Anti-p16 protein antibody as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological detection, in particular to an anti-p16 protein antibody as well as a preparation method and application thereof. The anti-p16 protein antibody has CDRs as shown in SEQ ID NO. 2 to SEQ ID NO. 7 or CDRs as shown in SEQ ID NO. 8 to SEQ ID NO. 13. The anti-p16 protein antibody disclosed by the invention can be used for preparing the anti-p16 protein antibody. The invention provides a novel anti-p16 protein antibody which can be used for developing an in-vitro diagnostic kit and is applied to quantitative detection of human p16 protein in a clinical sample.

Description

Technical Field

[0001] The present application relates to the field of biological detection technology, and in particular to an anti-p16 protein antibody and a preparation method and application thereof. Background Art

[0002] 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 are accompanied by abnormal gene changes during their occurrence and development, including activation of oncogenes, inactivation of tumor suppressor genes, abnormal expression of growth factors and abnormal apoptosis proteins, which lead to abnormal expression of related downstream genes, abnormal cell proliferation and differentiation, and ultimately tumor formation. p16 protein is highly expressed in high-grade cervical intraepithelial neoplasia and tumors infected with high-risk HPV.

[0003] The detection of p16 protein in cervical lesion tissues is helpful for the early diagnosis of cervical cancer and can be used as one of the important indicators for predicting the prognosis of cervical cancer. Therefore, adding the detection of p16 protein in cells or tissues to the existing cervical lesion screening methods can improve the detection rate or accuracy of cervical precancerous lesions and cervical cancer, and play a certain role in preventing cervical precancerous lesions, avoiding excessive diagnosis and treatment of cervical cancer, and reducing the referral rate of colposcopy. Therefore, the development of new p16 protein antibodies plays an important role in clinical diagnosis.

[0004] In view of this, this application is hereby filed. Summary of the invention

[0005] One or more embodiments of the present application provide an anti-p16 protein antibody and a preparation method and application thereof, including the following technical solutions:

[0006] One or more embodiments of the present 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] The antigen binding domain a includes the following CDRs:

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

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

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

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

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

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

[0015] The antigen binding domain b includes the following CDRs:

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

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

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

[0019] VH-CDR1: amino acid sequence as 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 the present 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 the present 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 the present application, the amino acid sequence of the light chain variable region of the anti-p16 protein antibody is as shown in SEQ ID NO.16.

[0025] In some embodiments of the present 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 the present 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 the present application, the species of the constant region of the anti-p16 protein antibody is mouse, cow, horse, pig, sheep, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose or human.

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

[0029] One or more embodiments of the present application also provide a vector, which includes the nucleic acid.

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

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

[0032] One or more embodiments of the present application further provide a p16 protein detection kit, wherein the p16 protein detection kit comprises the anti-p16 protein antibody.

[0033] In some embodiments of the present application, the p16 protein detection kit comprises 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 the present application, the detection kit includes a detection test strip, the detection test strip includes a bottom plate and a sample pad, a marking pad, a detection membrane and an absorption pad sequentially arranged on the bottom plate, and the detection membrane is provided with a detection line;

[0035] Of the label 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 the present 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 the present application further 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 the present application, the production method is used to produce anti-p16 protein antibodies, and the anti-p16 protein antibodies are prepared into a p16 protein detection kit.

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

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

[0041] The details of one or more embodiments of the present application are set forth in the description which follows, and other features, objects, and advantages of the present application will be apparent from the description and its claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0043] Figure 1 This is the map of the pcDNA3.4 expression vector;

[0044] Figure 2 This is the SDS-PAGE result of p16 antigen;

[0045] Figure 3 This is a map of the antibody light chain expression vector;

[0046] Figure 4 This is a map of the antibody heavy chain expression vector;

[0047] Figure 5 This is the kinetic curve of affinity detection of p16-Ab01;

[0048] Figure 6 This is the kinetic curve of p16-Ab02 affinity detection. DETAILED DESCRIPTION

[0049] The present application will be further described in detail below in conjunction with the accompanying drawings, embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate the present application and are not used to limit the scope of the present application. The purpose of providing these embodiments and examples is to make the understanding of the disclosure of the present application more thorough and comprehensive. It should also be understood that the present application can be implemented in many different forms, is not limited to the embodiments and examples described herein, and those skilled in the art can make various changes or modifications without violating the connotation of the present application, and the equivalent form obtained also falls within the protection scope of the present application. In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the present application, and 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 those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing implementation modes and embodiments and are not intended to limit this application.

[0051] the term

[0052] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0053] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the 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 that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of 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, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0054] In the present application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a number greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0055] As used herein, "combination thereof", "any combination thereof", "any combination thereof" etc. include all suitable combinations of any two or more of the listed items.

[0056] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc., shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0057] Herein, “preferred”, “better”, “more preferred” and “suitable” are merely used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute limitations on the scope of protection of this application.

[0058] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0059] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0060] In the present application, the terms "first", "second", "third", "fourth", etc. in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0061] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0062] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0063] The temperature parameters in this application, unless otherwise specified, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0064] In the present 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 documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited with all contents and all purposes. When the cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the cited documents are involved in this application, the examples and preferred methods of the cited relevant technical features can also be incorporated into this application as references, but are limited to the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0066] At present, the p16 protein ELISA test kit can be used to detect p16 protein in tissues or cells. The tissues of patients clinically diagnosed with cervical cancer were tested using ELISA kits. Some patients' tissues can be identified by ELISA results, but some patients cannot be identified, and it cannot be used for early screening, indicating that the specificity and sensitivity of the antibody pair in the kit need to be improved.

[0067] In a first aspect of the embodiments of the present application, an anti-p16 protein antibody is provided, wherein the anti-p16 protein antibody has one of an antigen binding domain a and an antigen binding domain b;

[0068] in,

[0069] The antigen binding domain a includes the following CDRs:

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

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

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

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

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

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

[0076] The antigen binding domain b includes the following CDRs:

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

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

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

[0080] VH-CDR1: amino acid sequence as 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] The present application provides a novel anti-p16 protein antibody, which can be used for the development of an in vitro diagnostic kit and applied to the quantitative detection of human p16 protein in clinical samples.

[0084] The anti-p16 protein antibody of the present application may have the above-mentioned CDRs, or may be a derivative fragment of the above-mentioned CDRs. The derivative fragment is formed by amino acid substitution ("conservative modification" or "conservative substitution or replacement") at no more than 6 sites relative to its corresponding CDR, and retains the biological activity consistent with its corresponding complementary determining region. For example, the derivative fragment is substituted at 1, 2, 3, 4, 5 or 6 sites relative to its corresponding complementary determining region, and one amino acid may be replaced by another amino acid, or one amino acid may be replaced by multiple (for example, 2) amino acids.

[0085] In the CDRs provided in the present application, the derivative fragment (conservative variant) refers to a polypeptide formed by replacing 1, 2, or 3 amino acids with amino acids of similar or similar properties compared to the amino acid sequence of the antibody of the present application. These conservative variant polypeptides are preferably generated by amino acid substitution according to Table A.

[0086] Table A

[0087] Initial residue Representative replacement Preferred substitutions 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 or replacement" 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, etc.), so that changes can be made frequently without changing the biological activity of the protein. Those skilled in the art know that, in general, single amino acid replacements in non-essential regions of a polypeptide do not substantially change the biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th edition)). In addition, replacement of amino acids with similar structure or function is unlikely to destroy biological activity.

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

[0090] The "antibody" mentioned in this application refers to immunoglobulin. A complete antibody is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and arrangement order of the constant region of the immunoglobulin heavy chain are different, so its antigenicity is also different. Based on this, immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are μ chain, δ chain, γ chain, α chain, and ε chain respectively. The same type of Ig can be divided into different subclasses according to the difference in the amino acid composition of its hinge region and the number and position of the heavy chain disulfide bonds, such as IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chain is divided into κ chain or λ chain by the difference in the constant region. Each of the five types of Ig can have κ chain or λ chain.

[0091] The sequences of about 110 amino acids near the N-terminus of the antibody heavy and light chains vary greatly, which is the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable, which is the constant region. The variable region includes three hypervariable regions (HVRs) and four relatively conservative framework regions (FRs). The three hypervariable regions determine the specificity of the antibody, also known as the complementarity determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDR regions and four FR regions, arranged in the 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 antibody having the above CDRs may be a murine antibody (such as p16-Ab01 and p16-Ab02 in the examples of the present application), a chimeric antibody, or a humanized antibody.

[0093] The term "mouse antibody" in this application refers to an antibody against p16 protein prepared according to the knowledge and skills in the art. During the preparation, the p16 protein or its epitope is used as an antigen to inject the test subject, and then the hybridoma expressing the antibody with the desired sequence or functional properties is isolated. In some examples, the murine anti-p16 antibody or its antigen-binding fragment may further contain a light chain constant region of a murine κ, λ chain or a variant thereof, or further contain a heavy chain constant region of a murine IgG1, IgG2, IgG3 or a variant thereof.

[0094] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a mouse antibody with the constant region of a human antibody, which can reduce the immune response induced by the mouse antibody. To establish a chimeric antibody, a hybridoma that secretes mouse-specific monoclonal antibodies must first be established, and then the variable region genes must be cloned from the mouse hybridoma cells, and then the constant region genes of the human antibody must be cloned as needed, and the mouse variable region genes must be connected to the human constant region genes to form a chimeric gene and inserted into an expression vector, and finally the chimeric antibody molecule is expressed in a eukaryotic system or a prokaryotic system. For example, the antibody light chain of the chimeric antibody further comprises a light chain constant region of a human κ, λ chain, or a variant thereof. The antibody heavy chain of the chimeric antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4, or a variant thereof, preferably comprising a human IgG1, IgG2, or IgG4 heavy chain constant region, or an IgG1, IgG2, or IgG4 variant using amino acid mutations (e.g., L234A and / or L235A mutations, and / or S228P mutations).

[0095] The term "humanized antibody", also known as CDR-grafted antibody, refers to an antibody produced by transplanting the CDR sequence of a non-human species into the antibody variable region framework of a human, i.e., different types of human germline antibody framework sequences. The heterologous reaction induced by chimeric antibodies due to carrying a large amount of heterologous protein components can be overcome. Such framework sequences can be obtained from public DNA databases including germline antibody gene sequences or published references. For example, the germline DNA sequences of human heavy chain and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet www.mrccpe.com.ac.uk / vbase), and in Kabat, EA et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition. In order to avoid the decrease in activity caused by the decrease in immunogenicity, the human antibody variable region framework sequence can be subjected to minimal reverse mutation or back mutation to maintain activity.

[0096] In the present application, the antibody or its antigen-binding fragment may further comprise a light chain constant region of a human or mouse κ, λ chain or a variant thereof, or further comprise a heavy chain constant region of a human or mouse IgG1, IgG2, IgG3, IgG4 or a variant thereof; preferably comprise a human IgG1, IgG2 or IgG4 heavy chain constant region, or an IgG1, IgG2 or IgG4 variant using amino acid mutations (e.g., L234A and / or L235A mutations, and / or S228P mutations).

[0097] The "variants" of the heavy chain constant region of the human antibody and the light chain constant region of the human antibody described in the present application refer to the variants of the heavy chain constant region or light chain constant region that are derived from humans and do not change the structure and function of the antibody variable region, which have been disclosed in the prior art. Exemplary variants include IgG1, IgG2, IgG3 or IgG4 heavy chain constant region variants that undergo site-directed modification and amino acid substitutions in the heavy chain constant region; specific substitutions such as YTE mutations, L234A and / or L235A mutations, S228P mutations, and / or mutations that obtain knob-into-hole structures (so that the antibody heavy chain has a combination of knob-Fc and hole-Fc), which have been shown to give antibodies new properties, but do not change the function of the antibody variable region.

[0098] "HuMAb", "humanized antibody", "fully human antibody", "fully human antibody" are used interchangeably and may be an antibody derived from a human or an antibody obtained from a transgenic organism that has been "engineered" to produce specific human antibodies in response to antigenic stimulation, and may be produced by any method known in the art. In certain techniques, human heavy and light chain loci elements are introduced into cell lines derived from embryonic stem cell lines in which endogenous heavy and light chain loci are targeted for disruption. The transgenic organism can synthesize human antibodies specific for human antigens, and the organism can be used to produce hybridomas that secrete human antibodies. A human antibody may also be an antibody in which the heavy and light chains are encoded by nucleotide sequences derived from one or more human DNA sources. Fully human antibodies may also be constructed by gene or chromosome transfection methods and phage display technology, or by in vitro activated B cells, all of which are known in the art.

[0099] The terms "full-length antibody", "intact antibody", "complete antibody" and "whole antibody" are used interchangeably herein to refer to an antibody in substantially intact form, as distinguished from an antigen-binding fragment as defined below. The term specifically refers to an antibody that contains constant regions in both the light and heavy chains. The disclosure "antibody" encompasses "full-length antibodies" and antigen-binding fragments thereof.

[0100] The full-length antibody of the present application includes a full-length antibody formed by connecting the light chain variable region and the light chain constant region in the combination of the light and heavy chain variable regions in the following table, and connecting the heavy chain variable region and the heavy chain constant region. Those skilled in the art can select the light chain constant region and the heavy chain constant region of different antibody sources according to actual needs, such as the light chain constant region and the heavy chain constant region of human antibody sources.

[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 to perform the antigen-binding function of an antibody. Examples of binding fragments included in the term "antigen-binding fragment" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments connected by a disulfide bridge on the hinge region, (iii) a Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment consisting of the VH and VL domains of a single arm of an antibody; (v) a dsFv, an antigen-binding fragment formed by VH and VL via an interchain disulfide bond; (vi) diabodies, bispecific antibodies, and multispecific antibodies comprising fragments such as scFv, dsFv, and Fab. In addition, although the two domains of the Fv fragment, VL and VH, are connected by a synthetic linker, they can be produced as a single protein chain in which the VL and VH regions are paired to form a monovalent molecule (called single-chain Fv (scFv); 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 the fragments are screened for functionality in the same manner as for intact antibodies. Antigen-binding portions can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins. The antibody can be an antibody of different isotypes, for example, an IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), an IgA1, an IgA2, an IgD, an IgE or an IgM antibody.

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

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

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

[0105] Alternatively, the Fab' fragment can be produced by inserting a DNA encoding the Fab' fragment into an expression vector and introducing the vector into a host.

[0106] The term "single-chain antibody", "single-chain Fv" or "scFv" means a molecule comprising an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker. Such scFv molecules can have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Other linkers useful in the present disclosure are described by, 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] Diabodies are antibody fragments in which scFv or Fab is dimerized, and are antibody fragments having bivalent antigen-binding activity. In the bivalent antigen-binding activity, the two antigens may 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 a polypeptide in which one amino acid residue in each of VH and VL is substituted by a cysteine ​​residue via a disulfide bond between the cysteine ​​residues. The amino acid residue substituted by the cysteine ​​residue can be selected based on the three-dimensional structure prediction of the antibody according to a known method (e.g., Protein Engineering, 7, 697 (1994)).

[0110] The term "amino acid difference" or "amino acid mutation" refers to the presence of amino acid changes or mutations in a variant protein or polypeptide compared to the original protein or polypeptide, including the insertion, deletion or substitution of 1, 2, 3 or more amino acids based on the original protein or polypeptide.

[0111] The term "antibody framework" or "FR region" refers to a portion of a variable domain VL or VH that serves as a scaffold for the antigen binding loops (CDRs) of the variable domain. In essence, it is a variable domain without CDRs.

[0112] The term "complementarity determining region", "CDR" or "hypervariable region" refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contributes to antigen binding. Typically, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region. The amino acid sequence boundaries of the CDRs may be determined using any of a variety of well-known schemes, including the "Kabat" numbering convention (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 convention (see Al-Lazikani et al., (1997) JMB 273:927-948) and the ImmunoGenTics (IMGT) numbering convention (see Lecture 1, 1996). MP, Immunologist, 7, 132-136 (1999); Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003) etc. For example, for the classical format, following the Kabat rule, 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 the Chothia rule, the CDR amino acid residues in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2) and 95-102 (HCDR3); and the CDR amino acid residues in VL are numbered 26-32 (HCDR1), 52-56 (HCDR2) and 95-102 (HCDR3). The amino acid residues are numbered 26-32 (LCDR1), 50-52 (LCDR2) and 91-96 (LCDR3). By combining the CDR definitions of Kabat and Chothia, the CDRs are 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 rule, the CDR amino acid residues in VH are numbered approximately 26-35 (CDR1), 51-57 (CDR2) and 93-102 (CDR3), and the CDR amino acid residues in VL are numbered approximately 27-32 (CDR1), 50-52 (CDR2) and 89-97 (CDR3).Following the IMGT rules, the CDR regions of an antibody can be determined using the program IMGT / DomainGap Align.

[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). An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-consecutive amino acids in a unique spatial conformation. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996).

[0114] The terms "specific binding", "selective binding", "selectively binds" and "specifically binds" refer to the binding of an antibody to a predetermined epitope on an antigen. Typically, the antibody binds with an affinity (KD) of about less than 10-8M, such as about less than 10-9M, 10-10M, 10-11M, 10-12M or less.

[0115] The term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction. Typically, the antibodies of the present disclosure bind to TrKA or an epitope thereof with a dissociation equilibrium constant (KD) of less than about 10-7 M, such as less than about 10-8 M or 10-9 M. For example, in the present disclosure, the affinity of the antibody to the cell surface antigen is determined by FACS method to determine the KD value.

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

[0117] In a 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 no less than 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 a 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 no less than 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 a 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 no less than 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 a 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 no less than 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 between two polypeptides. When the positions in the two comparison sequences are occupied by the same base or amino acid monomer subunit, for example, if every position of the 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 × 100. For example, when the sequences are optimally aligned, if 6 of the 10 positions in the two sequences are matched or homologous, then the two sequences are 60% homologous; if 95 of the 100 positions in the two sequences are matched or homologous, then the two sequences are 95% homologous. Typically, when two sequences are aligned, comparison is made to give the maximum percentage identity. For example, the comparison can be performed by the BLAST algorithm, in which the parameters of the algorithm are selected to give the maximum match between each sequence over the entire length of each reference sequence. The following references relate to BLAST algorithms frequently used for 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 conventional BLAST algorithms such as those provided by NCBI BLAST are also well known to those skilled in the art.

[0120] According to a second aspect of the embodiments of the present application, a nucleic acid is provided, wherein the nucleic acid encodes the anti-p16 protein antibody.

[0121] The term "nucleic acid molecule" as used in this application 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. A nucleic acid is "operably linked" when it is 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 connected to the coding sequence.

[0122] The nucleic acid molecules of the present application mainly refer to isolated nucleic acid molecules. "Isolated" refers to molecules that are substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates or other materials, such as cell debris and growth medium. Generally, the term "isolated" is not intended to refer to the complete absence of these materials or the absence of water, buffer or salts, unless they are present in an amount that significantly interferes with the experimental or therapeutic use of the compounds as described herein.

[0123] According to a second aspect of the embodiments of the present application, a vector is provided, wherein the vector comprises the nucleic acid.

[0124] Optionally, the vector is selected from mammalian cell virus, bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus or a combination thereof.

[0125] The term "vector" is also "expression vector" and refers to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. In one embodiment, the vector is a "plasmid", which refers to a circular double-stranded DNA loop into which another DNA segment can be connected. In another embodiment, the vector is a viral vector, in which another DNA segment can be connected to a viral genome. The vector disclosed herein can be autonomously replicated in a host cell introduced therein (e.g., a bacterial vector and an additional mammalian vector having a bacterial origin of replication) or can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating with the host genome (e.g., a non-additional mammalian vector). On the whole, the vector can be selected from, but is not limited to, a mammalian cell virus, a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, or a combination thereof.

[0126] According to a third aspect of the embodiments of the present application, a cell is provided, wherein the cell comprises the nucleic acid or the vector.

[0127] The term "cell" or "host cell" refers to a cell into which an expression vector has been introduced. Host cells may include bacteria, microorganisms, plants or animal cells. Easily transformed bacteria include members of the family Enterobacteriaceae, such as strains of Escherichia coli or Salmonella; Bacillaceae, 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] As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such names include progeny. Thus, "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom, without regard to the number of passages. It should also be understood that all progeny may not be exactly identical in terms of DNA content, due to intentional or unintentional mutations. Mutant progeny having the same function or biological activity as screened for in the originally transformed cell are included. Where different names are intended, this is clear from the context.

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

[0130] The antibodies of the present application can be prepared and purified by conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors. The recombinant immunoglobulin expression vector can stably transfect host cells. As a more recommended prior art, mammalian expression systems can lead to glycosylation of antibodies, especially at the highly conserved N-terminal site in the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to p16 protein. Positive clones are expanded and cultured in bioreactors to produce antibodies. The culture fluid that secretes antibodies can be purified by conventional techniques. For example, purification is performed using an A or G Sepharose FF column containing an adjusted buffer. Non-specifically bound components are washed away. The bound antibodies are then eluted using a pH gradient method, and the antibody fragments are detected by SDS-PAGE and collected. The antibodies can be filtered and concentrated using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieves and ion exchange. The obtained product needs to be immediately frozen, such as -70°C, or lyophilized.

[0131] Methods for producing and purifying antibodies and antigen-binding fragments are well known in the prior art, such as the Cold Spring Harbor Guide to Antibody Experimental Techniques, Chapters 5-8 and 15. The resulting antibodies can be renatured, purified, and amino acid sequenced by conventional methods. Antigen-binding fragments can also be prepared by conventional methods. The antibodies or antigen-binding fragments described in the invention use genetic engineering methods to add one or more human FR regions to the non-human CDR region. Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) website http: / / imgt.cines.fr by comparing the IMGT human antibody variable region germline gene database and MOE software, or from the Journal of Immunoglobulins, 2001 ISBN012441351.

[0132] According to a fifth aspect of the embodiments of the present application, a p16 protein detection kit is provided, wherein the p16 protein detection kit comprises the anti-p16 protein antibody.

[0133] The antibodies in the detection kit of the present application can compete and bind to the p16 protein in the sample to be tested.

[0134] When the term "competes" is used in the context of antigen binding proteins that compete for the same epitope, it means competition between antigen binding proteins, which is determined by an assay in which the antigen binding protein to be tested (e.g., an antibody or a functional fragment thereof) prevents or inhibits (e.g., reduces) specific binding of a reference antigen binding protein (e.g., a ligand or a reference antibody) to a common antigen (e.g., 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, such as: solid phase direct or indirect radioimmunoassays (RIA), solid phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253); solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619), solid phase direct label assays, solid phase direct label sandwich assays (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); using I-1 2 Solid phase direct labeling RIA with 5-label (see, e.g., Morel et al., 1988, Molec. Immunol. 25: 7-15); solid phase direct biotin-avidin EIA (see, e.g., Cheung, et al., 1990, Virology 176: 546-552); and direct labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32: 77-82). Typically the assay involves the use of purified antigen bound to a solid surface or cells bearing either an unlabeled test 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 cells in the presence of the test antigen binding protein. Typically the test antigen binding protein is present in excess. Antigen binding proteins identified by competitive assays (competing 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 adjacent epitopes sufficiently close to the binding epitope of the reference antigen binding protein that the two epitopes sterically interfere with each other for binding. Additional details on methods for determining competitive binding are provided in the Examples herein. Typically, when the competing antigen binding protein is present in excess, 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 certain instances, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

[0135] The present application relates to a method for immunodetection or determination of a target antigen (e.g., p16 protein), a reagent for immunodetection or determination of a target antigen (e.g., p16 protein), a method for immunodetection or determination of cells expressing a target antigen (e.g., p16 protein), and a diagnostic agent for diagnosing a disease associated with cells positive for the target antigen (e.g., p16 protein), which comprises the antibody or antibody fragment of the present application that specifically recognizes the target antigen (e.g., p16 protein) and binds to the amino acid sequence of the extracellular region or its three-dimensional structure as an active ingredient.

[0136] In the present application, the method for detecting or measuring the amount of target antigen (e.g., p16 protein) can be any known method. For example, it includes immunoassay or assay. Immunoassay or assay is a method for using labeled antigen or antibody to detect or measure antibody amount or antigen amount. Examples of immunoassay or assay include radioactive material labeled immunoantibody method (RIA), enzyme immunoassay (EIA or ELISA), fluorescent immunoassay (FIA), luminescent immunoassay, protein immunoblotting, physicochemical methods, etc.

[0137] In order to detect cells expressing the polypeptide, known immunodetection methods can be used, and preferably immunoprecipitation, fluorescent cell staining, immunohistostaining, etc. In addition, fluorescent antibody staining using FMAT8100 HTS system (Applied Biosystem) can be used.

[0138] In the application, there is no particular limitation on the sample to be tested for detecting or measuring the target antigen (e.g., p16 protein), as long as it has the possibility of containing cells expressing the target antigen (e.g., p16 protein), such as tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid or culture fluid.

[0139] According to the desired detection method, the detection product containing the monoclonal antibody or its antibody fragment of the present application may also contain reagents for performing antigen-antibody reactions or reagents for detecting reactions. Reagents for performing antigen-antibody reactions include buffers, salts, etc. Reagents for detection include reagents commonly used in immunoassays or assays, such as a second antibody that recognizes a label of the monoclonal antibody, its antibody fragment, or its conjugate, and a substrate corresponding to the label, etc.

[0140] In one example, 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. That is, two antibodies are used in combination.

[0141] In one example, the detection kit includes a test strip, which includes a base plate and a sample pad, a marking pad, a detection membrane and an absorption pad sequentially arranged on the base plate, and a detection line is arranged on the detection membrane; one of the marking 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 marking pad is coated with the anti-p16 protein antibody 2, and the detection line is coated with the anti-p16 protein antibody 1. The detection kit of the present application is detected by colloidal gold method, and the anti-p16 protein antibody 2 on the marking pad forms a complex with gold nanoparticles.

[0142] In a fifth aspect of the embodiments of the present application, a method for producing the p16 protein detection kit is provided, the method comprising:

[0143] The anti-p16 protein antibody is used to prepare a p16 protein detection kit;

[0144] Optionally, the production method is used to produce anti-p16 protein antibodies, and the anti-p16 protein antibodies are prepared into a p16 protein detection kit.

[0145] In a sixth aspect of the embodiments of the present application, a method for detecting p16 protein in a sample is provided, the detection method comprising using the anti-p16 protein antibody or the p16 protein detection kit to detect the p16 protein in the sample.

[0146] The detection method of the present 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), and can also be used for the purpose of determining tumor prognosis.

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

[0148] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0149] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0150] The p16 protein is normally expressed in normal cervical tissues and overexpressed in tissue cells of cervical cancer patients. The p16 protein can be obtained by lysing cervical cancer tissues or cells, and then the p16 protein is detected by a double antibody sandwich method, which plays a role in screening and preventing cervical cancer. The present invention immunizes Balb / c mice after preparing p16 recombinant antigens, extracts RNA from spleen tissues, quickly obtains anti-p16 antibodies by phage display technology, and obtains antibody pairs against p16 protein by double antibody sandwich pairing ELISA detection. The library capacity and diversity of the phage display library can screen more antibodies, and a large number of antibody pairing tests can be performed at the same time, thereby ensuring the sensitivity and specificity of the final available antibody pairs; the colloidal gold method is used to detect cervical cancer tissue samples, which is simple to operate and quickly produces results, has low detection costs, and can be quickly promoted and used in clinical detection.

[0151] Embodiment 1

[0152] 1. Immunogen Preparation

[0153] The amino acid sequence of p16 protein (P42771-2) was obtained from the Uniprot database. The full length was selected and fused with 3×flag-tag at the C-terminus to obtain the amino acid sequence of p16 immunogen (SEQ ID NO.1). A gene synthesis company was commissioned to synthesize the entire gene sequence and construct it into the pcDNA3.4 expression vector (see Figure 1 ) to obtain the expression plasmid containing the target gene p16-3×flag-tag. The plasmid was chemically transformed into the competent E. coli Top10, and a single clone was picked and inoculated into LB medium. After culturing at 37°C and 200 rpm for 16-20 hours, the bacteria were collected by centrifugation. The recombinant plasmid was prepared using an endotoxin-free plasmid extraction kit to obtain A 260 / A 280 =1.8-2.0 plasmid sample and under the action of transfection reagent PEI, the recombinant plasmid was transiently transfected into mammalian cells HEK293F, and the expression product was obtained by suspension culture at 37°C and 120rpm for 48-72h. After pretreatment, the expression product was subjected to anti-flag affinity chromatography and ion exchange chromatography to obtain p16 recombinant antigen, and the collected eluted sample was dialyzed into PBS buffer to obtain the immunogen, and the identification was seen Figure 2 .

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

[0155] MEPAAGSSMEPSADWLATAAARGRVEEVRALLEAGALPNAPNSYGRRPIQVMMMGSARVAELLLLHGA

[0156] EPNCADPATLTRPVHDAAREGFLDTLVVLHRAGARLDVRDAWGRLPVDLAEELGHRDVARYLRAAAGGTRGSNHARIDAAEGPSDIPD DYKDDDDKGDYKDDDDKIDYKDDDDK .

[0157] 2. Mouse immunization and acquisition of scFv (single chain antibody fragment)

[0158] The p16 antigen obtained in item 1 was used to immunize 8-12 week old Balb / c mice, and the antigen was emulsified with Freund's adjuvant and immunized subcutaneously. The tail blood of the mice was tested by the indirect ELISA method until the titer of the tail blood reached 10 5Immunization was stopped at 4 pm. Three days before extracting mouse RNA, the immunogen was injected intraperitoneally for booster immunization. The mouse RNA was extracted using a commercially available kit, and the purity, concentration and integrity of the RNA extraction were confirmed by agarose gel electrophoresis. The light chain variable region and heavy chain variable region of the mouse were amplified using specific primers to obtain VL and VH; finally, the scFv single-chain antibody was obtained by overlap. The scFv was seamlessly cloned into the pComb3XSS vector and electrotransferred to TG1 competent cells. The phages were screened using the double antibody sandwich method (M13 phage displayed VL, T4 phage displayed VH, and the specific antibody of the T4 phage capsid protein gp23 was fixed on the ELISA plate. The T4 phage library was added to the wells of the ELISA plate and incubated at 37°C for 1 hour to fix the T4 phage on the ELISA plate. The ELISA plate was washed to remove unbound phages, and the ELISA plate was added to the wells of the ELISA plate. p16 antigen, incubate at 37℃ for 1h, add M13 phage library to the wells of the ELISA plate, incubate at 37℃ for 1h, incubate the light chain displayed by M13 phage and the heavy chain displayed by T4 phage with p16 antigen, wash the ELISA plate to remove unbound phage, add specific antibodies against M13 phage capsid protein pVIII to the wells of the ELISA plate, incubate at 37℃ for 1h), and perform at least three rounds of panning to obtain the phage library bound to the p16 antigen. After coating the plate, pick the monoclonal culture to obtain the scFv single-chain antibody, and detect the activity of scFv in the culture supernatant by indirect ELISA. The results are shown in Table 1. To ensure that the screened scFv antibody is specific to the p16 antigen, set up experimental group 1 without coating antigen and directly blocking casein and experimental group 2 with coating p16 antigen and then casein blocking.

[0159] Table 1. Indirect ELISA to detect the activity of scFv ("+" indicates that the OD value exceeds the measurement range of the microplate reader)

[0160]

[0161] 3. Preparation of Recombinant Antibodies

[0162] The bacterial liquid of 40 monoclonal clones with OD values ​​less than 0.2 in experimental group 1 and greater than 2.0 in experimental group 2 under item 2 above was sent to Bio-Tech for sequencing. According to the sequencing results, the sequence of scFv, including the light chain variable region and heavy chain variable region of the antibody, was obtained. The sequences of the light and heavy chain variable regions were inserted into the eukaryotic vector containing the mouse light and heavy chain constant regions ( Figure 3 and Figure 4 ), transfected into mammalian cells HEK293F for expression, and the activity of the cell expression supernatant was detected by indirect ELISA method as shown in Table 2; the cell expression product with OD value greater than 2.0 was purified by Protein A affinity chromatography and ion exchange chromatography to obtain complete anti-p16 antibody.

[0163] For p16-positive tissue samples, a total of 30 antibodies with OD values ​​greater than 2.0 were screened using the double antibody sandwich method, and the optimal antibody pairing combination was obtained as shown in combination 2 in Table 3: p16-25 and p16-11. p16-25 was named p16-Ab01, and p16-11 was named p16-Ab02.

[0164] Table 2. ELISA test results of cell expression supernatant ("+" indicates that the OD value exceeds the measurement range of the microplate reader)

[0165]

[0166]

[0167] Table 3. Results of ELISA screening of candidate antibodies

[0168]

[0169]

[0170] 4. Antibody affinity testing

[0171] The affinity test of the antibody was performed according to the following protocol:

[0172] 4.1 Prepare anti-p16 antibodies: p16-Ab01 and p16-Ab02 according to the description in item 3 above;

[0173] 4.2 The prepared p16-Ab01 and p16-Ab02 were chemically coupled and immobilized on a Biacore T200 detection chip;

[0174] 4.3 Using p16 antigen as the analyte, different concentrations of 50nM, 25nM, 12.5nM, 6.25nM, and 3.125nM were used to react with the immobilized antibody;

[0175] 4.4 According to the reaction procedure: capture 30s, binding 30s, dissociation 600s, regeneration Gly2.25 30s, the binding rate ka, dissociation rate kd and reaction response value of each antibody were obtained, and then the affinity KD of each antibody was calculated (see Table 4, Figure 5 and Figure 6 ).

[0176] Table 4. Antibody affinity test results

[0177] 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 ]]>

[0178] Figure 5 and Figure 6In the figure, the black line represents the kinetic curve fitted by the instrument software, and the other colored curves are the kinetic curves detected by the machine at concentrations of 3.126nM, 6.25nM, 12.5nM, 25nM, and 50nM from bottom to top.

[0179] 5. p16 protein colloidal gold method for detection of clinical cervical tissue samples

[0180] 5.1 Preparation of colloidal gold test strips

[0181] (1) Preparation of NC membrane coated with antibody p16-Ab01

[0182] The coating antibody was prepared into 1.0 mg / mL NC coating solution using NC membrane coating diluent, and coated on the NC membrane attached to the PVC board using a film scribing machine, and finally dried in a 50° C. drying oven for 24 h to prepare the test line T.

[0183] In the same way, goat anti-mouse IgG antibody was coated on the NC membrane to prepare quality control line C.

[0184] (2) Preparation of labeling pad for labeled antibody p16-Ab02

[0185] Measure colloidal gold into a clean beaker and set the speed so that there is a small vortex in the center; add 0.1MK 2 CO 3 , adjust to the corresponding pH range with a pH meter, and stir evenly; then slowly and evenly add the labeled antibody to a final concentration of 0.01 mg / mL, and stir for 20 minutes after the addition is complete; add 20wt% BSA in an amount of 1% of the volume of colloidal gold during blocking, and continue stirring for 25 minutes; centrifuge after balancing the solution; collect the precipitate (add a small amount of 1× labeling solution to rinse the centrifuge bottle), and finally use the gold labeling solution to make up the volume to prepare a colloidal gold labeled antibody complex mixture. The colloidal gold labeled antibody complex mixture is coated with a glass cellulose membrane, and the colloidal gold labeled anti-sheep anti-mouse IgG antibody is coated with the glass cellulose membrane in the same way to prepare a labeling pad.

[0186] (3) Assembling the test strips

[0187] The marking pad prepared in step (2), the prepared sample pad and the absorption pad are assembled on a PVC plate to prepare a test strip.

[0188] 5.2 Tissue sample processing

[0189] The collected clinically positive tissue samples and normal tissue samples were processed as follows:

[0190] First, weigh the tissue sample; then use scissors to cut the tissue into pieces, add it to a clean glass grinder and grind it for 1 minute. Note that the glass grinder should be pre-cooled in ice in advance, and the entire grinding process should be carried out on ice; prepare the amount of PBS buffer according to the ratio of 1g tissue: 9mL PBS pH7.4, first add a certain amount of PBS, grind for 1 minute, continue to add a certain amount of PBS and grind for 1 minute, leave a little PBS to clean the tube wall again, and finally aspirate the ground lysate; the lysate is centrifuged at 4℃3000rpm for 20 minutes, and the supernatant is taken for detection.

[0191] 5.3 Sample testing

[0192] The colloidal gold method was used to detect positive tissue samples (cervical cancer tissue samples diagnosed clinically) and normal tissue samples. The results of positive and negative observation by naked eye are shown in Table 5. The conclusion is that the test results of clinical positive tissue samples are positive, and the test results of clinical normal tissue samples are negative. This shows that the developed test strip can be used for the detection of clinical tissue samples of p16 protein and provide a certain basis for clinical diagnosis.

[0193] Table 5. Results of colloidal gold method for tissue samples

[0194] sample Test results Positive tissue sample 1 + Positive tissue sample 2 + Positive tissue sample 3 + Positive tissue sample 4 + Positive tissue sample 5 + Positive tissue sample 6 + Positive tissue sample 7 + Positive tissue sample 8 + Positive tissue sample 9 + Positive tissue samples 10 + Normal tissue sample 1 - Normal tissue sample 2 - Normal tissue sample 3 - Normal tissue sample 4 - Normal tissue samples 5 - Normal tissue samples6 - Normal tissue samples7 - Normal tissue samples8 - Normal tissue samples9 - Normal tissue samples 10 -

[0195] Table 6. CDRs of p16-Ab01 and p16-Ab02 (CDRs defined using the Kabat system)

[0196] 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)

[0197] The amino acid sequence of the light chain variable region of p16-Ab01 (SEQ ID NO.14):

[0198] DVLMTQTPLSLPVSLGDQASISC RSSQSIVHSNGNTYLE WYLQKPGQSPKLLIY KVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC FQGSHVPQT FGGGTNLEIK.

[0199] The amino acid sequence of the heavy chain variable region of P16-Ab01 (SEQ ID NO.15):

[0200] QVQLQQSAAELARPGASVKMSCKASGYSFT GYTMH WVKQRPGQGLEWIG YINPSSGYTDYNQKF KD KTTLTADKSSSTAYMQLSSSLTSEDSAVYYCAR RVYGSLPY WGQGTLVTVSA.

[0201] The amino acid sequence of the light chain variable region of p16-Ab02 (SEQ ID NO.16):

[0202] DIQMTQTTSSLSASLGDRVTISC RASQDISNYLN WYQQKPDGTVKFLIY YTSRLHS GVPSRFSGSGSGTDYSLTISNLEQEDIATYFC QQGNTLPPTF GGGTKLEIK.

[0203] The amino acid sequence of the heavy chain variable region of P16-Ab02 (SEQ ID NO.17):

[0204] QVQLQQSGAELVRPGTSVKVSCKASGYAFT NYLIE WMYQRPGQGLEWIG VINPGSGINNYNEKFKG KATLTADKSSSTAYMQLSSSLTSDDSAVYFCAR FYDRYYAMDY WGQGTSVTVSS.

[0205] In general, the present invention uses phage display technology to obtain antibodies against p16 protein. Compared with traditional hybridoma technology, the development cycle is shortened to 3 months (including immunization time); 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 present invention can detect clinical tissue samples by colloidal gold method to quickly obtain the result of whether p16 protein is overexpressed.

[0206] The technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. To make the description concise, not all possible combinations of the technical features in the above-mentioned implementation modes 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.

[0207] The above-described embodiments only express several implementation methods of the present application, which is convenient for understanding the technical solution of the present application in detail, but it cannot be understood as a limitation on the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several deformations and improvements can be made, which all belong to the protection scope of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, the technicians in this field can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by the technicians in this field through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the protection scope of the claims attached to the present application. Therefore, the protection scope of the patent of the present application shall be based on the content of the attached claims, and the description and drawings can be used to explain the content of the claims.

Claims

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

13.

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

15.

3. The anti-p16 protein antibody according to claim 1, characterized in that: 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 constant region sequence of the anti-p16 protein antibody is selected from any one of the constant region sequences 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 of the constant region of the anti-p16 protein antibody is mouse, cow, 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 according to any one of claims 1 to 5.

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

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

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

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

11. The p16 protein detection kit according to claim 10, characterized in that: The p16 protein detection kit comprises 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.

12. The p16 protein detection kit according to claim 11, characterized in that: The p16 protein detection kit comprises a detection test strip, which comprises a bottom plate and a sample pad, a marking pad, a detection membrane and an absorption pad arranged on the bottom plate in sequence, and a detection line is arranged on the detection membrane; Of the label 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.

13. The p16 protein detection kit according to claim 12, characterized in that: 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.

14. A method for producing the p16 protein detection kit according to any one of claims 10 to 13, characterized in that: The production method comprises: A p16 protein detection kit is prepared using the anti-p16 protein antibody according to any one of claims 1 to 5; Optionally, the anti-p16 protein antibody is produced by the production method of claim 9, and the anti-p16 protein antibody is used to prepare a p16 protein detection kit.

15. A method for detecting p16 protein in a sample, characterized in that: The detection method comprises: The p16 protein in the sample is detected 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.

16. The method for detecting p16 protein in a sample according to claim 15, characterized in that: The sample is a cell sample or a tissue sample.

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