Anti-p16 protein antibodies, methods of making and using the same

Abstract

The application relates to the technical field of biological detection, in particular to an anti-p16 protein antibody and a preparation method and application thereof. The anti-p16 protein antibody has CDRs shown in SEQ ID NO. 2-SEQ ID NO. 7 or has CDRs shown in SEQ ID NO. 8-SEQ ID NO. 13. The application provides a new anti-p16 protein antibody which can be used for development of an in vitro diagnosis kit and applied to quantitative detection of human p16 protein in a clinical sample.

Description

Technical Field

[0001] This application relates to the field of biodetection technology, and in particular to an anti-p16 protein antibody, its preparation method, and its application. Background Technology

[0002] The p16 protein, also known as p16INK4A, MST1, and CDKN2, is located on human chromosome 9P21. It is an inhibitor of cyclin-dependent kinases and a product of tumor suppressor genes. Most tumors undergo abnormal gene alterations during their occurrence and development, including activation of oncogenes, inactivation of tumor suppressor genes, abnormal expression of growth factors, and abnormalities in related apoptosis proteins. These alterations lead to abnormal expression of downstream genes, causing abnormal cell proliferation and differentiation, ultimately resulting in tumor formation. The p16 protein is highly expressed in high-grade cervical intraepithelial neoplasia and tumors infected with high-risk HPV.

[0003] Detection of p16 protein in cervical lesion tissue is helpful for the early diagnosis of cervical cancer and can serve as an important indicator for predicting cervical cancer prognosis. Therefore, adding p16 protein detection in cells or tissues to existing cervical lesion screening methods can improve the detection rate or accuracy of precancerous lesions and cervical cancer, playing a role in preventing precancerous lesions, avoiding overdiagnosis and treatment of cervical cancer, and reducing the colposcopy referral rate. Therefore, the development of new p16 protein antibodies plays a crucial role in clinical diagnosis.

[0004] In view of the above, this application is hereby submitted. Summary of the Invention

[0005] This application provides one or more embodiments of an anti-p16 protein antibody, its preparation method, and its application. The technical solutions include the following:

[0006] One or more embodiments of this application provide an anti-p16 protein antibody, wherein the anti-p16 protein antibody has one of an antigen-binding domain a and an antigen-binding domain b;

[0007] in,

[0008] Antigen-binding domain a includes the following CDRs:

[0009] VL-CDR1: The amino acid sequence is shown in SEQ ID NO.2.

[0010] VL-CDR2: The amino acid sequence is shown in SEQ ID NO.3.

[0011] VL-CDR3: The amino acid sequence is shown in SEQ ID NO.4.

[0012] VH-CDR1: The amino acid sequence is shown in SEQ ID NO.5.

[0013] VH-CDR2: The amino acid sequence is shown in SEQ ID NO.6, and,

[0014] VH-CDR3: The amino acid sequence is shown in SEQ ID NO.7;

[0015] Antigen-binding domain b includes the following CDRs:

[0016] VL-CDR1: The amino acid sequence is shown in SEQ ID NO.8.

[0017] VL-CDR2: The amino acid sequence is shown in SEQ ID NO.9.

[0018] VL-CDR3: The amino acid sequence is shown in SEQ ID NO.10.

[0019] VH-CDR1: The amino acid sequence is shown in SEQ ID NO.11.

[0020] VH-CDR2: The amino acid sequence is shown in SEQ ID NO.12, and,

[0021] VH-CDR3: The amino acid sequence is shown in SEQ ID NO.13.

[0022] In some embodiments of this application, the amino acid sequence of the light chain variable region of the anti-p16 protein antibody is shown in SEQ ID NO.14.

[0023] In some embodiments of this application, the amino acid sequence of the heavy chain variable region of the anti-p16 protein antibody is as shown in SEQ ID NO.15.

[0024] In some embodiments of this application, the amino acid sequence of the light chain variable region of the anti-p16 protein antibody is shown in SEQ ID NO.16.

[0025] In some embodiments of this application, the amino acid sequence of the heavy chain variable region of the anti-p16 protein antibody is as shown in SEQ ID NO.17.

[0026] In some embodiments of this application, the sequence of the constant region of the anti-p16 protein antibody is selected from the sequence of any one of the constant regions of IgG, IgA, IgM, IgE and IgD.

[0027] In some embodiments of this application, the species source of the constant region of the anti-p16 protein antibody is rat, cow, horse, pig, sheep, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, or human.

[0028] One or more embodiments of this application also provide a nucleic acid that encodes the anti-p16 protein antibody.

[0029] One or more embodiments of this application also provide a vector comprising the aforementioned nucleic acid.

[0030] One or more embodiments of this application also provide a cell, said cell comprising the nucleic acid or the vector described herein.

[0031] One or more embodiments of this application also provide a method for producing the anti-p16 protein antibody, wherein the method uses the host cell to produce the anti-p16 protein antibody.

[0032] One or more embodiments of this application also provide a p16 protein detection kit, the p16 protein detection kit comprising the aforementioned anti-p16 protein antibody.

[0033] In some embodiments of this application, the p16 protein detection kit includes an anti-p16 protein antibody 1 having the antigen-binding domain a and an anti-p16 protein antibody 2 having the antigen-binding domain b.

[0034] In some embodiments of this application, the test kit includes a test strip, the test strip including a base plate and a sample pad, a marking pad, a detection membrane and an absorption pad sequentially disposed on the base plate, the detection membrane having a detection line;

[0035] Of the labeling pad and the detection line, one is coated with the anti-p16 protein antibody 1, and the other is coated with the anti-p16 protein antibody 2.

[0036] In some embodiments of this application, the label pad is coated with the anti-p16 protein antibody 2, and the detection line is coated with the anti-p16 protein antibody 1.

[0037] One or more embodiments of this application also provide a method for producing the p16 protein detection kit, the method comprising: preparing the p16 protein detection kit using the anti-p16 protein antibody.

[0038] In some embodiments of this application, the production method described above is used to produce anti-p16 protein antibodies, and the anti-p16 protein antibodies are prepared into p16 protein detection kits.

[0039] One or more embodiments of this application also provide a method for detecting p16 protein in a sample, the method comprising using the anti-p16 protein antibody or the p16 protein detection kit described above to detect p16 protein in the sample.

[0040] In some embodiments of this application, the sample is a cell sample or a tissue sample.

[0041] Details of one or more embodiments of this application are set forth in the following description, and other features, objects, and advantages of this application will become apparent from the specification and its claims. Attached Figure Description

[0042] 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.

[0043] Figure 1 Map of the pcDNA3.4 expression vector;

[0044] Figure 2 The results of SDS-PAGE of p16 antigen;

[0045] Figure 3 Diagram of antibody light chain expression vector;

[0046] Figure 4 Diagram of antibody heavy chain expression vector;

[0047] Figure 5 The kinetic curve for the affinity detection of p16-Ab01;

[0048] Figure 6 The kinetic curve for the affinity test of p16-Ab02. Detailed Implementation

[0049] 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.

[0050] 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.

[0051] the term

[0052] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0053] 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 using 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").

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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℃.

[0064] 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.

[0065] 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.

[0066] Currently, p16 protein ELISA kits can detect p16 protein in tissues or cells. When used to detect tissues from patients clinically diagnosed with cervical cancer, some tissues can be identified by the ELISA results, but others cannot, thus failing to serve as an early screening tool. This indicates that the specificity and sensitivity of the antibody pairs in the kit need to be improved.

[0067] A first aspect of the present application provides an anti-p16 protein antibody, wherein the anti-p16 protein antibody has one of an antigen-binding domain a and an antigen-binding domain b.

[0068] in,

[0069] Antigen-binding domain a includes the following CDRs:

[0070] VL-CDR1: The amino acid sequence is shown in SEQ ID NO.2.

[0071] VL-CDR2: The amino acid sequence is shown in SEQ ID NO.3.

[0072] VL-CDR3: The amino acid sequence is shown in SEQ ID NO.4.

[0073] VH-CDR1: The amino acid sequence is shown in SEQ ID NO.5.

[0074] VH-CDR2: The amino acid sequence is shown in SEQ ID NO.6, and,

[0075] VH-CDR3: The amino acid sequence is shown in SEQ ID NO.7;

[0076] Antigen-binding domain b includes the following CDRs:

[0077] VL-CDR1: The amino acid sequence is shown in SEQ ID NO.8.

[0078] VL-CDR2: The amino acid sequence is shown in SEQ ID NO.9.

[0079] VL-CDR3: The amino acid sequence is shown in SEQ ID NO.10.

[0080] VH-CDR1: The amino acid sequence is shown in SEQ ID NO.11.

[0081] VH-CDR2: The amino acid sequence is shown in SEQ ID NO.12, and,

[0082] VH-CDR3: The amino acid sequence is shown in SEQ ID NO.13.

[0083] This application provides a novel antibody against p16 protein, which can be used in the development of in vitro diagnostic kits for the quantitative detection of human p16 protein in clinical samples.

[0084] The anti-p16 protein antibody of this application may have the aforementioned CDRs, or derivative fragments having the aforementioned CDRs. The derivative fragments are formed by replacing amino acids at no more than six sites relative to their corresponding CDRs (“conservative modification” or “conservative substitution”), retaining the biological activity consistent with their corresponding complementarity-determining regions. For example, the derivative fragments 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 their corresponding complementarity-determining regions.

[0085] 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.

[0086] Table A

[0087] The initial residues Representative substitution Preferred replacement Ala(A) Val; Leu; Ile Val Arg(R) Lys;Gln;Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg;Gln;Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

[0088] "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.

[0089] In this application, the anti-p16 protein antibody may also be an antigen-binding fragment of an antibody or a small modular immunodrug. Further, it may be a monoclonal antibody, an F(ab')2 fragment, a Fab' fragment, a Fab fragment, an Fv fragment, a scFv fragment, a biclonal antibody, a multispecific antibody, a microantibody, a chelated recombinant antibody, an internal antibody, a nanobody, a domain-binding immunoglobulin fusion protein, or a small modular immunodrug.

[0090] 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.

[0091] 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.

[0092] The antibodies containing the above-mentioned CDRs can be murine antibodies (such as p16-Ab01 and p16-Ab02 in the embodiments of this application), chimeric antibodies, or humanized antibodies.

[0093] In this application, the term "mouse antibody" refers to an antibody against the p16 protein prepared in accordance with the knowledge and skills in the art. Preparation involves using the p16 protein 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 said mouse anti-p16 antibody or its antigen-binding fragment may further include a light chain constant region of a mouse κ, λ chain or its variants, or further include a heavy chain constant region of mouse IgG1, IgG2, IgG3 or its variants.

[0094] 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, which can alleviate the immune response induced by murine antibodies. To establish a chimeric antibody, a hybridoma secreting a murine-specific monoclonal antibody is first created. Then, the variable region gene is cloned from murine hybridoma cells, followed by the cloning of the constant region gene of a human antibody 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. For example, the antibody light chain of the chimeric antibody further includes the light chain constant region of a human κ, λ chain, or a variant thereof. The antibody heavy chain of the chimeric antibody further includes the heavy chain constant region of human IgG1, IgG2, IgG3, IgG4, or a variant thereof, preferably including the heavy chain constant region of human IgG1, IgG2, or IgG4, or using IgG1, IgG2, or IgG4 variants with amino acid mutations (e.g., L234A and / or L235A mutations, and / or S228P mutations).

[0095] 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 germline 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.

[0096] 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).

[0097] 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.

[0098] The terms "human antibody" (HuMAb), "human-derived antibody," "fully human antibody," and "completely human antibody" are used interchangeably. A human antibody can be derived from a genetically modified organism (GMO) that is "engineered" 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 GMO 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.

[0099] 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. The term specifically refers to antibodies containing constant regions in both the light and heavy chains. This disclosure of “antibody” includes “full-length antibodies” and their antigen-binding fragments.

[0100] The full-length antibody of this application includes full-length antibodies formed by linking light chain variable regions with light chain constant regions and heavy chain variable regions with heavy chain constant regions in the light and heavy chain variable region combinations in the table below. Those skilled in the art can select light chain constant regions and heavy chain constant regions from different antibody sources according to actual needs, such as light chain constant regions and heavy chain constant regions derived from human antibodies.

[0101] 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 (e.g., TrKA). 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 an antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and are screened for functionality in the same manner as for intact antibodies. The antigen-binding moiety 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.

[0102] Fab is an antibody fragment with antigen-binding activity obtained by treating IgG antibody molecules with an enzyme of the same activity as papain.

[0103] F(ab')2 is an antibody fragment with antigen-binding activity obtained by digesting IgG with an enzyme of the same activity as pepsin.

[0104] Fab' is an antibody fragment with antigen-binding activity obtained by cleaving the above-mentioned F(ab')2.

[0105] 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.

[0106] 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 disclosure 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.

[0107] A biantibody is an antibody fragment in which scFv or Fab is dimerized, and it is an antibody fragment with bivalent antigen-binding activity. In bivalent antigen-binding activity, the two antigens can be the same or different.

[0108] Bispecific antibodies and multispecific antibodies are antibodies that can bind to two or more antigens or antigenic determinants.

[0109] 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)).

[0110] 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.

[0111] 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.

[0112] 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 facilitates antigen binding. Typically, three CDRs (HCDR1, HCDR2, HCDR3) exist in each heavy chain variable region, and three CDRs (LCDR1, LCDR2, LCDR3) exist in each light chain variable region. 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 ed., Public Health Service, National Institutes of Health, Bethesda, MD), the “Chothia” numbering rule (see Al-Lazikani et al., (1997) JMB 27 3: 927-948), and the Im Muno Gen Ti Cs (IMGT) numbering rule (Leefran). cM.P., Immunologist, 7, 132-136 (1999); Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003), etc. For example, for the classical 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); 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 VL... The amino acid residues in the VH are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). Combining the CDR definitions from Kabat and Chothia, the CDR consists of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) from human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) from 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 region of an antibody can be determined using the IMGT / DomainGap Align procedure.

[0113] The term "epitope" or "antigenic determinant" refers to a site on an antigen that is bound by an immunoglobulin or antibody (e.g., a specific site on a TrKA molecule). 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 in Methods in Molecular Biology, Vol. 66, GEMorris, Ed. (1996).

[0114] The terms "specific binding," "selective binding," "selective binding," and "specific binding" refer to the binding of an antibody to an epitope on a pre-defined antigen. Typically, antibodies bind with an affinity (KD) of approximately less than 10⁻⁸ M, such as approximately less than 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, 10⁻¹² M, or even less.

[0115] The term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction. Typically, the antibodies of this disclosure bind to TrKA or its epitopes with a dissociation equilibrium constant (KD) of less than about 10⁻⁷ M, for example less than about 10⁻⁸ M or 10⁻⁹ M. For example, in this disclosure, the affinity of the antibody for the cell surface antigen is determined using the FACS method to determine the KD value.

[0116] Optionally, the species origin of the heavy chain variable region and the light chain variable region of the antibody in this application is not particularly limited, and each can be independently selected from, but not limited to: rat, human, cow, horse, pig, sheep, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck or goose.

[0117] In one specific example, the amino acid sequence of the light chain variable region of the anti-p16 protein antibody is as shown in SEQ ID NO. 14, or its light chain sequence 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 99.9%) identity with SEQ ID NO. 14. In one specific example, the amino acid sequence of the heavy chain variable region of the anti-p16 protein antibody is as shown in SEQ ID NO. 15, or its heavy chain sequence 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 99.9%) identity with SEQ ID NO. 15. Further, the amino acid sequence of the light chain variable region of the anti-p16 protein antibody is as shown in SEQ ID NO. 14, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO. 15.

[0118] In one specific example, the amino acid sequence of the light chain variable region of the anti-p16 protein antibody is as shown in SEQ ID NO. 16, or its light chain sequence 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 99.9%) identity with SEQ ID NO. 16. In one specific example, the amino acid sequence of the heavy chain variable region of the anti-p16 protein antibody is as shown in SEQ ID NO. 17, or its heavy chain sequence 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 99.9%) identity with SEQ ID NO. 17. Further, the amino acid sequence of the light chain variable region of the anti-p16 protein antibody is as shown in SEQ ID NO. 16, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO. 17.

[0119] "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 frequently used in sequence analysis: BLAST ALGORITHMS: Altschul, SF et al., (1990) J. Mol. Biol. 215: 403-410; Gish, W. et al., (1993) Nature Genet. 3: 266-272; Madden, TL et al., (1996) Meth. Enzymol. 266: 131-141; Altschul, SF 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.

[0120] A second aspect of this application provides a nucleic acid that encodes the anti-p16 protein antibody.

[0121] As used in this application, the term "nucleic acid molecule" refers to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, preferably double-stranded DNA or single-stranded mRNA or modified mRNA. When a nucleic acid is placed in a functional relationship with another nucleic acid sequence, the nucleic acid is "effectively linked." 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.

[0122] 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.

[0123] A second aspect of this application provides a carrier comprising the aforementioned nucleic acid.

[0124] Optionally, the vector is selected from mammalian cell viruses, bacterial plasmids, bacteriophages, yeast plasmids, or combinations thereof.

[0125] 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.

[0126] A third aspect of this application provides a cell, the cell comprising the nucleic acid or the vector described above.

[0127] 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, and 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.

[0128] 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.

[0129] A fourth aspect of this application provides a method for producing the anti-p16 protein antibody, wherein the method uses the host cell to produce the anti-p16 protein antibody.

[0130] The antibodies of this application can be prepared and purified using conventional methods. For example, cDNA sequences encoding the 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 antibodies that specifically bind to the p16 protein. 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 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, e.g., at -70°C, or lyophilized.

[0131] 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 Laboratory Techniques, Chapters 5-8 and 15. The resulting antibodies can be renatured, 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 region. Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) website http: / / imgt.cines.fr by comparing against the IMGT Human Antibody Variable Region Germplasm Database and MOE software, or from the journal *Immunoglobulins*, 2001 ISBN012441351.

[0132] A fifth aspect of this application provides a p16 protein detection kit, the p16 protein detection kit comprising the aforementioned anti-p16 protein antibody.

[0133] The antibody in the detection kit of this application can competitively bind to the p16 protein in the sample to be tested.

[0134] When the term "competition" is used in the context of competing antigen-binding proteins for the same epitope, it refers to competition between antigen-binding proteins, which is determined by an assay in which the antigen-binding protein to be detected (e.g., an antibody or a functional fragment thereof) prevents or inhibits (e.g., reduces) the specific binding of a reference antigen-binding protein (e.g., a ligand or a reference antibody) to a common antigen (e.g., a TrKA antigen or a fragment thereof). Numerous types of competitive binding assays can be used to determine whether one antigen-binding protein competes with another. These assays include: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition assays (see, for example, Stahli et al., 1983, Methods in Enzymology 9: 242-253); solid-phase direct biotin-avidin EIA (see, for example, Kirkland et al., 1986, J. Immunol. 137: 3614-3619), solid-phase direct labeling assays, and solid-phase direct labeling sandwich assays (see, for example, Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); and assays using I-1 2. 5. Solid-phase direct labeling of RIAs (see, for example, Morel et al., 1988, Molec. Immunol. 25: 7-15); solid-phase direct biotin-avidin EIAs (see, for example, Cheung et al., 1990, Virology 176: 546-552); and directly labeled RIAs (Moldenhauer et al., 1990, Scand. J. Immunol. 32: 77-82). Typically, the assay involves using a solid surface or cell to bind purified antigen loaded with either an unlabeled detection antigen-binding protein or a labeled reference antigen-binding protein. Competitive inhibition is measured by measuring the amount of label bound to the solid surface or cell in the presence of the detection antigen-binding protein. Typically, the detection antigen-binding protein is present in excess. Antigen-binding proteins identified by competitive assays (competitive antigen-binding proteins) include: antigen-binding proteins that bind to the same epitope as a reference antigen-binding protein; and antigen-binding proteins that bind to a neighboring epitope sufficiently close to the binding epitope of the reference antigen-binding protein, wherein the two epitopes spatially prevent each other from binding. Further details regarding methods for determining competitive binding are provided in the embodiments herein. Typically, when an excess of a competing antigen-binding protein is present, it will inhibit (e.g., reduce) at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more of the specific binding of the reference antigen-binding protein to the common antigen.In some cases, the binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

[0135] This application relates to methods for immunodetection or determination of target antigens (e.g., p16 protein), reagents for immunodetection or determination of target antigens (e.g., p16 protein), methods for immunodetection or determination of cells expressing target antigens (e.g., p16 protein), and diagnostic agents for diagnosing diseases associated with cells positive for target antigens (e.g., p16 protein), comprising, as active ingredients, antibodies or antibody fragments that specifically recognize the target antigen (e.g., p16 protein) and bind to the amino acid sequence or its three-dimensional structure in the extracellular region.

[0136] In this application, the method for detecting or determining the amount of a target antigen (e.g., p16 protein) can be any known method. For example, it includes immunoassay or assay methods. Immunoassay or assay methods are methods that use labeled antigens or antibodies to detect or determine the amount of antibody or antigen. Examples of immunoassay or assay methods include radiolabeled antibody immunoassays (RIA), enzyme immunoassays (EIA or ELISA), fluorescence immunoassays (FIA), luminescent immunoassays, Western blotting, physicochemical methods, etc.

[0137] To detect cells expressing peptides, known immunoassay methods can be used, with immunoprecipitation, fluorescent cell staining, and immunohistochemical staining being preferred. Alternatively, fluorescent antibody staining using the FMAT8100HTS (Applied Biosystem) can be employed.

[0138] The application does not impose any particular restrictions on the test sample used to detect or determine the target antigen (e.g., p16 protein), as long as it has the potential to contain cells expressing the target antigen (e.g., p16 protein), such as tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid, or culture medium.

[0139] Depending on the required detection method, detection products containing the monoclonal antibody or antibody fragment thereof of this application may also contain reagents for performing antigen-antibody reactions or reagents for detecting the reaction. Reagents for performing antigen-antibody reactions include buffers, salts, etc. Reagents for detection include those commonly used in immunoassay or assay methods, such as labeled second antibodies that recognize the monoclonal antibody, its antibody fragments, or conjugates, and substrates corresponding to the labeled antibodies.

[0140] In one example, the p16 protein detection kit includes anti-p16 protein antibody 1 having the antigen-binding domain a and anti-p16 protein antibody 2 having the antigen-binding domain b. That is, the two antibodies are used in combination.

[0141] In one example, the test kit includes a test strip comprising a base plate and, sequentially disposed on the base plate, a sample pad, a labeling pad, a detection membrane, and an absorbent pad, wherein the detection membrane has a detection line; one of the labeling pad and the detection line is coated with the anti-p16 protein antibody 1, and the other is coated with the anti-p16 protein antibody 2. Further, the labeling pad is coated with the anti-p16 protein antibody 2, and the detection line is coated with the anti-p16 protein antibody 1. The test kit of this application uses a colloidal gold method for detection, where the anti-p16 protein antibody 2 on the labeling pad forms a complex with gold nanoparticles.

[0142] A fifth aspect of this application provides a method for producing the p16 protein detection kit, the method comprising:

[0143] A p16 protein detection kit was prepared using the aforementioned anti-p16 protein antibody;

[0144] Optionally, the anti-p16 protein antibody is produced using the aforementioned production method, and the anti-p16 protein antibody is then prepared into a p16 protein detection kit.

[0145] A sixth aspect of this application provides a method for detecting p16 protein in a sample, the method comprising using the anti-p16 protein antibody or the p16 protein detection kit to detect p16 protein in the sample.

[0146] The detection method described in this application can be for non-diagnostic purposes or for diagnostic purposes, such as diagnosing tumors (cervical cancer, oropharyngeal cancer, esophageal squamous cell carcinoma, colorectal cancer), autoimmune diseases (such as systemic lupus erythematosus), neurodegenerative diseases (such as Alzheimer's disease), or for the purpose of tumor prognosis.

[0147] The definition of the test sample in the detection method refers to the definition of "sample to be tested" in the fourth aspect.

[0148] 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.

[0149] 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.

[0150] p16 protein is normally expressed in normal cervical tissue but overexpressed in the cells of cervical cancer patients. p16 protein can be obtained by lysing cervical cancer tissue or cells, and then detected using a double-antibody sandwich ELISA to screen for and prevent cervical cancer. This invention involves immunizing Balb / c mice with a recombinant p16 antigen, extracting RNA from their spleen tissue, and rapidly obtaining anti-p16 antibodies using phage display technology. Antibody pairs targeting p16 protein are then obtained through a double-antibody sandwich ELISA. The large capacity and diversity of the phage display library allow for screening more antibodies and enables extensive antibody pairing detection, ensuring the sensitivity and specificity of the final usable antibody pairs. The colloidal gold method for detecting cervical cancer tissue samples is simple, provides rapid results, and has low cost, allowing for rapid promotion and use in clinical testing.

[0151] Example 1

[0152] 1. Immunogen preparation

[0153] The amino acid sequence of p16 protein (P42771-2) was obtained from the Uniprot database. The full-length sequence was selected and fused with a 3×flag-tag at the C-terminus to obtain the amino acid sequence of the p16 immunogen (SEQ ID NO.1). This sequence was then synthesized in its entirety by a gene synthesis company and constructed into the pcDNA3.4 expression vector (see [link to gene sequence]). Figure 1 In a study, an expression plasmid containing the target gene p16-3×flag-tag was obtained. This plasmid was chemically transformed into *E. coli* competent cells Top10. Single colonies were picked and inoculated into LB medium, cultured at 37°C and 200 rpm for 16-20 h, and then collected by centrifugation. Recombinant plasmids were prepared using an endotoxin-free plasmid extraction kit to obtain A… 260 / A 280 Plasmid samples with a density of 1.8-2.0 g were transiently transfected into HEK293F mammalian cells using PEI transfection reagent. The cells were then cultured in suspension at 37°C and 120 rpm for 48-72 h to obtain the expression product. After pretreatment, the expression product was subjected to anti-flag affinity chromatography and ion exchange chromatography to obtain the p16 recombinant antigen. The collected eluted samples were dialyzed against PBS buffer to obtain the immunogen, which was then identified. Figure 2 .

[0154] SEQ ID NO.1: Human p16 protein amino acid sequence (156aa), C-terminal fused 3×flag-tag

[0155] MEPAAGSSMEPSADWLATAAARGRVEEVRALLEAGALPNAPNSYGRRPIQVMMMGSARVAELLLLHGAEPNCADPATLTRPVHDAAREGFLDTLVVLHRAGARLDVRDAWGRLPVDLAEELGHRDVARYLRAAAGGTRGSNHARIDAAEGPSDIPD DYKDDDDKGDYKDDDDKIDYKDDDDK .

[0156] 2. Mouse immunization and the acquisition of scFv (single chain antibody fragment).

[0157] Balb / c mice were immunized with the p16 antigen obtained in Section 1 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. 5Immunization 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. Mouse RNA was extracted using a commercially available kit, and the purity, concentration, and integrity of the extracted RNA were confirmed by agarose gel electrophoresis. The variable regions of the light and heavy chains of mice were amplified using specific primers to obtain VL and VH; finally, scFv single-chain antibodies were obtained through overlap. scFv was seamlessly cloned into the pComb3XSS vector, electroporated into TG1 competent cells, and phages were selected using a double-antibody sandwich method (M13 phage displayed VL, T4 phage displayed VH). A specific antibody against the T4 phage capsid protein gp23 was immobilized on an ELISA plate, and a T4 phage library was added to the wells. The plates were incubated at 37°C for 1 hour to immobilize the T4 phages. The ELISA plate was washed to remove unbound phages, and the remaining phages were added to the wells. p16 antigen was incubated at 37°C for 1 hour. M13 phage library was added to the wells of an ELISA plate and incubated at 37°C for 1 hour, allowing the light chain displayed by M13 phage and the heavy chain displayed by T4 phage to co-incubate with the p16 antigen. The ELISA plate was washed to remove unbound phages. A specific antibody against the M13 phage capsid protein pVIII was added to the wells and incubated at 37°C for 1 hour. This process was repeated at least three times to obtain the phage library bound to the p16 antigen. Single clones were picked and cultured after plating to obtain scFv single-chain antibodies. The activity of scFv in the culture supernatant was detected by indirect ELISA, and the results are shown in Table 1. To ensure the screened scFv antibodies specifically targeted the p16 antigen, experimental group 1 (uncoated casein directly blocked) and experimental group 2 (coated p16 antigen followed by casein blocking) were set up.

[0158] Table 1. Indirect ELISA detection of scFv activity ("+" indicates that the OD value is outside the measurement range of the microplate reader)

[0159]

[0160] 3. Preparation of recombinant antibodies

[0161] Forty single-clone bacterial cultures from experimental group 1 (OD value less than 0.2) and experimental group 2 (OD value greater than 2.0) were sent to Sangon Biotech for sequencing. The sequencing results yielded the scFv sequence, including the antibody's light chain variable region and heavy chain variable region. The sequences of the light and heavy chain variable regions were then inserted into a eukaryotic vector containing the mouse light and heavy chain constant regions. Figure 3 and Figure 4 HEK293F expression was transfected into mammalian cells, and the activity of the cell expression supernatant was detected by indirect ELISA as shown in Table 2. Cell expression products with OD values ​​greater than 2.0 were subjected to Protein A affinity chromatography and ion exchange chromatography to obtain intact anti-p16 antibodies.

[0162] Thirty antibodies with OD values ​​greater than 2.0 were paired and screened using a double-antibody sandwich method on p16-positive tissue samples. The optimal antibody pairing combination was obtained as shown in Table 3, combination 2: p16-25 and p16-11. p16-25 was named p16-Ab01, and p16-11 was named p16-Ab02.

[0163] Table 2. ELISA results of cell expression supernatant ("+" indicates that the OD value is outside the measurement range of the microplate reader)

[0164]

[0165]

[0166] Table 3. Results of candidate antibody paired ELISA screening

[0167]

[0168]

[0169] 4. Antibody affinity detection

[0170] The antibody affinity was tested according to the following protocol:

[0171] 4.1 Prepare anti-p16 antibodies p16-Ab01 and p16-Ab02 according to the description in section 3 above;

[0172] 4.2 The prepared p16-Ab01 and p16-Ab02 were chemically coupled and solidified onto the Biacore T200 detection chip;

[0173] 4.3 Using p16 antigen as the analyte, different concentrations of 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.125 nM were used to react with immobilized antibodies.

[0174] 4.4 Following the reaction procedure: capture 30s, binding 30s, dissociation 600s, regeneration Gly 2.25 30s, the binding rate ka, dissociation rate kd, and reaction response value of each antibody were obtained, and the affinity KD of each antibody was calculated (see Table 4). Figure 5 and Figure 6 ).

[0175] Table 4. Antibody Affinity Detection Results

[0176] name ka(1 / Ms) kd(1 / s) KD(M) p16-Ab01 <![CDATA[5.363×10 5 ]]> <![CDATA[2.671×10 -4 ]]> <![CDATA[4.98×10 -10 ]]> p16-Ab02 <![CDATA[6.930×10 5 ]]> <![CDATA[1.453×10 -4 ]]> <![CDATA[2.096×10 -10 ]]>

[0177] Figure 5 and Figure 6In the diagram, the black line represents the kinetic curve fitted by the instrument's software, while the other colored curves, from bottom to top, represent the kinetic curves detected by the machine at concentrations of 3.126 nM, 6.25 nM, 12.5 nM, 25 nM, and 50 nM.

[0178] 5. Detection of p16 protein in clinical cervical tissue samples using colloidal gold method

[0179] 5.1 Preparation of Colloidal Gold Test Strips

[0180] (1) Preparation of NC membranes coated with antibody p16-Ab01

[0181] The coated antibody was prepared into an NC coating solution with a concentration of 1.0 mg / mL using NC membrane coating diluent. The solution was then coated onto the NC membrane that had been attached to a PVC plate using a membrane coating machine. Finally, the membrane was dried in a 50°C drying oven for 24 hours to prepare the detection line T.

[0182] Using the same method, goat anti-mouse IgG antibody was coated onto an NC membrane to prepare control line C.

[0183] (2) Preparation of labeling pad for labeled antibody p16-Ab02

[0184] Measure colloidal gold into a clean beaker, setting the rotation speed to a point where a small vortex appears in the center. Add 0.1 Mk₂CO₃, adjust the pH to the appropriate range using a pH meter, and stir until homogeneous. Then, slowly and uniformly add the labeled antibody to a final concentration of 0.01 mg / mL, stirring for 20 min after the addition is complete. For blocking, add 20 wt% BSA (1% of the colloidal gold volume) and continue stirring for 25 min. Balance the solution and centrifuge. Collect the precipitate (rinse the centrifuge bottle with a small amount of 1× labeling solution), and finally dilute to volume with gold-labeled reconstitution solution to prepare a colloidal gold-labeled antibody complex mixture. Coat a glass fiber membrane with this colloidal gold-labeled antibody complex mixture, and then coat the glass fiber membrane with colloidal gold-labeled anti-goat and anti-mouse IgG antibody using the same method to prepare a labeled pad.

[0185] (3) Assemble the test strips

[0186] Assemble the labeling pad, the prepared sample pad, and the absorbent pad prepared in step (2) onto a PVC board to prepare a test strip.

[0187] 5.2 Processing of tissue samples

[0188] The collected clinically positive tissue samples and normal tissue samples were processed as follows:

[0189] First, weigh the tissue sample. Then, use scissors to cut the tissue into small pieces and add it to a clean glass grinder. Grind for 1 minute, making sure to pre-cool the glass grinder on ice beforehand. The entire grinding process should be performed on ice. Prepare the PBS buffer solution according to the ratio of 1g tissue to 9mL PBS pH 7.4. First, add a certain amount of PBS and grind for 1 minute. Then, add a certain amount of PBS and grind for another minute. Leave a small amount of PBS to rinse the tube wall again. Finally, aspirate the lysate. Centrifuge the lysate at 3000rpm for 20 minutes at 4℃ and take the supernatant for analysis.

[0190] 5.3 Sample Testing

[0191] The colloidal gold method was used to detect positive tissue samples (clinically diagnosed cervical cancer tissue samples) and normal tissue samples. The results of visual observation of positive and negative results are shown in Table 5. The conclusion is that the test results for clinically positive tissue samples are positive, and the test results for clinically normal tissue samples are negative. This indicates that the developed test strip can be used for the detection of p16 protein in clinical tissue samples and provides some basis for clinical diagnosis.

[0192] Table 5. Results of tissue sample analysis using colloidal gold method

[0193] sample Test results Positive tissue sample 1 + Positive tissue sample 2 + 3 positive tissue samples + 4 positive tissue samples + 5 positive tissue samples + 6 positive tissue samples + 7 positive tissue samples + 8 positive tissue samples + 9 positive tissue samples + 10 positive tissue samples + Normal tissue sample 1 - Normal tissue sample 2 - 3 normal tissue samples - 4 normal tissue samples - 5 normal tissue samples - 6 normal tissue samples - 7 normal tissue samples - 8 normal tissue samples - 9 normal tissue samples - 10 normal tissue samples -

[0194] Table 6. CDRs for p16-Ab01 and p16-Ab02 (CDRs defined using the Kabat system)

[0195] p16-Ab01 p16-Ab02 VL-CDR1 RSSQSIVHSNGNTYLE(SEQ ID NO.2) RASQDISNYLN (SEQ ID NO.8) VL-CDR2 KVSNRFS (SEQ ID NO.3) YTSRLHS(SEQ ID NO.9) VL-CDR3 FQGSHVPQT(SEQ ID NO.4) QQGNTLPPTF(SEQ ID NO.10) VH-CDR1 GYTMH (SEQ ID NO.5) NYLIE (SEQ ID NO.11) VH-CDR2 YINPSSGYTDYNQKFKD(SEQ ID NO.6) VINPGSGINNYNEKFKG(SEQ ID NO.12) VH-CDR3 RVYGSLPY (SEQ ID NO.7) FYDRYYAMDY (SEQ ID NO.13)

[0196] The amino acid sequence of the p16-Ab01 light chain variable region (SEQ ID NO.14):

[0197] DVLMTQTPLSLPVSLGDQASISC RSSQSIVHSNGNTYLE WYLQKPGQSPKLLIY KVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC FQGSHVPQT FGGGTNLEIK.

[0198] The sequence of amino acids in the variable region of the P16-Ab01 heavy chain (SEQ ID NO.15):

[0199] QVQLQQSAAELARPGASVKMSCKASGYSFT GYTMH WVKQRPGQGLEWIG YINPSSGYTDYNQKF KD KTTLTADKSSSTAYMQLSSSLTSEDSAVYYCAR RVYGSLPYWGQGTLVTVSA.

[0200] The amino acid sequence of the p16-Ab02 light chain variable region (SEQ ID NO.16):

[0201] DIQMTQTTSSLSASLGDRVTISC RASQDISNYLN WYQQKPDGTVKFLIY YTSRLHS GVPSRFSGSGSGTDYSLTISNLEQEDIATYFC QQGNTLPPTF GGGTKLEIK.

[0202] The sequence of amino acids in the variable region of the P16-Ab02 heavy chain (SEQ ID NO.17):

[0203] QVQLQQSGAELVRPGTSVKVSCKASGYAFT NYLIE WMYQRPGQGLEWIG VINPGSGINNYNEKFKG KATLTADKSSSTAYMQLSSSLTSDDSAVYFCAR FYDRYYAMDY WGQGTSVTVSS.

[0204] Overall, the embodiments of this application utilize phage display technology to obtain antibodies against the p16 protein, which shortens the development cycle to 3 months (including immunization time) compared to traditional hybridoma technology; the library capacity is large, and a large number of single-chain antibody sequences can be obtained through panning and identification. The antibodies provided in the embodiments of this application can rapidly obtain results on whether p16 protein is overexpressed by detecting clinical tissue samples using the colloidal gold method.

[0205] 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.

[0206] 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. An anti-p16 protein antibody, characterized in that, The anti-p16 protein antibody has one of antigen binding domain a and antigen binding domain b; wherein, The antigen binding domain a comprises the following CDRs: VL-CDR1: the amino acid sequence is shown as SEQ ID NO. 2, VL-CDR2: the amino acid sequence is shown as SEQ ID NO. 3, VL-CDR3: the amino acid sequence is shown as SEQ ID NO. 4, VH-CDR1: the amino acid sequence is shown as SEQ ID NO. 5, VH-CDR2: the amino acid sequence is shown as SEQ ID NO. 6, and VH-CDR3: the amino acid sequence is shown as SEQ ID NO. 7; The antigen binding domain b comprises the following CDRs: VL-CDR1: the amino acid sequence is shown as SEQ ID NO. 8, VL-CDR2: the amino acid sequence is shown as SEQ ID NO. 9, VL-CDR3: the amino acid sequence is shown as SEQ ID NO. 10, VH-CDR1: the amino acid sequence is shown as SEQ ID NO. 11, VH-CDR2: the amino acid sequence is shown as SEQ ID NO. 12, and VH-CDR3: the amino acid sequence is shown as SEQ ID NO.

13.

2. The anti-p16 protein antibody according to claim 1, characterized by The amino acid sequence of the light chain variable region of the anti-p16 protein antibody is shown as SEQ ID NO. 14, or / and the amino acid sequence of the heavy chain variable region of the anti-p16 protein antibody is shown as SEQ ID NO.

15.

3. The anti-p16 protein antibody according to claim 1, characterized by The amino acid sequence of the light chain variable region of the anti-p16 protein antibody is shown as SEQ ID NO. 16, or / and the amino acid sequence of the heavy chain variable region of the anti-p16 protein antibody is shown as SEQ ID NO.

17.

4. The anti-p16 protein antibody according to any one of claims 1 to 3, characterized in that, The sequence of the constant region of the anti-p16 protein antibody is selected from the sequence of any one of the constant regions of IgG, IgA, IgM, IgE and IgD.

5. The anti-p16 protein antibody according to any one of claims 1 to 3, characterized in that, The species origin of the constant region of the anti-p16 protein antibody is mouse, bovine, horse, pig, sheep, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose or human.

6. A nucleic acid, characterized in that, The nucleic acid encodes the anti-p16 protein antibody of any one of claims 1 to 5.

7. A vector, characterized in that, The vector comprises the nucleic acid of claim 6.

8. A cell, comprising: The cell comprises the nucleic acid of claim 6 or the vector of claim 7; The cell is a microorganism or an animal cell.

9. A method for producing the anti-p16 protein antibody according to any one of claims 1 to 5, characterized by, The production method comprises: producing the anti-p16 protein antibody by using the cell of claim 8.

10. A p16 protein detection kit, characterized by, The p16 protein detection kit comprises the anti-p16 protein antibody of any one of claims 1 to 5.

11. The p16 protein detection kit according to claim 10, characterized by, The p16 protein detection kit comprises the anti-p16 protein antibody 1 with the antigen binding domain a and the anti-p16 protein antibody 2 with the antigen binding domain b.

12. The p16 protein detection kit according to claim 11, characterized by, The p16 protein detection kit comprises a test strip, the test strip comprising a base plate and, sequentially arranged on the base plate, a sample pad, a label pad, a detection membrane and an absorption pad, the detection membrane being provided with a detection line. One of the marker pad and the detection line is coated with the anti-p16 protein antibody 1, and the other is coated with the anti-p16 protein antibody 2.

13. The p16 protein detection kit according to claim 12, characterized by, The marker pad is coated with the anti-p16 protein antibody 2, and the detection line is coated with the anti-p16 protein antibody 1.

14. A method for producing the p16 protein detection kit according to any one of claims 10 to 13, characterized by, The production method comprises: The p16 protein detection kit is prepared by using the anti-p16 protein antibody according to any one of claims 1 to 5.

15. The method for producing the p16 protein detection kit according to claim 14, characterized in that, The anti-p16 protein antibody is produced by using the production method according to claim 9, and the p16 protein detection kit is prepared by using the anti-p16 protein antibody.

16. A method of detecting p16 protein in a sample, comprising: The detection method comprises: The p16 protein in the sample is detected by using the anti-p16 protein antibody according to any one of claims 1 to 5 or the p16 protein detection kit according to any one of claims 10 to 13. The detection method is not directly aimed at obtaining a disease diagnosis result or a health condition.

17. The method of claim 16, wherein the sample is a tissue sample. The sample is a cell sample or a tissue sample.

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