Specific binding protein of AFP as well as preparation method and application of specific binding protein

Abstract

The invention relates to an AFP specific binding protein and a preparation method and application thereof in the technical field of immunodetection. The AFP specific binding protein has an AFP binding structural domain a or / and an AFP binding structural domain b; the AFP binding structural domain a comprises VH CDR1-3 as shown in a sequence SEQ ID NO. 7 to 9 and VL CDR1-3 as shown in a sequence SEQ ID NO. 10 to 12; the AFP binding structural domain b comprises VH CDR1-3 as shown in a sequence SEQ ID NO. 13-15, VH CDR1-3 as shown in a sequence SEQ ID NO. 16-18 of the VL CDR1-3 is shown as the sequence SEQ ID NO. 16-18 of the VL CDR1-3. Compared with the prior art, the invention provides a novel anti-AFP antibody, the effective detection of AFP can be realized by adopting the antibody, and a novel scheme is provided for the detection of AFP.

Description

Technical Field

[0001] This application relates to the field of immunoassay technology, and particularly relates to a specific binding protein of AFP, a preparation method thereof, and an application thereof. Background Art

[0002] Tumor markers refer to substances that exist in blood, body fluids, and tissues and can be detected and are related to the occurrence and development of tumors. The amount produced in tumor tissues far exceeds that in normal tissues. The presence and quantity changes of such substances can play a role in indicating the nature of tumors and play an auxiliary role in the diagnosis, classification, prognosis judgment, and clinical treatment guidance of tumors.

[0003] Among many tumor markers, alpha-fetoprotein (AFP) is widely used clinically. Alpha-fetoprotein is a special glycoprotein synthesized by the liver and yolk sac in the early stage of fetal development.

[0004] AFP consists of 609 amino acids and has a molecular weight of approximately 69 kDa. AFP consists of an N-terminal domain (domain I), a C-terminal domain (domain III), and a central domain (domain II). These three domains of AFP are connected by disulfide bonds to form a V-shaped structure, and each domain exhibits different biological activities. Domain I can bind to the phosphatase domain of PTEN, thereby affecting PTEN activity; domain II has high flexibility and is easily digested by proteases; domain III is the most conserved domain, which consists of several hydrophobic amino acid continuous sequences to form a leucine zipper-like structure. Domain III is responsible for binding to signaling proteins and receptors, thereby regulating their biological activities.

[0005] Under normal circumstances, AFP exists in the human body at a relatively low concentration level. However, during the embryonic period, AFP produced by the fetus in pregnant women will enter the mother's body through the umbilical cord, resulting in an increase in AFP levels. Normal adult hepatocytes lose the ability to synthesize AFP, so the AFP content in adult serum is relatively low, and the reference value is 0 - 20 μg / L.

[0006] When liver cells become cancerous, the AFP gene is activated and highly expressed, and the AFP level in serum increases significantly. This is an important indicator for clinically assisting in the diagnosis of primary liver cancer. Generally, the larger the tumor, the more numerous the tumors, and the higher the stage, the higher the AFP level in serum. In addition, the AFP levels in patients with acute and chronic hepatitis and cirrhosis are higher than those in normal people. In acute hepatitis or as the condition improves, the AFP level in their body often drops to the normal level within a short time, that is, the "transient increase" phenomenon of AFP. In patients with chronic liver disease and cirrhosis, the AFP level will show a downward or continuously low level, while in patients with liver cancer, it shows a gradually increasing trend.

[0007] In recent years, there have been numerous methods for detecting the levels of tumor markers. Among them, common ones include enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay (CLIA), radioimmunoassay (RIA), etc. The common methods for AFP level testing are chemiluminescent immunoassay and enzyme-linked immunosorbent assay. The testing of both methods requires specific antibodies. Therefore, the research and development of specific antibodies for AFP are crucial. Summary of the Invention

[0008] Based on this, one or more embodiments of the present application provide a specific binding protein of AFP, its preparation method and application. The technical solutions include:

[0009] One or more embodiments of the present application provide a specific binding protein of AFP, and the specific binding protein of AFP has an AFP binding domain a or / and an AFP binding domain b;

[0010] The AFP binding domain a includes:

[0011] VH CDR1 shown in sequence SEQ ID NO.7, VH CDR2 shown in sequence SEQ ID NO.8, and VH CDR3 shown in sequence SEQ ID NO.9; and,

[0012] VL CDR1 shown in sequence SEQ ID NO.10, VL CDR2 shown in sequence SEQ ID NO.11, and VL CDR3 shown in sequence SEQ ID NO.12;

[0013] The AFP binding domain b includes:

[0014] VH CDR1 shown in sequence SEQ ID NO.13, VH CDR2 shown in sequence SEQ ID NO.14, and VH CDR3 shown in sequence SEQ ID NO.15; and,

[0015] VL CDR1 shown in sequence SEQ ID NO.16, VL CDR2 shown in sequence SEQ ID NO.17, and VL CDR3 shown in sequence SEQ ID NO.18.

[0016] In some embodiments of the present application, the species sources of the framework region and the constant region of the specific binding protein of AFP are independently bovine, equine, porcine, ovine, murine, canine, feline, rabbit, camel, donkey, deer, mink, chicken, duck, goose or human.

[0017] In some embodiments of the present application, the sequence of the constant region of the AFP-specific binding protein is selected from the sequences of the constant regions of any one of IgG, IgA, IgM, IgE, and IgD.

[0018] In some embodiments of the present application, the sequence of the light chain variable region of the AFP-binding domain a is as shown in SEQ ID NO.20.

[0019] In some embodiments of the present application, the sequence of the heavy chain variable region of the AFP-binding domain a is as shown in SEQ ID NO.19.

[0020] In some embodiments of the present application, the sequence of the light chain variable region of the AFP-binding domain b is as shown in SEQ ID NO.22.

[0021] In some embodiments of the present application, the sequence of the heavy chain variable region of the AFP-binding domain b is as shown in SEQ ID NO.21.

[0022] In some embodiments of the present application, the heavy chain variable regions of the AFP-binding domain a and the AFP-binding domain b, and the light chain variable regions of the AFP-binding domain a and the AFP-binding domain b are each independently connected by a linker.

[0023] In some embodiments of the present application, the AFP-binding domain a is located at the N-terminus of the AFP-specific binding protein.

[0024] In some embodiments of the present application, signal peptides are independently connected to the N-termini of the heavy chain and the light chain of the AFP-specific binding protein.

[0025] In some embodiments of the present application, the sequence of the heavy chain variable region of the AFP-specific binding protein is as shown in SEQ ID NO.5 and / or the sequence of the light chain variable region is as shown in SEQ ID NO.6.

[0026] One or more embodiments of the present application provide a nucleic acid encoding the AFP-specific binding protein.

[0027] One or more embodiments of the present application provide a vector comprising the nucleic acid.

[0028] In some embodiments of the present application, the vector is a bacterial plasmid, phage, yeast plasmid, plant cell virus, or mammalian cell virus.

[0029] One or more embodiments of the present application provide a host cell comprising the nucleic acid or the vector.

[0030] In some embodiments of the present application, the host cell is a CHO cell, a COS cell, an NSO cell, a HeLa cell, a BHK cell or a HEK293 cell.

[0031] One or more embodiments of the present application provide a method for preparing an AFP-specific binding protein, and the preparation method uses the host cell described above to produce the AFP-specific binding protein.

[0032] One or more embodiments of the present application provide an application of the AFP-specific binding protein described above in the preparation of an AFP detection product.

[0033] One or more embodiments of the present application provide a detection kit, and the detection kit includes the AFP-specific binding protein described above.

[0034] In some embodiments of the present application, the detection kit includes a first antibody and a second antibody for detecting AFP. Among the first antibody and the second antibody, one has the AFP binding domain a and the AFP binding domain b, and the other has any one of the AFP binding domain a and the AFP binding domain b.

[0035] In some embodiments of the present application, among the first antibody and the second antibody, one is a bispecific antibody and the other is a chimeric antibody.

[0036] One or more embodiments of the present application provide a method for detecting AFP in a sample to be tested, and the detection method uses the AFP-specific binding protein described above or the detection kit described above.

[0037] Compared with the traditional technology, the present application provides a new AFP-specific binding protein. Using this specific binding protein can effectively detect AFP and provide a new solution for the detection of AFP. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more fully understand the present application and its beneficial effects, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present application. For those skilled in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0039] Figure 1 It is a full-length gel diagram of AFP;

[0040] Figure 2 It is a gel diagram of an AFP fragment;

[0041] Figure 3It is the vector map of pFUSE-CHIg-mG1;

[0042] Figure 4 It is the vector map of pFUSE2-CLIg-mk;

[0043] Figure 5 It is the SDS-PAGE electrophoresis diagram of the bispecific antibody;

[0044] Figure 6 It is the schematic diagram for the construction of the bispecific antibody. Specific implementation manners

[0045] The present application will be further described in detail below with reference to the accompanying drawings, implementation manners and examples. It should be understood that these implementation manners and examples are only used to illustrate the present application and not to limit the scope of the present application. The purpose of providing these implementation manners and examples is to make the understanding of the disclosed content of the present application more thorough and comprehensive. It should also be understood that the present application can be implemented in many different forms and is not limited to the implementation manners and examples described herein. Those skilled in the art can make various changes or modifications without departing from the connotation of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. In addition, in the following description, a large number of specific details are given to provide a more thorough understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this application belongs. The terms used in the description of this application herein are only for the purpose of describing the implementation manners and examples and are not intended to limit this application.

[0047] Terms

[0048] Unless otherwise stated or there is a contradiction, the terms or phrases used herein have the following meanings:

[0049] As used herein, the alternative scopes of the terms "and / or", "or / and", and "and / or" include any one of two or more related listed items, and also include any and all combinations of the related listed items. The said any and all combinations include combinations of any two related listed items, any more related listed items, or 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, this technical solution undoubtedly includes the technical solution connected by "logical AND", and undoubtedly also includes the technical solution connected by "logical OR". For example, "A and / or B" includes three parallel solutions: A, B, and A + B. Another example, for the technical solution of "A, and / or, B, and / or, C, and / or, D", it includes any one of A, B, C, and D (that is, the technical solution connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, it includes combinations of any two or any three of A, B, C, and D, and also includes the combination of the four items A, B, C, and D (that is, the technical solution connected by "logical AND").

[0050] In this application, the terms "multiple", "multiple types", "multiple times", "multiple elements", etc., unless otherwise specified, mean greater than or equal to 2 in quantity. For example, "one or more types" means one type or greater than or equal to two types.

[0051] As used herein, "their combinations", "any combination thereof", "any combination mode thereof", etc. include all suitable combination modes of any two or more of the listed items.

[0052] In this article, the "suitable" in "suitable combination mode", "suitable mode", "any suitable mode", etc. is subject to being able to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0053] In this article, "preferred", "better", "more preferable", "preferably" are only used to describe the embodiments or examples with better effects, and it should be understood that they do not constitute a limitation on the protection scope of this application.

[0054] In this application, "further", "even further", "especially", etc. are used for descriptive purposes, indicating differences in content, but should not be construed as a limitation on the protection scope of this application.

[0055] In this application, "optionally", "optional", "optional" mean that it can be there or not, that is, it refers to any one of the two parallel solutions of "yes" or "no". If "optional" appears in a technical solution in multiple places, unless otherwise specified and there is no contradiction or mutual restriction relationship, each "optional" is independent of each other.

[0056] In this application, in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are only for descriptive purposes, and cannot be construed as indicating or implying relative importance or quantity, nor can they be construed 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 enumerative description, and it should be understood that they do not constitute a closed limitation on quantity.

[0057] In this application, among the technical features described in an open-ended manner, there are included closed technical solutions composed of the listed features, as well as open technical solutions containing the listed features.

[0058] In this application, regarding numerical intervals (i.e., numerical ranges), unless otherwise specified, the selectable numerical values are considered continuous within the above numerical intervals, and include the two numerical endpoints (i.e., the minimum value and the maximum value) of the numerical range, as well as each numerical value between these two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as each integer between the two endpoints. In this article, it is equivalent to directly listing each integer. For example, t is an integer selected from 1 to 10, which means that t is any integer selected from the integer group 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 in this article should be understood to include any and all sub-ranges subsumed therein.

[0059] The temperature parameter in this application, unless otherwise specified, allows both constant temperature treatment and variation within a certain temperature range. It should be understood that the so-called constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuation within a range such as ±5°C, ±4°C, ±3°C, ±2°C, ±1°C is allowed.

[0060] In this application, %(w / w) and wt% both represent weight percentages, %(v / v) refers to volume percentages, and %(w / v) refers to mass-volume percentages.

[0061] All documents mentioned in this application are cited herein by reference as if each document were individually cited by reference. Unless it conflicts with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited in their entirety and for all purposes. When this application involves cited documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When this application involves cited documents, the examples and preferred methods of the relevant technical features cited can also be incorporated into this application as references, but only to the extent that this application can be implemented. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or be modified adaptively according to the description in this application.

[0062] In the first aspect of this embodiment, an AFP-specific binding protein is provided, and the AFP-specific binding protein has an AFP binding domain a or / and an AFP binding domain b;

[0063] The AFP binding domain a includes:

[0064] VH CDR1 shown in sequence SEQ ID NO.7, VH CDR2 shown in sequence SEQ ID NO.8, and VH CDR3 shown in sequence SEQ ID NO.9; and,

[0065] VL CDR1 shown in sequence SEQ ID NO.10, VL CDR2 shown in sequence SEQ ID NO.11, and VL CDR3 shown in sequence SEQ ID NO.12;

[0066] The AFP binding domain b includes:

[0067] VH CDR1 shown in sequence SEQ ID NO.13, VH CDR2 shown in sequence SEQ ID NO.14, and VH CDR3 shown in sequence SEQ ID NO.15; and,

[0068] VL CDR1 shown in sequence SEQ ID NO.16, VL CDR2 shown in sequence SEQ ID NO.17, and VL CDR3 shown in sequence SEQ ID NO.18.

[0069] This application provides a new AFP-specific binding protein, and the effective detection of AFP can be achieved by using it, providing a new solution for the detection of AFP.

[0070] The AFP-specific binding protein of the present application may have the AFP binding domain a, or may have the AFP binding domain b, or may have both the AFP binding domain a. Using it to detect the APF protein can not only improve the sensitivity and detection rate, but also improve the specificity and reduce false positives. In addition, it can be prepared by recombinant expression and purification, which is convenient for controlling the batch-to-batch difference through the process, controlling the batch-to-batch difference of raw materials, and reducing costs.

[0071] In some embodiments, the binding protein is an antibody, an antigen-binding fragment of an antibody or a small modular immunopharmaceutical. Optionally, the binding protein is a monoclonal antibody, F(ab’)2 fragment, Fab’ fragment, Fab fragment, Fv fragment, scFv fragment, linear antibody, multispecific antibody (such as bispecific, trispecific or multispecific antibody), minibody, chelated recombinant antibody, intrabody, nanobody, binding domain immunoglobulin fusion protein, small modular immunopharmaceutical, camelized antibody or an antibody containing VHH. In some embodiments, the antigen-binding fragment is derived from a complete antibody molecule, and specifically further includes a heavy chain framework region, a light chain framework region, a constant region, etc., such as monoclonal antibodies and bispecific antibodies. The preparation methods are known in the art.

[0072] As is well known in the art, the binding specificity and affinity of an antibody are mainly determined by the CDR sequences. According to the mature and well-known existing technologies, the amino acid sequences of non-CDR regions can be easily changed to obtain variants with similar biological activities. Therefore, the present invention also includes "functional derivatives" of the binding protein. A "functional derivative" refers to a variant with amino acid substitutions, and a functional derivative retains detectable binding protein activity. A "functional derivative" may include "variants" and "fragments", and because it has the same CDR sequences as the binding protein described in the present invention, it has similar biological activities.

[0073] The binding proteins described herein may contain amino acids with one or more substitutions, deletions or insertions relative to the above CDR sequences, for example, the number of amino acid insertions, substitutions or deletions does not exceed 3, preferably 1. Substitutions, deletions or insertions can be introduced into the nucleic acid molecules encoding the binding proteins of the present application by conventional techniques such as site-directed mutagenesis or PCR-mediated mutagenesis. In some embodiments, conservative amino acid substitutions are made at one or more positions. "Conservative amino acid substitution" is the situation where one amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acids with similar side chains have been defined in the prior art and include basic side chains (such as lysine, arginine, histidine), acidic side chains (such as aspartic acid, glutamic acid), uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β side chains (such as threonine, valine, isoleucine) and aromatic side chains (such as tyrosine, phenylalanine, tryptophan, histidine).

[0074] "Variable region" or "variable domain" refers to the amino-terminal domain of the heavy or light chain of an antibody. The variable domain of the heavy chain can be referred to as "VH". The variable domain of the light chain can be referred to as "VL". These domains are generally the most variable parts of the antibody and contain the antigen-binding sites. The variable region of the light or heavy chain (VL or VH) is composed of framework regions interrupted by three hypervariable regions called "complementary determining regions" or "CDRs". The framework regions of the antibody, that is, the framework regions constituting the combination of the light and heavy chains of the components, play a role in positioning and aligning the CDRs, which are mainly responsible for binding to the antigen.

[0075] As used herein, the "framework", "framework region" or "FR" region means the region outside the regions defined as CDRs in the variable domain of the antibody. Each antibody variable domain framework can be further divided into adjacent regions (FR1, FR2, FR3 and FR4) separated by CDRs. Generally, the variable regions VL / VH of the heavy and light chains can be obtained by arranging and connecting the following numbered CDRs and FRs in the following combination: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0076] Based on the above description of CDRs, the present application does not particularly limit the species origin of the framework region and the constant region, nor the sequence of the constant region. Optionally, the sequence of the constant region is selected from the sequences of any one of IgG (including IgG1, IgG2, IgG3, IgG4), IgA, IgM, IgE, and IgD. Optionally, the species origin of the framework region and the constant region can be independently bovine, equine, porcine, ovine, rat, mouse, guinea pig, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, or human.

[0077] The binding proteins, antibodies, or antigen-binding fragments of antibodies described herein are chimeric because they contain at least one human constant region. For example, the constant region of an antibody produced by the hybridomas of the present application can be replaced (partially or completely) with a human constant region. Relative to non-chimeric antibodies, chimeric antibodies generally have lower immunogenicity in humans and can therefore be used in the development of antibody drugs in certain cases. In some embodiments, the chimeric antibodies described herein contain IgG constant regions. Those skilled in the art know various human constant regions. Methods for preparing chimeric antibodies are known in the art. In in vitro diagnostic clinical applications, the selection of the Fc segment of an antibody can often directly affect the specificity of clinical detection, while the species origin of the antibody plays a crucial role in the affinity of the antibody and determines the sensitivity of the reagent in clinical detection.

[0078] In some embodiments, the binding proteins, antibodies, or fragments described herein are humanized because they contain at least one murine framework region; further optionally, the sequences of the heavy chain framework region and the light chain framework region are all or partially the antibody framework region sequences derived from mice. For example, one or more (e.g., one, two, three, four, five, or six) framework regions of an antibody produced by the hybridomas of the present application can be replaced with one or more (e.g., one, two, three, four, five, or six) human framework regions. Those skilled in the art know various murine framework regions. Methods for preparing humanized antibodies are known in the art.

[0079] In some embodiments of the present application, the sequence of the constant region of the AFP-specific binding protein is selected from the sequences of any one of IgG, IgA, IgM, IgE, and IgD.

[0080] In some embodiments of the present application, the sequence of the light chain variable region of the AFP-binding domain a is as shown in SEQ ID NO. 20 or a sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9%) identity with SEQ ID NO. 20.

[0081] In some embodiments of the present application, the sequence of the heavy chain variable region of the AFP-binding domain a is as shown in SEQ ID NO. 19 or is a sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9%) identity to SEQ ID NO. 19.

[0082] In some embodiments of the present application, the sequence of the light chain variable region of the AFP-binding domain b is as shown in SEQ ID NO. 22 or is a sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9%) identity to SEQ ID NO. 22.

[0083] In some embodiments of the present application, the sequence of the heavy chain variable region of the AFP-binding domain b is as shown in SEQ ID NO. 21 or is a sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9%) identity to SEQ ID NO. 21.

[0084] In some embodiments of the present application, the heavy chain variable regions of the AFP-binding domain a and the AFP-binding domain b, and the light chain variable regions of the AFP-binding domain a and the AFP-binding domain b are each independently linked by a linker. The present application does not make special limitations on the linker, including but not limited to the linkers in SEQ ID NO. 5 and SEQ ID NO. 6.

[0085] The AFP-binding domain a of the present application may be located at the N-terminus of the AFP-specific binding protein or at the C-terminus of the AFP-specific binding protein. In some embodiments of the present application, the AFP-binding domain a is located at the N-terminus of the AFP-specific binding protein.

[0086] In the present application, the N-terminals of the heavy chain and light chain of the AFP-specific binding protein may or may not be linked to signal peptides. In some embodiments of the present application, the N-terminals of the heavy chain and light chain of the AFP-specific binding protein are each independently linked to a signal peptide. The present application does not particularly limit the sequence of the signal peptide, including but not limited to the signal peptides in SEQ ID NO.5 and SEQ ID NO.6.

[0087] In some embodiments of the present application, the sequence of the heavy chain variable region of the AFP-specific binding protein is as shown in SEQ ID NO.5 or a sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9%) identity to SEQ ID NO.5.

[0088] In some embodiments of the present application, the sequence of the light chain variable region of the AFP-specific binding protein is as shown in SEQ ID NO.6 or a sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9%) identity to SEQ ID NO.6.

[0089] In a second aspect of the embodiments of the present application, there is provided a nucleic acid encoding the AFP-specific binding protein described above.

[0090] This nucleic acid can be used to construct an expression vector, such as an expression vector containing the above nucleic acid sequence. This nucleic acid sequence or expression vector can also be transfected into a host cell, such as a host cell transfected or transformed with the above expression vector or the above nucleic acid sequence. Herein, the nucleic acid sequence includes its variants with conservative substitutions (e.g., substitutions of degenerate codons) and complementary sequences. The terms "nucleic acid" and "polynucleotide" are synonymous and include genes, cDNA molecules, mRNA molecules and their fragments such as oligonucleotides.

[0091] In a third aspect of the embodiments of the present application, there is provided a vector including the nucleic acid described above.

[0092] The nucleic acid therein is operably linked to at least one regulatory sequence. "Operably linked" means that the coding sequence is linked to the regulatory sequence in a manner that allows the expression of the coding sequence. The regulatory sequence is selected to direct the expression of the target protein in a suitable host cell and includes promoters, enhancers and other expression regulatory elements.

[0093] In this article, a vector can refer to a molecule or reagent that contains the nucleic acid of the present invention or a fragment thereof, is capable of carrying genetic information, and can deliver the genetic information into cells. Typical vectors include plasmids, viruses, bacteriophages, cosmids, and minichromosomes. A vector can be a cloning vector (i.e., a vector used to transfer genetic information into cells, which can propagate the cells and can select the cells with or without the genetic information) or an expression vector (i.e., a vector that contains the necessary genetic elements to allow the genetic information of the vector to be expressed in cells). Therefore, a cloning vector can contain a selection marker and an origin of replication that matches the cell type specified by the cloning vector, while an expression vector contains regulatory elements necessary for affecting expression in the specified target cells.

[0094] The nucleic acid of this article or a fragment thereof can be inserted into a suitable vector to form a cloning vector or an expression vector carrying the nucleic acid fragment of the present invention. This new vector is also part of the present invention. The vector can include plasmids, bacteriophages, cosmids, minichromosomes, or viruses, and also includes naked DNA that is only transiently expressed in specific cells. The cloning vectors and expression vectors of the present invention can replicate spontaneously, and thus can provide a high copy number for high-level expression or high-level replication purposes for subsequent cloning. An expression vector can include a promoter for driving the expression of the nucleic acid fragment of the present invention, an optional nucleic acid sequence encoding a signal peptide that enables the peptide expression product to be secreted or integrated into the membrane, the nucleic acid fragment of the present invention, and an optional nucleic acid sequence encoding a terminator. When operating an expression vector in a production strain or cell line, the vector can be integrated into the genome of the host cell when introduced into the host cell, or it may not be integrated into the host cell genome. The vector usually carries a replication site and a marker sequence that can provide phenotypic selection in transformed cells.

[0095] There is no particular limitation on the type of vector in this article. For example, it can be a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, or a mammalian cell virus.

[0096] In the fourth aspect of the embodiments of this application, a host cell is provided, and the host cell includes the nucleic acid or the vector described above.

[0097] Host cells also include cells from multicellular organisms such as fungi, insect cells, plant cells, or mammalian cells, preferably cells from mammals, such as CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, or HEK293 cells, etc. The host cell can replicate the nucleic acid fragment of this application. When recombinantly preparing the binding protein of this application, the binding protein can be secreted into the culture medium or carried on the surface of the host cell.

[0098] In the fifth aspect of the embodiments of the present application, a method for preparing an AFP-specific binding protein is provided, and the host cell as described above is used in the preparation method to produce the AFP-specific binding protein.

[0099] In the sixth aspect of the embodiments of the present application, an application of the above-mentioned AFP-specific binding protein in the preparation of an AFP detection product is provided. The present application does not particularly limit the type of the detection product, which may be a reagent, a chip, a test strip, etc.

[0100] In the seventh aspect of the embodiments of the present application, a detection kit is provided, and the detection kit includes the above-mentioned AFP-specific binding protein.

[0101] The present application does not particularly limit the detection mechanism of the kit. For example, the sandwich immunoassay method can be used, and specifically, it can be immunochromatography, chemiluminescence immunoassay, immunoturbidimetry, etc. In some embodiments, the development of the antibody provided by the present application can be applied to chemiluminescence kits and immunofluorescence chromatography kits; the chemiluminescence kit has the characteristics of being fully automated, high-throughput, and highly sensitive. The developed kit can detect whole blood, serum, and plasma. In particular, the application of whole blood can greatly shorten the detection time, provide good assistance for the diagnosis and treatment of acute aortic syndrome, and win valuable time for the diagnosis and treatment of this disease.

[0102] The detection kit of the present application based on the sandwich immunoassay method includes a first antibody and a second antibody for detecting AFP. Among the first antibody and the second antibody, one has the AFP binding domain a and the AFP binding domain b, and the other has any one of the AFP binding domain a and the AFP binding domain b.

[0103] In some embodiments of the present application, among the first antibody and the second antibody, one is a bispecific antibody and the other is a chimeric antibody.

[0104] In the eighth aspect of the embodiments of the present application, a method for detecting AFP in a sample to be tested is provided, and the above-mentioned AFP-specific binding protein or the above-mentioned detection kit is used in the detection method.

[0105] In some embodiments, the detection method uses the sandwich immunoassay method. Optionally, in the sandwich immunoassay method, one antibody is the binding protein a as defined above, and the other antibody is the binding protein b as defined above. Optionally, among the first antibody and the second antibody, one is a bispecific antibody and the other is a chimeric antibody.

[0106] The present application does not particularly limit the types of biological samples, including but not limited to whole blood, serum, and plasma.

[0107] The implementation plan of this application will be described in detail below in combination with embodiments. It should be understood that these embodiments are only used to illustrate this application and not to limit the scope of this application. For the experimental methods without specific conditions noted in the following embodiments, the guidance given in this application is preferably referred to, and it can also be carried out according to the experimental manuals or conventional conditions in this field, or according to the conditions recommended by the manufacturer, or by referring to the experimental methods known in this field.

[0108] In the following specific embodiments, regarding the measurement parameters of raw material components, if there is no special indication, there may be slight deviations within the weighing accuracy range. Regarding temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0109] Example 1

[0110] 1. Immunogen preparation

[0111] The full-length AFP gene fragment was respectively constructed into the vector pcDNA3.1(+) expression vector, and after recombinant expression in 293F cells, it was prepared by affinity chromatography and ion exchange chromatography to obtain the immunogen.

[0112] The full-length AFP gel pattern is shown in the appendix Figure 1 The AFP fragment gel pattern is shown in the appendix Figure 2 , Figure 2 In it, from left to right are marker, AFP fragment 1, AFP fragment 2, and AFP fragment 3 in turn.

[0113] The amino acid sequence composition is as follows:

[0114] (1) The full-length AFP is as shown in SEQ ID NO.1.

[0115] (2) AFP fragment 1 is as shown in SEQ ID NO.2.

[0116] (3) AFP fragment 2 is as shown in SEQ ID NO.3.

[0117] (4) AFP fragment 3 is as shown in SEQ ID NO.4.

[0118] 2. Screening of mouse monoclonal antibodies against AFP

[0119] The prepared full-length APF was used to immunize 6-8-week-old BalB / C mice. The full-length AFP and AFP protein fragments were respectively coated to detect the mouse tail blood by ELISA until the titer reached 10 5Stop immunization, remove the spleens of the mice, process them and fuse them with the mouse myeloma cell line Sp2 / 0, and screen for positive hybridoma cell lines by the limiting dilution method. During the screening of positive hybridomas, different AFP fragments were used as coating materials to specifically screen for antibodies against different domains, and antibodies AFP-01 against the full length of AFP, AFP-02 against AFP fragment 1, AFP-03 against AFP fragment 2, and AFP-04 against AFP fragment 3 were obtained.

[0120] Table 1. Detection results of monoclonal antibody cell supernatants (coated with the full length of AFP)

[0121] Dilution factor AFP-01 AFP-02 AFP-03 AFP-04 Original (+) (+) (+) (+) 10 (+) (+) 3.409 (+) 100 (+) 3.592 1.731 2.701 1000 2.235 1.703 0.235 0.373 10000 0.268 0.168 0.021 0.026 100000 0.033 0.023 0.029 0.021 1000000 0.021 0.019 0.015 0.017 Negative Control 0.022 0.026 0.023 0.024

[0122] Note: The dilution factor refers to the dilution gradient of the monoclonal antibody cell supernatant; Negative control refers to the negative control (i.e., the background); (+) represents a strong positive value, indicating that the reading of this reaction has exceeded the upper limit of the microplate reader.

[0123] Table 2. Detection results of monoclonal antibody cell supernatants (coated with AFP fragment 1)

[0124]

[0125]

[0126] Note: The dilution factor refers to the dilution gradient of the monoclonal antibody cell supernatant; Negative control refers to the negative control (i.e., the background); (+) represents a strong positive value, indicating that the reading of this reaction has exceeded the upper limit of the microplate reader.

[0127] Table 3. Detection results of monoclonal antibody cell supernatants (coated with AFP fragment 2)

[0128] Dilution factor AFP-01 AFP-02 AFP-03 AFP-04 Original 0.063 0.066 (+) 0.055 10 0.037 0.032 (+) 0.042 100 0.039 0.041 (+) 0.031 1000 0.041 0.036 0.903 0.035 10000 0.028 0.027 0.089 0.022 100000 0.022 0.021 0.037 0.029 1000000 0.018 0.023 0.023 0.024 Negative Control 0.024 0.019 0.025 0.021

[0129] Note: The dilution factor refers to the dilution gradient of the monoclonal antibody cell supernatant; Negative control refers to the negative control (i.e., the background); (+) represents a strong positive value, indicating that the reading of this reaction has exceeded the upper limit of the microplate reader.

[0130] Table 4. Detection results of monoclonal antibody cell supernatants (coated with AFP fragment 3)

[0131] Dilution factor AFP-01 AFP-02 AFP-03 AFP-04 Original 0.063 0.066 0.066 (+) 10 0.037 0.032 0.032 (+) 100 0.039 0.041 0.041 (+) 1000 0.041 0.036 0.036 2.328 10000 0.028 0.021 0.021 0.258 100000 0.022 0.021 0.021 0.023 1000000 0.025 0.029 0.023 0.023 Negative Control 0.024 0.019 0.019 0.028

[0132] Note: The dilution factor refers to the dilution gradient of the monoclonal antibody cell supernatant; Negative control refers to the negative control (i.e., the background); (+) represents a strong positive value, indicating that the reading of this reaction has exceeded the upper limit of the microplate reader.

[0133] Coat the ELISA plate with AFP full-length protein, AFP fragment 1, AFP fragment 2, and AFP fragment 3 at a concentration of 1 μg / mL respectively, and test the activity of the supernatant of each hybridoma cell line.

[0134] Table 5. Detection results of the sensitivity of monoclonal antibodies labeled with HRP

[0135] Dilution factor AFP-01-HRP AFP-02-HRP AFP-03-HRP AFP-04-HRP 1 / 500 (+) (+) (+) (+) 1 / 1000 (+) (+) (+) (+) 1 / 2000 (+) (+) (+) (+) 1 / 4000 (+) (+) (+) (+) 1 / 5000 (+) (+) 1.572 (+) 1 / 10000 1.665 (+) 0.697 (+) 1 / 20000 0.832 (+) 0.359 (+) 1 / 50000 0.103 (+) 0.076 (+) Negative Control 0.052 0.079 0.071 0.062

[0136] Note: The dilution factor refers to the dilution gradient of the monoclonal antibody sample labeled with HRP; Negative control refers to the negative control (i.e., the background); (+) represents a strong positive value, indicating that the reading of this reaction has exceeded the upper limit of the microplate reader.

[0137] Coat the ELISA plate with AFP full-length protein at a concentration of 1 μg / mL, and test the reactivity of each antibody labeled with HRP (horseradish peroxidase) with AFP full-length protein. AFP-02 and AFP-04 showed positive results for all gradients. Therefore, coat AFP-Ab01 and AFP-03 on the ELISA plate at a concentration of 1 μg / mL respectively to test the specificity of AFP-02-HRP and APF-04-HRP. The AFP-02 and AFP-04 antibodies labeled with HRP have strong non-specificity, so they are not suitable for the construction of subsequent bispecific antibodies.

[0138] Table 6. Detection results of the sensitivity of monoclonal antibody pairing tests

[0139]

[0140] Note: The dilution factor refers to the dilution gradient of the AFP02 and AFP04 antibody samples labeled with HRP; Negative control refers to the negative control (i.e., the background); (+) represents a strong positive value, indicating that the reading of this reaction has exceeded the upper limit of the microplate reader.

[0141] Antibody pair 1: AFP-01 (capture antibody) + APF-02-HRP; Antibody pair 2: AFP-01 (capture antibody) + APF-04-HRP; Antibody pair 3: AFP-02 (capture antibody) + APF-02-HRP; Antibody pair 4: AFP-02 (capture antibody) + APF-02-HRP; Antibody pair 5: AFP-03 (capture antibody) + APF-02-HRP; Antibody pair 6: AFP-03 (capture antibody) + APF-04-HRP; Antibody pair 7: AFP-04 (capture antibody) + APF-02-HRP; Antibody pair 8: AFP-04 (capture antibody) + APF-04-HRP.

[0142] 3. Construction of bispecific antibodies against APF

[0143] Amplify the corresponding positive hybridoma cell lines, and use RT-PCR method and appropriate primers to amplify the gene fragments of their variable regions, named: VH1 (heavy chain variable region of AFP-01), VL1 (light chain variable region of AFP-01); VH2 (heavy chain variable region of AFP-03), VL2 (light chain variable region of AFP-03).

[0144] Construct VH1 and VH2 into the heavy chain expression vector pFUSE-CHIg-mG1 simultaneously (the vector map is as Figure 3 shown), and construct VL1 and VL2 into the light chain expression vector pFUSE2-CLIg-mk simultaneously (the vector map is as Figure 4 shown).

[0145] Use an endotoxin-free plasmid extraction kit to obtain plasmids containing light and heavy chains, and then transiently transfect 293F cells at an appropriate ratio. After about 48 h, collect the cell culture supernatant, concentrate it, and purify it by Protein A affinity chromatography and ion exchange chromatography to obtain the bispecific antibody AFP-Ab-13. The SDS-PAGE electrophoresis pattern of the prepared bispecific antibody is as Figure 5 shown, Figure 6 which is a schematic diagram of the construction of the bispecific antibody. Similarly, construct VH1 and VH2 into the heavy chain expression vector pFUSE-CHIg-mG1 respectively, and construct VL1 and VL2 into the light chain expression vector pFUSE2-CLIg-mk respectively to obtain the chimeric antibodies AFP-c01 and AFP-c03.

[0146] 4. Verification of the activity of bispecific antibodies against APF

[0147] Coat AFP-Ab13, AFP-c01, AFP-c03, AFP-01, and AFP-03 onto the ELISA plate at a concentration of 1 μg / mL. After blocking with BSA, react with different concentrations of APF, and then add an appropriate concentration of HRP-labeled rabbit polyclonal antibody. Use TMB for color development. Among them, the rabbit polyclonal antibody was prepared according to the conventional polyclonal antibody preparation method:

[0148] The first immunization: Use the prepared full-length APF protein and AFP protein fragments (AFP fragments 1 to 3 shown in SEQ ID NO.2 to 4) as immunogens to perform subcutaneous immunization injections on New Zealand white rabbits. Select the immunization site at a location with rich lymph nodes, such as the back of the neck, and perform antigen immunization injections in a multi-point manner with a single injection.

[0149] The second serum titer test: After 3 immunizations, collect blood from the marginal ear vein of the rabbit for serum titer testing. Coat the full-length AFP and AFP protein fragments respectively and detect the tail blood of mice by ELISA until the titer reaches 10 6 Stop immunization and collect rabbit blood when it reaches the above level.

[0150] The third step of polyclonal antibody purification: Purify the rabbit polyclonal antibody by antigen-immunity affinity chromatography. First, centrifuge and filter the collected rabbit blood, then perform ammonium sulfate precipitation, collect the precipitate after centrifugation, and resuspend it with 1×PBS. The resuspended supernatant is dialyzed overnight at 4°C into 1×PBS. After dialysis, the supernatant is filtered and purified with a medium conjugated with the antigen. The obtained eluted sample is processed to form a finished product with a concentration ≥ 3.0 mg / mL. After aliquoting, it is stored frozen at -80°C.

[0151] Table 7. Detection results of the sensitivity of each AFP antibody-antigen test

[0152]

[0153]

[0154] Note: Negative control refers to the negative control (i.e., the background); (+) represents a strong positive value, indicating that the reading of this reaction has exceeded the upper limit of the ELISA reader.

[0155] Coat AFP-Ab13, AFP-c01, and AFP-c03 onto the ELISA plate at a concentration of 1 μg / mL. After blocking with BSA, react with different concentrations of APF protein, and then add an appropriate concentration of HRP-labeled AFP-Ab13, AFP-c01, and AFP-c03 antibodies. Use TMB for color development.

[0156] Table 8. Detection results of the sensitivity of each AFP antibody-paired antigen test

[0157] APF protein Antibody pair 9 Antibody pair 10 Antibody pair 11 Antibody pair 12 Antibody pair 13 Antibody pair 14 100 ng / mL (+) (+) (+) (+) (+) (+) 20 ng / mL (+) (+) (+) (+) (+) (+) 4 ng / mL (+) (+) (+) 3.412 (+) (+) 0.8 ng / mL 3.093 2.483 1.219 0.716 1.646 0.896 0.16 ng / mL 0.618 0.436 0.237 0.148 0.340 0.163 0.032 ng / mL 0.147 0.087 0.056 0.031 0.065 0.032 0.0064 ng / mL 0.029 0.035 0.029 0.032 0.029 0.031 0.0013 ng / mL 0.031 0.032 0.022 0.035 0.025 0.030 Negative Control 0.026 0.030 0.024 0.032 0.028 0.031

[0158] Note: Negative control refers to the negative control (i.e., the background); (+) represents a strong positive value, indicating that the reading of this reaction has exceeded the upper limit of the microplate reader.

[0159] Antibody pair 9: AFP-Ab13 (capture antibody) + AFP-c01-HRP;

[0160] Antibody pair 10: AFP-Ab13 (capture antibody) + AFP-c03-HRP;

[0161] Antibody pair 11: AFP-c01 (capture antibody) + AFP-Ab13-HRP;

[0162] Antibody pair 12: AFP-c01 (capture antibody) + AFP-c03-HRP;

[0163] Antibody pair 13: AFP-c03 (capture antibody) + AFP-Ab13-HRP;

[0164] Antibody pair 14: AFP-c03 (capture antibody) + AFP-c01-HRP.

[0165] The summary of the example sequences is as follows:

[0166] SEQ ID NO.1:

[0167] MKWVESIFLIFLLNFTESRTLHRNEYGIASILDSYQCTAEISLADLATIFFAQFVQEATYKEVSKMVKDALTAIEKPTGDEQSSGCLENQLPAFLEELCHEK

[0168] EILEKYGHSDCCSQSEEGRHNCFLAHKKPTPASIPLFQVPEPVTSCEAYEEDRETFMNKFIYEIARRHPFLYAPTILLWAARYDKIIPSCCKAENAVECFQT

[0169] KAATVTKELRESSLLNQHACAVMKNFGTRTFQAITVTKLSQKFTKVNFTEIQKLVLDVAHVHEHCCRGDVLDCLQDGEKIMSYICSQQDTLSNKITEC

[0170] CKLTTLERGQCIIHAENDEKPEGLSPNLNRFLGDRDFNQFSSGEKNIFLASFVHEYSRRHPQLAVSVILRVAKGYQELLEKCFQTENPLECQDKGEEELQ

[0171] KYIQESQALAKRSCGLFQKLGEYYLQNAFLVAYTKKAPQLTSSELMAITRKMAATAATCCQLSEDKLLACGEGAADIIIGHLCIRHEMTPVNPGVGQCC

[0172] TSSYANRRPCFSSLVVDETYVPPAFSDDKFIFHKDLCQAQGVALQTMKQEFLINLVKQKPQITEEQLEAVIADFSGLLEKCCQGQEQEVCFAEEGQKLISKTRAALGVHHHHHH*

[0173] SEQ ID NO.2:

[0174] MKWVESIFLIFLLNFTESRTLHRNEYGIASILDSYQCTAEISLADLATIFFAQFVQEATYKEVSKMVKDALTAIEKPTGDEQSSGCLENQLPAFLEELCHEK

[0175] EILEKYGHSDCCSQSEEGRHNCFLAHKKPTPASIPLFQVPEPVTSCEAYEEDRETFMNKFIYEIARRHPFLYAPTILLWAARYDKIIPSCCKAENAVECFQT KAATVT HHHHHH*

[0176] SEQ ID NO.3:

[0177] MKWVESIFLIFLLNFTESKELRESSLLNQHACAVMKNFGTRTFQAITVTKLSQKFTKVNFTEIQKLVLDVAHVHEHCCRGDVLDCLQDGEKIMSYICSQ

[0178] QDTLSNKITECCKLTTLERGQCIIHAENDEKPEGLSPNLNRFLGDRDFNQFSSGEKNIFLASFVHEYSRRHPQLAVSVILRVAKGYQELLEKCFQTENPLE CQDKGEEELQHHHHHH*

[0179] SEQ ID NO.4:

[0180] MKWVESIFLIFLLNFTESKYIQESQALAKRSCGLFQKLGEYYLQNAFLVAYTKKAPQLTSSELMAITRKMAATAATCCQLSEDKLLACGEGAADIIIGHL

[0181] CIRHEMTPVNPGVGQCCTSSYANRRPCFSSLVVDETYVPPAFSDDKFIFHKDLCQAQGVALQTMKQEFLINLVKQKPQITEEQLEAVIADFSGLLEKCCQ GQEQEVCFAEEGQKLISKTRAALGVHHHHHH*

[0182] Amino acid sequence of the heavy chain variable region of AFP-Ab-13 (SEQ ID NO.5)

[0183] MGWSWIFLFLLSGTAGVLSEVQVQLQQSGDDLVKPGVSVKISCKGSGYTFTGYTFQNYWITWVKQRPGQGLEWIG RIAPGDSTSYYNEMFKG KAT

[0184] LTVDTSSSTAYIQLTSLSSEDSAVYFCAR MGNYGFSY WGQGTLVTVSAAKGGGSGGGSGGGSGGGSQLQQSGPELVKPGASVKISCKTSGYTFT EY

[0185] SFTGYMN WVKQSHGKSLEWLG GLINPYNGGTRYNQKFKG KATLTVDKSSSTAYMELRSLTSEDSAVYYCAR FGRYGTISYALDY WGQGTTLTVSSAKTTA*1aa - 21aa is the signal peptide.

[0186] Amino acid sequence of the light chain variable region of AFP-Ab-13 (SEQ ID NO.6):

[0187] MSVLTQVLALLLLWLTGARCDVVMTQSTPLTLVTIGDRVSISC SSQSLLFDFHWYG WFLQKPGQPPKRLIY LVSRLDSG VPDRFTGSGSGTDFTLTIS

[0188] NVQSEDLGVYYFCWQTHSYPLT FGGGTKLEIKRADAAPTVSSGGGSGGGSGGGSGGGSDIVMTQSQAFMSTSVGETVTITC KASGNVGTAVA WI QQKQGKSPKLLVY SASNKFT GVPSRFSGSGSGTDFTLTIKSLQPEDFGSYYCHYQWSIPTFGAGTKLEIKRADAA *1aa - 20aa is the signal peptide.

[0189] In the variable region of AFP - Ab - 13, the target corresponding to the amino acid sequence before Liner is the N protein, the target corresponding to the amino acid sequence after Liner is the full - length AFP, and the target corresponding to the sequence after linker is the AFP fragment 2. AFP - 01 - VH CDR1 (SEQID NO.7): GYTFQNYWIT.

[0190] AFP - 01 - VH CDR2 (SEQ ID NO.8): RIAPGDSTSYYNEMFKG.

[0191] AFP - 01 - VH CDR3 (SEQ ID NO.9): MGNYGFSY.

[0192] AFP - 01 - VL CDR1 (SEQ ID NO.10): SSQSLLFDFHWYG.

[0193] AFP - 01 - VL CDR2 (SEQ ID NO.11): LVSRLDSG.

[0194] AFP - 01 - VL CDR3 (SEQ ID NO.12): WQTHSYPLT.

[0195] AFP - 03 - VH CDR1 (SEQ ID NO.13): EYSFTGYMN.

[0196] AFP - 03 - VH CDR2 (SEQ ID NO.14): GLINPYNGGTRYNQKFKG.

[0197] AFP - 03 - VH CDR3 (SEQ ID NO.15): FGRYGTISYALDY.

[0198] AFP - 03 - VL CDR1 (SEQ ID NO.16): KASGNVGTAVA.

[0199] AFP - 03 - VL CDR2 (SEQ ID NO.17): SASNKFT.

[0200] AFP-03-VL CDR3 (SEQ ID NO.18): HYQWSIPT.

[0201] AFP-01-VH (SEQ ID NO.19):

[0202] QVQLQQSGDDLVKPGVSVKISCKGSGYTFTGYTFQNYWITWVKQRPGQGLEWIGRIAPGDSTSYYNEMFKGKATLTVDTSSSTAYIQLTSLSSEDSAV YFCARMGNYGFSYWGQGTLVTVSAAK.

[0203] AFP-01-VL (SEQ ID NO.20):

[0204] DVVMTQSTPLTLVTIGDRVSISCSSQSLLFDFHWYGWFLQKPGQPPKRLIYLVSRLDSGVPDRFTGSGSGTDFTLTISNVQSEDLGVYYFCWQTHSYPL TFGGGTKLEIKRADAAPTVSS.

[0205] AFP-03-VH (SEQ ID NO.21):

[0206] QLQQSGPELVKPGASVKISCKTSGYTFTEYSFTGYMNWVKQSHGKSLEWLGGLINPYNGGTRYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAV YYCARFGRYGTISYALDYWGQGTTLTVSSAKTTA.

[0207] AFP-03-VL (SEQ ID NO.22):

[0208] DIVMTQSQAFMSTSVGETVTITCKASGNVGTAVAWIQQKQGKSPKLLVYSASNKFTGVPSRFSGSGSGTDFTLTIKSLQPEDFGSYYCHYQWSIPTFG AGTKLEIKRADAA.

[0209] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered to be within the scope described in this specification.

[0210] The above-described embodiments merely represent several implementation manners of the present application, which are convenient for understanding the technical solutions of the present application specifically and in detail, but should not be construed as a limitation on the scope of patent protection of the application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several deformations and improvements can still be made, and these all belong to the protection scope of the present application. In addition, it should be understood that after reading the above teachings of the present application, those skilled in the art 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 those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided by the present application are all within the protection scope of the appended claims of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the content of the appended claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. An AFP-specific binding protein, characterized in that, The AFP-specific binding protein has an AFP binding domain a or / and an AFP binding domain b; The AFP binding domain a includes: VH CDR1 shown in SEQ ID NO.7, VH CDR2 shown in SEQ ID NO.8, and VH CDR3 shown in SEQ ID NO.9; and, VL CDR1 shown in SEQ ID NO.10, VL CDR2 shown in SEQ ID NO.11, and VL CDR3 shown in SEQ ID NO.12; The AFP binding domain b includes: VH CDR1 shown in SEQ ID NO.13, VH CDR2 shown in SEQ ID NO.14, and VH CDR3 shown in SEQ ID NO.15; and, VL CDR1 shown in SEQ ID NO.16, VL CDR2 shown in SEQ ID NO.17, and VL CDR3 shown in SEQ ID NO.

18.

2. The AFP-specific binding protein according to claim 1, wherein The species origin of the framework region and constant region of the AFP-specific binding protein is independently bovine, equine, porcine, ovine, murine, canine, feline, rabbit, camel, donkey, deer, mink, chicken, duck, goose or human; or / and, the sequence of the constant region of the AFP-specific binding protein is selected from the sequences of any one of the constant regions of IgG, IgA, IgM, IgE and IgD.

3. The AFP-specific binding protein according to claim 2, characterized in that, The AFP-specific binding protein satisfies one or more of the following conditions: (1) The sequence of the light chain variable region of the AFP binding domain a is as shown in SEQ ID NO.20, or / and, the sequence of the heavy chain variable region of the AFP binding domain a is as shown in SEQ ID NO.19; and, (2) The sequence of the light chain variable region of the AFP binding domain b is as shown in SEQ ID NO.22, or / and, the sequence of the heavy chain variable region of the AFP binding domain b is as shown in SEQ ID NO.

21.

4. The AFP-specific binding protein according to any one of claims 1 to 3, characterized in that, The heavy chain variable regions of the AFP binding domain a and the AFP binding domain b and the light chain variable regions of the AFP binding domain a and the AFP binding domain b are independently connected by a linker; Optionally, the AFP binding domain a is located at the N-terminus of the AFP-specific binding protein; Optionally, signal peptides are independently connected to the N-termini of the heavy chain and light chain of the AFP-specific binding protein.

5. The AFP-specific binding protein according to any one of claims 1 to 3, characterized in that, The sequence of the heavy chain variable region of the AFP-specific binding protein is as shown in SEQ ID NO.5 or / and the sequence of the light chain variable region is as shown in SEQ ID NO.

6.

6. A nucleic acid, characterized in that, The nucleic acid encodes the AFP-specific binding protein according to any one of claims 1 to 5.

7. A carrier, characterized in that, The vector includes the nucleic acid according to claim 6; Optionally, the vector is a bacterial plasmid, phage, yeast plasmid, plant cell virus or mammalian cell virus.

8. A host cell, characterized in that, The host cell comprises the nucleic acid as claimed in claim 6 or the vector as claimed in claim 7; Optionally, the host cell is a CHO cell, a COS cell, an NSO cell, a HeLa cell, a BHK cell or a HEK293 cell.

9. A method for preparing an AFP-specific binding protein, characterized in that, The preparation method uses the host cell as claimed in claim 8 to produce the AFP specific binding protein.

10. Use of the AFP specific binding protein as claimed in any one of claims 1 to 5 in the preparation of an AFP detection product.

11. A detection kit, characterized in that, The detection kit comprises the AFP specific binding protein as claimed in any one of claims 1 to 5; Optionally, the detection kit comprises a first antibody and a second antibody for detecting AFP, and among the first antibody and the second antibody, one has the AFP binding domain a and the AFP binding domain b, and the other has any one of the AFP binding domain a and the AFP binding domain b; Optionally, among the first antibody and the second antibody, one is a bispecific antibody and the other is a chimeric antibody.

12. A detection method for AFP in a sample to be tested, characterized in that, The detection method uses the AFP specific binding protein as claimed in any one of claims 1 to 5 or the detection kit as claimed in claim 11.

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