chain constant region and the heavy chain variable region with the heavy chain constant region in the light and heavy chain variable region combination in the following embodiments. Those skilled in the art can select different antibody sources of light chain constant regions and heavy chain constant regions according to actual needs, such as light chain constant regions and heavy chain constant regions derived from human antibodies.

[0066] The term "antigen-binding fragment" or "functional fragment" of an antibody refers to one or more fragments that retain the ability to specifically bind to an antigen. It has been shown that fragments of full-length antibodies can be used for antigen-binding function. Examples of binding fragments included in the term "antigen-binding fragment" of an antibody include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments connected by disulfide bridges on the hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VH and VL domains on a single arm of the antibody; (v) dsFv, an antigen-binding fragment formed by interchain disulfide bonds between VH and VL; and (vi) bispecific, bispecific, and multispecific antibodies containing fragments such as scFv, dsFv, and Fab. Furthermore, although the two domains VL and VH of the Fv fragment are linked by a synthetic linker, enabling it to produce a single protein chain (referred to as a single-chain Fv (scFv) where the VL and VH regions pair to form a monovalent molecule; see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also included in the term "antigen-binding fragment" of antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and fragments are screened for functionality in the same manner as for intact antibodies. Antigen-binding moieties can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0067] Fab is an antibody fragment with antigen-binding activity obtained by treating IgG antibody molecules with an enzyme of the same activity as papain.

[0068] F(ab')2 is an antibody fragment with antigen-binding activity obtained by digesting IgG with an enzyme of the same activity as pepsin.

[0069] Fab' is an antibody fragment with antigen-binding activity obtained by cleaving the above-mentioned F(ab')2.

[0070] In addition, the Fab' can be produced by inserting DNA encoding the Fab' fragment into an expression vector and then introducing the vector into a host.

[0071] The terms "single-chain antibody," "single-chain Fv," or "scFv" refer to molecules that contain a variable domain (or region; VH) of the antibody heavy chain and a variable domain (or region; VL) of the antibody light chain linked by a linker. Such scFv molecules can have a generic structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Other connectors that may be used in this application are described in the following literature, for example, but not limited to: Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Alfthan et al. (1995), Protein Eng. 8:725-731; Choi et al. (2001), Eur. J. Immunol. 31:94-106; Hu et al. (1996), Cancer Res. 56:3055-3061; Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol.

[0072] dsFv is obtained by linking polypeptides in which one amino acid residue in each VH and VL is replaced by a cysteine ​​residue via disulfide bonds between cysteine ​​residues. The amino acid residues to be replaced by cysteine ​​residues can be selected based on the prediction of the antibody's three-dimensional structure using known methods (e.g., Protein Engineering, 7, 697 (1994)).

[0073] The term "amino acid difference" or "amino acid mutation" refers to an alteration or mutation of amino acids in a variant protein or polypeptide compared to the original protein or polypeptide. This includes the insertion, deletion, or substitution of one, two, three, or more amino acids in the original protein or polypeptide.

[0074] The term "antibody framework" or "FR region" refers to a portion of the variable domain VL or VH that serves as a scaffold for the antigen-binding loop (CDR) of that variable domain. Essentially, it is a variable domain without a CDR.

[0075] The terms "complementarity-determining region," "CDR," or "hypervariant region" refer to one of the six hypervariable regions within the variable domain of an antibody that primarily facilitate antigen binding. Typically, each heavy chain variable region contains three CDRs (HCDR1, HCDR2, HCDR3), and each light chain variable region contains three CDRs (LCDR1, LCDR2, LCDR3). The amino acid sequence boundaries of CDRs can be determined using any of a variety of well-known schemes, including the “Kabat” numbering rule (see Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), the “Chothia” numbering rule (see B Al-Lazikani et al., JMol Biol. 1997 Nov 7;273(4):927-48.), and the ImMunoGeneTics (IMGT) numbering rule (Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, M.P. et al., Dev Comp Immunol. 2003 Jan;27(1):55-77., etc.). For example, in the classic format, following Kabat rules, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Following Chothia rules, the CDR amino acids in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). By combining the CDR definitions of Kabat and Chothia, the CDR is composed of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL.Following the IMGT rules, the CDR amino acid residues in VH are approximately numbered 26-35 (CDR1), 51-57 (CDR2), and 93-102 (CDR3), while those in VL are approximately numbered 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3). Following the IMGT rules, the CDR regions of antibodies can be determined using the IMGT / DomainGap Align procedure.

[0076] The term "epitope" or "antigenic determinant" refers to a site on an antigen that is bound by immunoglobulins or antibodies. Epitopes typically consist of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a distinctive spatial conformation. See, for example, Epitope Mapping Protocols: Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996).

[0077] The terms "specific binding," "selective binding," "selective binding," and "specific binding" refer to the binding of an antibody to a pre-defined epitope on an antigen. Typically, antibodies bind at a concentration of approximately less than 10... -8 M, for example, approximately less than 10 -9 M, 10 - 10 M, 10 -11 M, 10 -12 M or lower affinity (KD) binding.

[0078] The term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction. Typically, the antibodies in this application have a dissociation equilibrium constant of less than approximately 10. -7 M, for example, less than approximately 10 -8 M or 10 -9 The dissociation equilibrium constant (KD) of M binds to the antigen or its epitope. For example, in this application, the affinity of the antibody for the cell surface antigen is determined by the FACS method to determine the KD value.

[0079] In some embodiments of this application, the species origin of the heavy chain variable region and the light chain variable region backbone region of the CP-IgM antibody is independently mouse, human, cattle, horse, pig, sheep, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck or goose.

[0080] In some embodiments of this application, the amino acid sequence of the light chain variable region of the CP-IgM antibody is as shown in SEQ ID NO.5, or its light chain variable region has at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with this sequence.

[0081] In some embodiments of this application, the amino acid sequence of the heavy chain variable region of the CP-IgM antibody is as shown in SEQ ID NO.10, or its heavy chain variable region has at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with this sequence.

[0082] In some embodiments of this application, the CP-IgM antibody includes a chimeric antibody;

[0083] Optionally, the species source of the light chain constant region and heavy chain constant region of the CP-IgM antibody includes humans.

[0084] In some embodiments of this application, the amino acid sequence of the light chain of the CP-IgM antibody is as shown in SEQ ID NO. 6, or its light chain has at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with this sequence.

[0085] In some embodiments of this application, the amino acid sequence of the heavy chain of the CP-IgM antibody is as shown in SEQ ID NO. 11, or its heavy chain has at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with this sequence.

[0086] A second aspect of this application provides a CP-IgM antibody, wherein the amino acid sequence of the heavy chain constant region of the CP-IgM antibody is shown as positions 118 to 540 of SEQ ID NO.11, and has a sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with the sequence.

[0087] "Identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. When positions in two compared sequences are occupied by the same base or amino acid monomer subunit—for example, if every position in two DNA molecules is occupied by adenine—then the molecules are homologous at that position. The percentage of identity between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared multiplied by 100. For example, in optimal sequence alignment, if 6 out of 10 positions in two sequences match or are homologous, then the two sequences are 60% homologous; if 95 out of 100 positions in two sequences match or are homologous, then the two sequences are 95% homologous. Typically, comparisons are made when aligning two sequences to give the maximum percentage of identity. For example, comparisons can be performed using the BLAST algorithm, where the algorithm's parameters are chosen to give the maximum match between the sequences over the entire length of each reference sequence. The following references relate to the BLAST algorithm commonly used in sequence analysis: BLAST ALGORITHMS: Altschul, S.F. et al., (1990) J. Mol. Biol. 215:403-410; Gish, W. et al., (1993) Nature Genet. 3:266-272; Madden, T.L. et al., (1996) Meth. Enzymol. 266:131-141; Altschul, S.F. et al., (1997) Nucleic Acids Res. 25:3389-3402; Zhang, J. et al., (1997) Genome Res. 7:649-656. Other common BLAST algorithms, such as those provided by NCBI BLAST, are also well-known to those skilled in the art.

[0088] A third aspect of this application provides a nucleic acid molecule that encodes the CP-IgM antibody.

[0089] As used herein, the term "nucleic acid molecule" refers to DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, preferably double-stranded DNA or single-stranded mRNA or modified mRNA. Nucleic acids are "effectively linked" when placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to said coding sequence.

[0090] The nucleic acid molecules in this application primarily refer to isolated nucleic acid molecules. "Isolated" means that the molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cell debris and growth media. Generally, the term "isolated" is not intended to mean the complete absence of these materials or the absence of water, buffers, or salts, unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the compounds described herein.

[0091] A fourth aspect of this application provides a carrier comprising the aforementioned nucleic acid molecule.

[0092] The term "vector," also known as "expression vector," refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one embodiment, the vector is a "plasmid," which refers to a circular double-stranded DNA loop to which an additional DNA segment can be linked. In another embodiment, the vector is a viral vector, in which an additional DNA segment can be linked to a viral genome. The vectors disclosed herein are capable of autonomous replication in host cells upon introduction (e.g., bacterial vectors with bacterial origins of replication and augmented mammalian vectors) or can be integrated into the host cell's genome after introduction, thereby replicating along with the host genome (e.g., non-augmented mammalian vectors). In general, vectors may be selected from, but are not limited to, mammalian cell viruses, bacterial plasmids, bacteriophages, yeast plasmids, or combinations thereof.

[0093] A fifth aspect of this application provides a cell that expresses the CP-IgM antibody, the nucleic acid molecule, or the vector.

[0094] The term "cell," also known as "host cell," refers to a cell into which an expression vector has been introduced. Host cells can include bacteria, microorganisms, or animal cells. Easily transformable bacteria include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; members of the Bacillaceae family, such as Bacillus subtilis; Pneumococcus; Streptococcus; and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, or HEK293 cells.

[0095] The terms “cell,” “cell line,” and “cell culture” used herein are used interchangeably, and all such names include progeny. Therefore, “transformant” and “transformed cell” include primary test cells and cultures derived from them, regardless of passage number. It should also be understood that, due to intentional or unintentional mutations, all progeny cannot be exactly identical in DNA content. This includes mutant progeny with the same function or biological activity as those screened from the original transformed cells. Where different names are intended, the context will be clear.

[0096] A sixth aspect of this application provides a method for constructing the cell described above, the method comprising the step of introducing the nucleic acid molecule or the vector into a target cell.

[0097] One or more embodiments of this application provide a method for preparing the CP-IgM antibody, the method comprising the steps of culturing the cells and isolating the CP-IgM antibody from the resulting culture.

[0098] The antibody or antigen-binding fragments of this application can be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors. Recombinant immunoglobulin expression vectors can stably transfect host cells. As a more preferred prior art, mammalian expression systems lead to glycosylation of the antibody, particularly at the highly conserved N-terminal site in the Fc region. Stable clones are obtained by expressing specific binding proteins, and positive clones are scaled up in a bioreactor to produce antibodies. The culture medium secreting the antibody can be purified using conventional techniques, such as using an A or G Sepharose FF column with adjusted buffer. Non-specifically bound components are washed away. The bound antibody is then eluted using a pH gradient, and the antibody fragments are detected by SDS-PAGE and collected. The antibody can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieving or ion exchange. The resulting product should be immediately frozen, such as at -70°C, or lyophilized.

[0099] Methods for producing and purifying antibodies and antigen-binding fragments are well-known in the prior art, such as those described in Cold Spring Harbor's Guide to Antibody Experimentation, Chapters 5-8 and 15. For example, mice can be immunized with human antigens or fragments thereof, and the resulting antibodies can be refolded, purified, and sequenced using conventional methods. Antigen-binding fragments can also be prepared using conventional methods. The antibodies or antigen-binding fragments described in this invention utilize genetic engineering methods to add one or more human FR regions to a non-human CDR. Human FR germline sequences can be obtained by comparing the IMGT Human Antibody Variable Region Germplasm Database and MOE software, from the ImMunoGeneTics (IMGT) website http: / / imgt.cines.fr, or from the journal Immunoglobulins, 2001 ISBN012441351.

[0100] A seventh aspect of this application provides a quality control product, the quality control product comprising the CP-IgM antibody.

[0101] An eighth aspect of this application provides a reagent kit, the reagent kit including the aforementioned quality control material.

[0102] A ninth aspect of this application provides a method for detecting CP-IgM antibodies, wherein the method uses the CP-IgM antibody as a quality control antibody, or uses the quality control material, or uses the kit described above during the detection of CP-IgM antibodies in a sample to be tested.

[0103] The detection method described in this application can be for diagnostic purposes or for non-diagnostic purposes.

[0104] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0105] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0106] 1. Preparation of CP antigen

[0107] The specific antigen of Chlamydia pneumoniae is its major outer membrane protein (MOMP), which is also the main target site of human specific humoral immune responses. The amino acid sequence of the CP antigen (MOMP sequence number: P27455) was obtained from the Uniprot database. Based on this sequence, a gene synthesis company was commissioned to synthesize the nucleic acid encoding the target antigen. During synthesis, the signal peptide sequence (aa1~23) was removed, and a 10×His-tag, EcoRI, and Sal I restriction sites were fused to the C-terminus. The resulting plasmid was then ligated into the pET-28a(+) vector (Novagen) to obtain an expression plasmid containing the target gene CP-10×His-tag.

[0108] The plasmid was transformed into Escherichia coli BL21(DE3) competent cells, plated on LB agar plates containing kanamycin (50 μg / mL), and cultured at 37°C for 16 h. After that, clones were picked and inoculated into LB medium. When the OD600 reached about 0.8, a final concentration of 1 mM IPTG was added, and expression was induced at 16°C for 20 h. After centrifugation, the bacterial cells were collected, sonicated, and purified by nickel column affinity chromatography and ion exchange chromatography.

[0109] SDS-PAGE Figure 1 The constructed CP antigen (SEQ ID NO.1) has a molecular weight of approximately 40 kDa (consistent with the theoretical value) and a purity >95%. The activity of the CP antigen was verified by an indirect ELISA method. The CP antigen was coated and then CP clinical positive serum samples (gradual dilution) and secondary antibody (anti-human IgM-HRP) were added. The OD values ​​showed a gradient (Table 1), indicating that the CP antigen can be used for immunization in mice.

[0110]

[0111] 2. Mouse immunization and screening of single-chain antibodies (scFv)

[0112] Bal B / c mice were immunized with the obtained CP antigen for 8-12 weeks. Tail blood was detected by indirect ELISA using Freund's adjuvant-antigen emulsification and subcutaneous immunization until the tail blood titer reached 10. 5 Immunization was stopped at the designated time. Three days prior to RNA extraction from mice, a booster immunization was administered via intraperitoneal injection of the immunogen. After obtaining mouse spleens, lymphocytes were isolated, and RNA was extracted using an RNA extraction kit. Agarose gel electrophoresis was used to confirm the purity, concentration, and integrity of the extracted RNA. The mouse light chain variable region and heavy chain variable region were amplified using specific primers to obtain VL and VH; finally, scFv was obtained through overlap. The scFv was seamlessly cloned into the pComb3XSS vector and electroporated into TG1 competent cells to obtain a mouse immunoglobulin library (library size ≥ 5 × 10⁻⁶). 8 CFU (insertion rate > 90%). Magnetic beads coated with CP antigen were used for liquid chromatography washing, followed by at least three rounds of washing with helper phage VCSM13 to obtain a phage library specifically binding to the CP antigen. Single clones were picked and cultured after plating, and the supernatant after centrifugation with helper phage was the phage-displayed scFv. The activity of scFv in the culture supernatant was detected by indirect ELISA to ensure that the selected scFv specifically targeted the CP antigen. Experimental group 1 (uncoated casein blocking) and experimental group 2 (CP antigen coated followed by casein blocking) were set up.

[0113] The results are shown in Table 2: 26 positive clones that reacted with the CP antigen and had an OD value higher than 2.5 were selected (OD values ​​of uncoated clones were less than 0.3). Sequencing confirmed 18 positive scFv sequences, with antibody numbers as follows: CP-2, CP-4, CP-8, CP-11, CP-14, CP-17, CP-19, CP-21, CP-22, CP-25, CP-27, CP-28, CP-32, CP-36, CP-39, CP-41, CP-44, and CP-46.

[0114]

[0115] 3. Preparation of CP-IgM antibody

[0116] The sequences of the 18 light and heavy chain variable regions obtained above were then synthesized by a gene synthesis company. The specific procedures are as follows:

[0117] ① The variable region of the mouse light chain in 18 scFvs is inserted into an expression vector containing the constant region of the human Kappa chain ( Figure 2 );

[0118] ② The variable regions of the mouse heavy chain in 18 scFvs were inserted into an expression vector containing the constant region of the human IgM chain. Figure 3 );

[0119] ③ The mouse heavy chain variable region + human IgM-CH1-CH3×3 base sequence from 18 scFvs was synthesized and inserted into the pCDNA3.4 expression vector.

[0120] After obtaining the light and heavy chain plasmids, mammalian 293F cells were co-transfected with PEI according to the ①+② combination (light chain mass ratio 2:1; control group) and the ①+③ combination (light chain mass ratio 2:1; experimental group). After 48 h of culture in a CO2 shaker at 37℃ and 120 rpm, the cells were fed with feed. After another 48 h, the expression products were collected. The activity of the expression supernatant was tested by the indirect ELISA method. The active expression supernatant was filtered through a 0.45 μm filter membrane and purified by Cytiva Capto L affinity chromatography to obtain the CP-IgM recombinant antibody.

[0121] Results: The supernatants of 12 recombinant antibody groups were found to be active using the indirect ELISA method (secondary antibody: anti-human IgM-HRP) (details are shown in Table 3). The OD values ​​of the experimental groups were all higher than those of the control group. The expression supernatants of the CP-8-IgM-1 control group and the CP-8-IgM-2 experimental group, which had the highest OD values, were selected for purification and gel electrophoresis. Figure 4 As shown.

[0122]

[0123] 4. CP-IgM antibody specificity detection

[0124] An indirect ELISA method was used for detection. The CP antigen, natural Mycoplasma pneumoniae (MP) protein, Mycoplasma hominis P120 protein, and human serum albumin (HSA) prepared in section 1 were coated onto 96-well plates (2 μg / mL, pH 9.6 carbonate buffer), with CP antigen as a positive control. CP-8-IgM-1 and CP-8-IgM-2 diluted 100-fold were added, and the plates were incubated at 37°C for 1 h. HRP-labeled anti-human IgM secondary antibody (TMB substrate) was then used for color development, and the A450 and A630 values ​​were read.

[0125] ELISA testing (see) Figure 5 Results: The CP-IgM antibody showed high specificity for both recombinant CP antigen and natural CP antigen.

[0126] 5. Application of CP-IgM antibody as a quality control material

[0127] The two antibodies were mixed in a serum matrix (human serum matrix lacking IgM, with endogenous IgM removed by Protein L affinity chromatography). Simultaneously, trehalose (0.5 wt%), mannitol (1 wt%), polysorbate 80 (0.1 wt%), and ProClin 300 (0.1 wt%) were added to a final concentration. The pH was adjusted to 7.4 ± 0.2. After aliquoting, a three-step lyophilization process was performed: ① The aliquoted quality control sample (0.5 mL / vial) was placed on the freeze dryer shelf; ① Pre-freezing at -40℃ for 2 hours to form uniform ice crystals; ② Drying at -20℃ for 10 hours (vacuum maintained at 0.1 mbar) to remove free water; ③ Stepwise heating to 25℃ for 6 hours (vacuum maintained at 0.01 mbar) to adsorb bound water, with a final residual moisture content ≤ 1.5%. After lyophilization, the final product was a white cake-like substance. After reconstitution (using ddH2O as the reconstitution solvent), the solution was clear and transparent. The reconstitution recovery rate was 98.2±2.1% (n=5), and the pH value remained stable at 7.4±0.2, which meets the requirements for clinical testing.

[0128] Two IgM quality control samples were adjusted to the same concentration (antibody concentration 1 mg / mL) and serially diluted simultaneously. The samples were then detected using the YHLO Chlamydia pneumoniae IgM antibody detection kit (chemiluminescence method) (catalog number C88014M). The results (see Table 4) showed that the signal of the CP-8-IgM-2 antibody was much higher than that of the CP-8-IgM-1 antibody. This reduces the amount of antibody used in the preparation of the quality control samples, ensuring a stable and weak matrix effect while improving the stability of the quality control samples.

[0129]

[0130] The sequences involved in the embodiments of this application are as follows:

[0131] >SEQ ID NO.1 (CP antigen amino acid sequence (aa24-389), C-terminal fused 10×His-tag):

[0132] MLPVGNPSDPSLLIDGTIWEGAAGDPCDPCATWCDAISLRAGFYGDYVFDRILKVDAPKTFSMGAKPTGSAAANYTTAVDRPNPAYNKHLHDAEWFTNAGFIALNIWDRFDVFCTLGASNGYIRGNSTAFNLVGLFGVKGTTVNANELPNVSLSNGVVELYTDTSFSWSVGARGALWECGCATLGAEFQYAQSKPKVEELNVICNVSQFSVNKPKGYKGVAFPLPTDAGVATATGTKSATINYHEWQVGASLSYRLNSLVPYIGVQWSRATFDADNIRIAQPKLPTAVLNLTAWNPSLLGNATALSTTDSFSDFMQIVSCQINKFKSRKACGVTVGATLVDADKWSLTAEARLINERAAHVSGQFRFHHHHHHHHHH*

[0133] >SEQ ID NO.2(CDR-L1): TASSSLSSSYLH*

[0134] >SEQ ID NO.3(CDR-L2): STSNLAS*

[0135] >SEQ ID NO.4(CDR-L3): HQYRRSGT*

[0136] >SEQ ID NO.5(light chain variable region):

[0137] QIVLTQSPAIMSASLGERVTMTCTASSSLSSSYLHWYQQKPGSSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISTMEAEDAATYYCHQYRRSGTFGGGTKLEIK*

[0138] >SEQ ID NO.6(light chain of CP-8-IgM-2 antibody):

[0139] QIVLTQSPAIMSASLGERVTMTCTASSSLSSSYLHWYQQKPGSSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISTMEAEDAATYYCHQYRRSGTFGGGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC *

[0140] >SEQ ID NO.7(CDR-H1): RHGIH*

[0141] >SEQ ID NO.8 (CDR-H2): VIWRGGSTDFNAAFMS*

[0142] >SEQ ID NO.9 (CDR-H3): NSDYYVLDY*

[0143] >SEQ ID NO.10 (Heavy chain variable region):

[0144] QVLLKQSGPGLVQPSQSLSITCTVSGFSLGRHGIHWIRQSPGKGLEWLGVIWRGGSTDFNAAFMSRLSITKDNSKGQVFFNMNSLQADDTAIYYCAKNSDYYVLDYWGQGTSVTVSS*

[0145] >SEQ ID NO.11 (Heavy chain):

[0146] QVLLKQSGPGLVQPSQSLSITCTVSGFSLGRHGIHWIRQSPGKGLEWLGVIWRGGSTDFNAAFMSRLSITKDNSKGQVFFNMNSLQADDTAIYYCAKNSDYYVLDYWGQGTSVTVSSGSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKG*

[0147] This application utilizes phage display technology to obtain antibodies against the CP protein, significantly shortening the development cycle compared to traditional hybridoma technology. It also offers a large library capacity, allowing for the acquisition of a large number of antibodies with high affinity and specificity through liquid chromatography washing and phase ELISA identification. The quality control prepared using the CP-IgM identified in this application can significantly improve the detection signal value. Meeting the quality control development requirements necessitates high-potency antibody raw materials, and minimizing the amount of antibody raw materials added to the serum matrix results in quality control materials that more closely resemble positive serum samples.

[0148] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0149] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A CP-IgM antibody, characterized in that, The CP-IgM antibody has amino acid sequences as shown in SEQ ID NO.2 to 4, namely CDR-L1 to CDR-L3, and amino acid sequences as shown in SEQ ID NO.7 to 9, namely CDR-H1 to CDR-H3.

2. The CP-IgM antibody according to claim 1, characterized in that, The species origin of the heavy chain variable region and the light chain variable region of the CP-IgM antibody are independently of mouse, human, cattle, horse, pig, sheep, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck or goose.

3. The CP-IgM antibody according to claim 2, characterized in that, The amino acid sequence of the light chain variable region of the CP-IgM antibody is shown in SEQ ID NO.5 and / or the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

10.

4. The CP-IgM antibody according to any one of claims 1 to 3, characterized in that, The CP-IgM antibody includes a chimeric antibody; Optionally, the species source of the light chain constant region and heavy chain constant region of the CP-IgM antibody includes humans.

5. The CP-IgM antibody according to claim 4, characterized in that, The amino acid sequence of the light chain of the CP-IgM antibody is shown in SEQ ID NO. 6 and / or the amino acid sequence of the heavy chain is shown in SEQ ID NO.

11.

6. A CP-IgM antibody, characterized in that, The amino acid sequence of the heavy chain constant region of the CP-IgM antibody is shown in positions 118 to 540 of SEQ ID NO.

11.

7. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the CP-IgM antibody as described in any one of claims 1 to 6.

8. A carrier, characterized in that, The carrier comprises the nucleic acid molecule as described in claim 7.

9. A cell, characterized in that, The cells express the CP-IgM antibody as described in any one of claims 1 to 6, including the nucleic acid molecule as described in claim 7 or the vector as described in claim 8.

10. The method for constructing cells according to claim 9, characterized in that, The construction method includes the step of introducing the nucleic acid molecule of claim 7 or the vector of claim 8 into target cells.

11. The method for preparing the CP-IgM antibody according to any one of claims 1 to 6, characterized in that, The preparation method includes the steps of culturing the cells of claim 9 and isolating the CP-IgM antibody from the resulting culture.

12. Quality control product, characterized in that, The quality control material includes the CP-IgM antibody according to any one of claims 1 to 6.

13. A reagent kit, characterized in that, The kit includes the quality control material as described in claim 12.

14. A method for detecting CP-IgM antibodies, characterized in that, The detection method uses the CP-IgM antibody as a quality control antibody in any one of claims 1 to 6, or the quality control product as described in claim 12, or the kit as described in claim 13, in the process of detecting CP-IgM antibody in the sample to be tested.

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