CN120137040A - Rabbit antibody against human TRAIL-R3 protein and its application - Google Patents

Abstract

The present invention belongs to the technical field of antibody preparation, and in particular to rabbit antibodies against human TRAIL-R3 protein and their applications. The amino acid sequences of CDR1-3 on the light chain variable region of the antibody are shown as SEQ ID NO.3-5, respectively, and the amino acid sequences of CDR1-3 on the heavy chain variable region are shown as SEQ ID NO.8-10, respectively. The antibody of the present invention has good specificity and strong anti-interference ability, can effectively identify TRAIL-R3 protein without specific reaction with other non-target antigens, and the test results are highly consistent with the actual situation, which is conducive to avoiding the occurrence of false positive or false negative results, improving the accuracy, reliability and signal-to-noise ratio of the test results, and has broad development potential in the development of TRAIL-R3 diagnostic reagents and anti-tumor preparations.

Description

Rabbit-derived antibody against human TRAIL-R3 protein and application thereof

Technical Field

The invention relates to the technical field of antibody preparation, in particular to a rabbit-derived antibody for resisting human TRAIL-R3 protein and application thereof.

Background

TRAIL-R3, i.e. tumor necrosis factor-related apoptosis-inducing ligand receptor 3 (Tumor Necrosis Factor related apoptosis inducing ligand receptor, TRAIL R3), also known as CD263, tumor necrosis factor receptor superfamily member 10C (TNFRSF 10C) or decoy receptor 1 (DcR 1), is a glycophospholipid anchored cell membrane protein consisting of 259 amino acids, belonging to the tumor necrosis factor receptor superfamily. TRAIL-R3 comprises an extracellular domain rich in cysteine (Cys) and a transmembrane domain, without intracellular Death Domain (DD), which structural feature distinguishes it functionally from other TRAIL receptors with complete death domains, such as death receptors TRAIL-R1 (DR 4), TRAIL-R2 (DR 5). As a decoy receptor, TRAIL-R3 is capable of binding to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and due to lack of cytoplasmic DD, is unable to transmit apoptosis signals, thereby reducing the binding opportunity of TRAIL to its functional receptors (e.g., DR4 and DR 5), blocking apoptosis induced by death receptors, protecting normal cells from escaping TRAIL-mediated cytotoxicity, and modulating TRAIL-induced apoptosis.

Apoptosis is a programmed cell death process, and deregulation of apoptosis is one of the important causes of tumorigenesis, and cancer cells often evade death by inhibiting the apoptotic pathway. Clinical studies indicate that TRAIL-R3 is often highly expressed in many tumor tissues, which helps tumor cells to develop resistance to TRAIL-induced apoptosis, thereby avoiding the killing of the immune system of the organism, promoting the survival, proliferation and metastasis of tumor cells, and the expression level of TRAIL-R3 is often inversely related to the survival rate of patients, and the tumor cells highly expressing TRAIL-R3 may be more resistant to the treatment of TRAIL and the like. For example, in lung and breast cancers, high expression of TRAIL-R3 is associated with tumor resistance to chemotherapeutic drugs and poor prognosis. In addition, in autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus, abnormal expression and function of TRAIL-R3 on immune cells may cause an imbalance in apoptosis of immune cells, so that the autoreactive immune cells cannot be cleared in time, thereby initiating and aggravating autoimmune inflammatory reactions. Therefore, detection of TRAIL-R3 expression levels in serum or tissue can help aid in early diagnosis, disease monitoring and prognosis evaluation of tumors. On the other hand, TRAIL-R3 becomes an important target for tumor treatment based on its role in tumor cell escape. By inhibiting the expression of TRAIL-R3 or blocking the combination of TRAIL, the sensitivity of tumor cells to TRAIL-induced apoptosis is hopeful to be restored, and the tumor treatment effect is enhanced.

Both detection and targeted therapeutic strategies for TRAIL-R3 expression levels rely on the development of their high performance antibodies. Therefore, the obtained monoclonal antibody with good biological activity and high-efficiency and specificity targeting and binding to TRAIL-R3 protein has important significance for developing related TRAIL-R3 diagnostic reagents and anti-tumor preparations.

Disclosure of Invention

Aiming at the problems of lack of monoclonal antibodies with high biological activity and capability of specifically binding with human TRAIL-R3 protein and the like in the prior art, the invention provides a rabbit antibody for resisting the human TRAIL-R3 protein, and further provides application of the rabbit antibody or an antibody conjugate thereof in preparation of a human TRAIL-R3 protein immunoassay kit and a related immunoassay kit. In order to achieve the above purpose, the present invention is specifically realized by the following technical scheme:

The first aspect of the invention provides a rabbit antibody against human TRAIL-R3 protein, comprising a light chain variable region and a heavy chain variable region, wherein the amino acid sequences of CDR1, CDR2 and CDR3 on the light chain variable region are respectively shown as SEQ ID NO.3-5, and the amino acid sequences of CDR1, CDR2 and CDR3 on the heavy chain variable region are respectively shown as SEQ ID NO. 8-10.

Further, the amino acid sequence of the light chain variable region is shown as SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 7.

Further, the amino acid sequence of the antibody light chain is shown as SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 6.

Further, the antibody is a full-length antibody or an antigen-binding region thereof, and the antigen-binding region is at least one selected from the group consisting of a Fab fragment, a F (ab) ₂ fragment, an Fv fragment, (Fv) ₂ fragment, an scFv fragment, and an sc (Fv) ₂ fragment.

In a second aspect, the invention provides an antibody conjugate comprising an antibody of rabbit origin against human TRAIL-R3 protein as described above and a detection label linked to said antibody.

In a third aspect the invention provides a nucleic acid molecule encoding a rabbit antibody as described above against a human TRAIL-R3 protein, a recombinant vector comprising said nucleic acid molecule or a host cell comprising said nucleic acid molecule.

Further, the nucleic acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO.12 or a sequence complementary thereto, and the nucleic acid sequence of the heavy chain variable region is shown as SEQ ID NO.14 or a sequence complementary thereto.

Further, the nucleic acid sequence of the light chain of the antibody is shown as SEQ ID NO.11 or the sequence complementary to the light chain, and the nucleic acid sequence of the heavy chain is shown as SEQ ID NO.13 or the sequence complementary to the heavy chain.

In a fourth aspect, the invention provides the use of a rabbit-derived antibody or antibody conjugate against human TRAIL-R3 protein as described above in the preparation of a human TRAIL-R3 protein immunoassay kit.

Further, the immunodetection kit is selected from an enzyme-linked immunosorbent kit, an immunohistochemical kit, an immunofluorescence kit, an immunoblotting kit or a flow cytometry kit.

In a fifth aspect, the invention provides a human TRAIL-R3 protein immunoassay kit comprising a rabbit-derived antibody or antibody conjugate as described above against a human TRAIL-R3 protein.

Further, the kit further comprises a fluorescence-coupled anti-rabbit IgG secondary antibody.

The invention has the advantages and positive effects that:

The antibody has good specificity and strong anti-interference capability, can effectively identify TRAIL-R3 protein without specific reaction with other non-target antigens, has high degree of coincidence between detection results and actual conditions, is favorable for avoiding false positive or false negative results, improves the accuracy, reliability and signal-to-noise ratio of the detection results, and has wide development potential in the development of TRAIL-R3 diagnostic reagents and anti-tumor preparations.

Drawings

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

FIG. 1 is a graph showing the results of immune serum titer detection after rabbits were immunized with human TRAIL-R3 protein according to example 1 of the present invention;

FIG. 2 is a graph showing the results of flow cytometry detection of samples of human chorionic tumor cells combined with immune serum from rabbits immunized with human TRAIL-R3 protein according to example 1 of the present invention;

FIG. 3 is a graph showing the results of flow cytometry detection of human leukemia chronic myelogenous cell samples combined with immune serum obtained after immunization of rabbits with human TRAIL-R3 protein according to example 1 of the present invention;

FIG. 4 is a diagram of a rabbit-derived antibody expression vector for constructing an anti-human TRAIL-R3 protein according to example 1 of the present invention, from left to right, pRB322 vector carrying in advance an antibody light chain constant region and a heavy chain constant region, respectively;

FIG. 5 is a graph showing the results of flow cytometry detection of samples of human chorionic tumor cells and human leukemia chronic myelogenous cells using a rabbit antibody against the human TRAIL-R3 protein in example 2 of the present invention;

FIG. 6 is a diagram showing the result of immunoblotting detection of a sample of tumor cell lysate of human chorionic villus combined with a rabbit-derived antibody against human TRAIL-R3 protein according to example 2 of the present invention;

FIG. 7 is a graph showing the results of immunohistochemical detection of samples of human spleen and human brain tissue sections using a rabbit-derived antibody against human TRAIL-R3 protein according to example 2 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the following examples. The examples described herein are intended to illustrate the invention only and are not intended to limit the invention.

Various modifications to the precise description of the invention will be readily apparent to those skilled in the art from the information contained herein without departing from the spirit or scope of the appended claims. It is to be understood that the scope of the invention is not limited to the defined processes, properties or components, as these embodiments, as well as other descriptions, are merely illustrative of specific aspects of the invention. Indeed, various modifications of the embodiments of the invention which are obvious to those skilled in the art or related fields are intended to be within the scope of the following claims.

For a better understanding of the present invention, and not to limit its scope, all numbers expressing quantities, percentages and other values used in the present invention are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. Each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

In addition, it is noted that unless otherwise defined, in the context of the present invention, scientific and technical terms used should have meanings commonly understood by one of ordinary skill in the art.

The terms "comprising," "including," "having," and the like are intended to be non-limiting, as other steps and other ingredients not affecting the result may be added.

The term "and/or" should be taken to refer to a specific disclosure of each of the two specified features or components with or without the other. For example, "A and/or B" is considered to include the cases of (i) A, (ii) B, and (iii) A and B.

The terms "rabbit monoclonal antibody", "rabbit antibody" and "rabbit monoclonal antibody" have the same meaning, and refer to antibodies that specifically bind to Human (Human) tumor necrosis factor-related apoptosis-inducing ligand receptor 3, unless otherwise specified. The terms "TRAIL-R3", "CD263", "TRAIL-R3/CD263", "TRAIL-related apoptosis-inducing ligand receptor 3", "TRAIL receptor 3", "DcR1/CD263", "TNFRSF10C", etc. have the same meaning. The modifier "rabbit" means that the Complementarity Determining Regions (CDRs) of the antibody are derived from a rabbit immunoglobulin sequence.

An antibody is an immunoglobulin molecule capable of specifically binding to an antigen or epitope of interest through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. In the present invention, the term "antibody" is to be interpreted in the broadest sense and includes different antibody structures, including but not limited to so-called full length antibodies, antibody fragments, and genetic or chemical modifications thereof, as long as they exhibit the desired antigen binding activity. An antibody fragment may be one or more portions or fragments of a full-length antibody that retains the ability of the antibody to specifically bind to an antigen of interest.

A typical antibody molecule (full length antibody) consists of two identical light chains (L) and two identical heavy chains (H). Light chains can be classified into two types, kappa and lambda, respectively, heavy chains can be classified into five types, mu, delta, gamma, alpha and epsilon, respectively, and antibodies are defined as IgM, igD, igG, igA and IgE, respectively. The amino acid sequences of the heavy and light chains near the N-terminus vary greatly, the other portions of the amino acid sequences are relatively constant, the region of the light and heavy chains near the N-terminus, where the amino acid sequences vary greatly, is referred to as the variable region (V), and the region near the C-terminus, where the amino acid sequences are relatively stable, is referred to as the constant region (C). Heavy chain variable regions (VH) and light chain variable regions (VL) are typically the most variable parts of antibodies and contain antigen recognition sites. The VH and VL regions can be further subdivided into hypervariable regions (hypervariable region, HVR) also known as Complementarity Determining Regions (CDRs) which are circular structures, and Framework Regions (FR) where the heavy and light chain CDRs are held closely together and cooperate with one another by the FR regions to form surfaces complementary to the three-dimensional structure of the antigen or epitope of interest, determining the specificity of the antibody, and are the sites for antibody recognition and binding to the antigen. The FR region is the more conserved part of VH and VL, which are generally in the β -sheet configuration, joined by three CDRs forming a connecting loop. Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

The light chain constant region (CL) and the heavy chain constant region (CH) are not directly involved in binding of antibodies to antigens, but they exhibit different effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC). CL lengths of different classes of igs (κ or λ) are substantially identical, but CH lengths of different classes of igs are different, e.g. IgG, igA and IgD include CH1, CH2 and CH3, while IgM and IgE include CH1, CH2, CH3 and CH4. The amino acid sequences of the antibody heavy and light chain constant regions are well known in the art and can be obtained by querying the IMGT database.

Full length antibodies are the most complete antibody molecular structure, having a typical Y-type molecular structure, and thus, "full length antibodies", "complete antibodies" and "Y-type antibodies" are used interchangeably in the context of the present invention.

An antibody fragment is one or more portions or fragments of a full length antibody that substantially retains the same biological function or activity as the full length form, in particular, an antibody fragment comprises at least the same CDR regions, more preferably the same variable regions, as the full length antibody, thereby retaining intact antigen recognition and binding sites capable of binding to the same antigen, particularly to the same epitope, as the full length antibody. In typical examples, antibody fragments include Fab, F (ab) ₂、Fab'、F(ab')₂、Fv、(Fv)₂、scFv、sc(Fv)₂, which can be obtained by techniques conventional in the art.

(I) Fab: monovalent fragments consisting of the complete light chain (variable and constant regions) and part of the heavy chain (variable and first constant regions) are obtained by proteolytic cleavage of full-length antibodies to give fragments such as Fab, F (ab ') ₂, fab'. For example, igG can be degraded into two Fab fragments and one Fc fragment by papain, and into one F (ab ') ₂ fragment and one pFC' fragment by pepsin. The F (ab ') ₂ fragment was further reduced to form two Fab' fragments. Because the Fab has an antigen binding region and a partial constant region, the Fab not only has the antibody-antigen affinity like scFv, excellent tissue penetrating power and the like, but also has a more stable structure.

(Ii) F (ab) ₂ A bivalent fragment comprising two Fab's linked by a disulfide bridge in the hinge region.

(Iii) Fv: the variable fragment (Fv) is located at the N-terminus of the antibody Fab fragment, contains only the variable region, and consists of one light chain and one heavy chain variable region, is a VH and one VL non-covalently bound dimer (VH-VL dimer), and the 3 CDRs of each variable region interact to form an antigen binding site on the surface of the VH-VL dimer, and has the ability to recognize and bind antigen, although with less affinity than the whole antibody.

(Iv) (Fv) ₂ consists of two Fv fragments covalently linked together.

(V) scFv A Single chain antibody (scFv) is an Fv fragment comprising a Single polypeptide chain, wherein a heavy chain variable region (VH) and a light chain variable region (VL) are linked via a flexible linker (linker, typically consisting of 10-25 amino acids) which retains the binding specificity of the original antibody to an antigen, and the linker in the present invention is not particularly limited as long as it does not interfere with the expression of the antibody variable regions linked at both ends thereof. Compared with full-length antibodies, scFv has the characteristic of small molecular weight, thus having higher penetrability and lower immune side reaction.

(Vi) The sc (Fv) ₂ fragment is formed by connecting two heavy chain variable regions and two light chain variable regions by a linker or the like.

In some embodiments, the full length sequence of an antibody or antibody fragment of the invention may comprise CDR regions and FR regions from a rabbit immunoglobulin sequence. In other embodiments, the antibodies may comprise amino acid residues encoded by non-rabbit immunoglobulin sequences, e.g., humanized antibodies, chimeric antibodies, etc., to reduce body rejection while maintaining the desired specificity, affinity. The term "chimeric antibody" refers to an antibody in which a portion is derived from a particular source or species, while the remainder is derived from a different source or species. The term "humanized antibody" is a chimeric antibody in which the CDR regions of a non-human antibody, such as a rabbit antibody, and the FR regions derived from a human, in some cases the variable regions of the non-human antibody bind to the constant regions of a human antibody, such as a human rabbit chimeric antibody, and in other cases the CDR regions of the non-human antibody bind to the FR regions and constant regions derived from human antibody sequences, i.e., the CDR regions of the non-human antibody are grafted onto human antibody Framework (FR) sequences derived from the FR sequences of a single or multiple other human antibody variable regions. In the present invention, the CDR regions in the chimeric or humanized antibody are derived from rabbit-derived CDR regions.

The terms "monoclonal antibody" or "mab" and the like are used interchangeably and refer to a homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for small amounts of mutations and/or post-translational modifications (e.g., isomerization, amidation) that may occur naturally. "monoclonal antibodies" are highly specific, exhibiting a single binding specificity and affinity for the same or substantially the same epitope on an antigen. The modifier "monoclonal" indicates the antibody is obtained from a substantially homogeneous population of antibodies and is not to be construed as limiting the source or manner of preparation of the antibody. The antibodies can be prepared by a variety of methods including, but not limited to, hybridoma methods, phage display methods, yeast display methods, recombinant DNA methods, single cell screening, or single cell sequencing methods.

The term "specific binding" is a term well known in the art that exhibits "specific binding," "specific binding," or is referred to as "preferential binding" if a molecule reacts more frequently, more rapidly, longer in duration, and/or with greater affinity to a particular antigen or epitope of interest than to other antigens or epitopes of interest, and does not necessarily require (although may include) exclusive binding. In order that the above-recited objects, features and advantages of the present invention will become more readily apparent, a more particular description of the invention briefly described above will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings.

The embodiment of the invention provides a rabbit antibody for resisting human TRAIL-R3 protein, which comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region comprise 3 Complementarity Determining Regions (CDRs) which are respectively named as CDR1, CDR2 and CDR3, the amino acid sequences of the CDR1, the CDR2 and the CDR3 on the light chain variable region are respectively shown as SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, and the amino acid sequences of the CDR1, the CDR2 and the CDR3 on the heavy chain variable region are respectively shown as SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO. 10.

The invention provides an antibody strain capable of specifically recognizing and combining human TRAIL-R3 protein, which has good applicability in a plurality of immune detection systems, in particular to an immune blotting, immune histochemistry and flow cytometry detection system. Specifically, in an immunoblotting detection system, the antibody can accurately and sensitively detect TRAIL-R3 single bands in cell lysate, and does not specifically bind with other irrelevant antigens, in an immunohistochemical detection system, positive cell groups of the antibody are accurately positioned in a tissue sample, no staining signals are generated in a place without target antigens and in a negative tissue, the positive detection signals are strong, the background interference is low, in a flow cell detection system, the signal transition of the antibody on the positive cells is obvious, the signal on the negative cells which do not express the target antigens is similar to a isotype control, and the positive and negative cell groups can be accurately distinguished. The results show that the antibody of the invention has good specificity and strong anti-interference capability, can effectively identify TRAIL-R3 protein without specific reaction with other non-target antigens, has high degree of coincidence between the detection result and the actual situation, is favorable for avoiding false positive or false negative results, improves the accuracy, reliability and signal-to-noise ratio of the detection result, and has wide development potential in the development of TRAIL-R3 diagnostic reagents and antitumor preparations.

Alternatively, the light chain variable region and the heavy chain variable region each comprise 4 Framework Regions (FR), 4 FR and 3 CDRs sequentially staggered to form the variable region. The amino acid sequence of the light chain variable region (VL) of the antibody is shown as SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region (VH) is shown as SEQ ID NO. 7.

Optionally, the antibodies of the invention further comprise a light chain constant region (CL) and a heavy chain constant region (CH), CL and VL constituting the complete light chain (FL), CH and VH constituting the complete heavy chain (FH). The constant region of the antibody is typically obtained by public interrogation, e.g., via IMGT online database (www.imgt.org), searching rabbit source IGG GAMMA C REIGN for CH, searching rabbit source IGG KAPPA C REIGN for CL.

Specifically, the amino acid sequence of the antibody light chain is shown as SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown as SEQ ID NO.6.

It should be noted that the monoclonal antibody of the present invention may be a full-length antibody (having a typical Y-type molecular structure) or an antigen-binding region of the full-length antibody, which refers to a polypeptide that substantially retains the same biological function or activity as the full-length form, specifically, the antigen-binding region includes CDR regions as described above, more preferably has variable regions as described above, thereby retaining intact antigen recognition and binding sites capable of binding to the same antigen, particularly to the same epitope, as the full-length antibody. Optionally, the antigen binding region is selected from at least one of Fab, F (ab) ₂、Fab'、F(ab')₂、Fv、(Fv)₂, scFv, and sc (Fv) ₂. These antigen binding regions can be obtained by techniques conventional in the art.

Yet another embodiment of the invention provides a nucleic acid molecule encoding a rabbit antibody as described above against a human TRAIL-R3 protein, a recombinant vector comprising said nucleic acid molecule or a host cell comprising said nucleic acid molecule.

The nucleic acid molecule may be in the form of DNA (e.g., cDNA or genomic DNA or synthetic DNA) or RNA (e.g., mRNA or synthetic RNA). The DNA may be single-stranded or double-stranded, or may be a coding strand or a non-coding strand.

The sequence of the nucleic acid molecule is deduced from the antibody AA sequence by conventional means such as codon encoding rules. The full-length sequence of the nucleic acid molecule or a fragment thereof can be obtained by PCR amplification, recombinant methods or artificial synthesis.

Illustratively, the antibody light chain variable region has a nucleic acid sequence as shown in or complementary to SEQ ID NO.12, and the heavy chain variable region has a nucleic acid sequence as shown in or complementary to SEQ ID NO. 14.

Illustratively, the antibody light chain has a nucleic acid sequence shown in or complementary to SEQ ID NO.11, and the heavy chain has a nucleic acid sequence shown in or complementary to SEQ ID NO. 13.

It will be appreciated by those skilled in the art that nucleic acid molecules other than those exemplified above may likewise be encoded to produce antibodies of the invention due to the degeneracy of the genetic code, and therefore the nucleic acid molecules exemplified above should not be taken as limiting the scope of the invention.

The original vector from which the recombinant vector is constructed is a variety of vectors conventional in the art, as long as it is capable of harboring the nucleic acid molecule. Typical vectors include plasmids (e.g., pBR322, pUC series, pET series, pGEX series), viral vectors, phages (e.g., λgt4λB, λ -Charon, λΔz1 and M13), cosmids and minichromosomes. The vector may be a cloning vector (i.e., for transferring the nucleic acid molecule into a host and for mass propagation in a host cell) or an expression vector (i.e., comprising the necessary genetic elements to allow expression of the nucleic acid molecule inserted into the vector in a host cell). The antibody can be obtained by inserting the nucleic acid molecule into a suitable vector to form a cloning vector or an expression vector carrying the nucleic acid molecule, introducing the vector into a host cell, and culturing the vector under specific conditions. This is well known in the art and will not be described in detail herein.

The nucleic acid molecules encoding antibodies FL and FH of the present invention may be inserted into two vectors, respectively, which may be introduced into the same or different host cells. When the heavy and light chains are expressed in different host cells, each chain may be isolated from the host cell in which it is expressed and the isolated heavy and light chains mixed and incubated under appropriate conditions to form the antibody. In other embodiments, antibodies FL and FH nucleic acid molecules can also be cloned into a vector, each nucleic acid sequence is connected to the appropriate promoter downstream, for example, the heavy chain and light chain encoding each nucleic acid sequence operably connected to different promoters, or the heavy chain and light chain encoding nucleic acid sequences can be connected to a single promoter, so that both heavy chain and light chain can be expressed by the same promoter. The choice of expression vector/promoter depends on the type of host cell used to produce the antibody.

Transfection or transformation of the recombinant vector into the host cell is performed using conventional techniques. When the host is a prokaryote such as E.coli, competent cells capable of absorbing DNA are obtained after the exponential growth phase, treated with CaCl ₂ or MgCl ₂, or by microinjection, electroporation or liposome packaging. When the host is eukaryote, the gene transfer can be realized by adopting a DNA transfection method such as a calcium phosphate coprecipitation method, a microinjection method, an electroporation method, liposome packaging or gene gun bombardment method.

The host cell may be a prokaryotic or eukaryotic cell. Examples of prokaryotic host cells useful in the present invention include, but are not limited to, E.coli (e.g., DH 5. Alpha., JM109, BL21, W3110), bacillus (e.g., bacillus subtilis, bacillus thuringiensis), enterobacteriaceae strains (e.g., salmonella typhimurium, serratia marcescens), and Pseudomonas. Examples of eukaryotic host cells that can be used for transformation include, but are not limited to, yeast, insect cells, and animal cells, such as Drosophila S2 or Sf9 cells, mammalian CHO, CHO DG44, CHO-S, COS-7, 293 series cells, hepG2, huh7, 3T3, RIN, MDCK, and HEK293 cell lines. After obtaining the host cell transfected or transformed with the recombinant vector as described above, the antibody can be expressed by culturing under appropriate conditions, and then isolated to obtain the purified antibody.

In a typical embodiment, the preparation method of the antibody comprises the steps of connecting the heavy chain gene and the light chain gene of the antibody with a signal peptide in series, respectively loading the heavy chain gene and the light chain gene on an expression vector pBR322, co-transfecting human kidney epithelial cells (293F), culturing 293F cells, collecting cell culture supernatant, and purifying to obtain the target antibody strain. The selection of the signal peptide is designed according to the host cell, and the present invention is not particularly limited thereto.

In a further embodiment, the invention provides application of the rabbit-derived antibody of the anti-human TRAIL-R3 protein or the antibody conjugate thereof in preparing a human TRAIL-R3 protein immunoassay kit, wherein the antibody conjugate comprises the rabbit-derived antibody of the anti-human TRAIL-R3 protein and a detection label connected with the antibody.

The application advantages of the rabbit-derived antibody or the antibody conjugate thereof for resisting the human TRAIL-R3 protein in preparing the human TRAIL-R3 protein immunoassay kit are the same as those of the rabbit-derived antibody for resisting the human TRAIL-R3 protein in the prior art, and are not repeated herein.

It is emphasized that the antibodies of the invention may be used alone or may be linked (covalently or non-covalently) to a detection label to form antibody conjugates. In some embodiments, the antibodies of the invention are used as antigen binding (or capture) antibodies that specifically recognize and bind to TRAIL-R3 protein in the sample to be tested, after which TRAIL-R3 is detected qualitatively or quantitatively by analysis of the detection label signal attached thereto, in other embodiments, the anti-TRAIL-R3 protein antibody (as primary or capture antibody) is not labeled, and the detection label is conjugated to a primary antibody (as detection antibody) or other molecule, e.g., if the anti-TRAIL-R3 antibody is a rabbit IgG antibody, the secondary antibody may be an anti-rabbit IgG antibody, whereby qualitative or quantitative TRAIL-R3 detection is achieved by analysis of the change in detection label signal generated after the specific binding of the secondary antibody, e.g., as in the multiple detection systems established in example 2 of the invention described below.

The detection mark is used for generating recognizable signal change so as to recognize the antibody or the secondary antibody thereof according to the signal change, and then the expression condition of TRAIL-R3 antigen in the sample to be detected is recognized through the specific reaction of the antigen-antibody. Detection labels include, but are not limited to, biotin, fluorescent dyes (e.g., umbelliferone, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride), fluorescent proteins (e.g., isophthalocyanin, phycoerythrin, perCP, and phycocyanin), enzymes (e.g., alkaline phosphatase, acid phosphatase, beta-galactosidase, glucose oxidase, horseradish peroxidase, acetylcholinesterase, avidin), colloidal gold, colored magnetic beads, latex particles, radionuclides, detection antibodies, or combinations thereof.

The immunodetection methods described above include, but are not limited to, enzyme-linked immunosorbent assay (Enzyme linked immunosorbent assay, ELISA), enzyme-linked immunosorbent assay (Enzyme-linked Immunospot, ELISPOT), immunohistochemistry (Immunohistochemistry, IHC), immunofluorescence (IF), immunoblotting (Western blot, WB), immunoprecipitation (Immunoprecipitation, IP) and Flow Cytometry (FC).

Based on the same inventive concept, the embodiment of the invention also provides a human TRAIL-R3 protein immunoassay kit, which comprises the rabbit-derived antibody or the antibody conjugate thereof for resisting the human TRAIL-R3 protein.

Optionally, the kit is an enzyme-linked immunosorbent kit, an immunohistochemical kit, an immunofluorescence kit, an immunoblotting kit or a flow cytometry kit.

Preferably, the kit is an immunoblotting kit, an immunohistochemical kit or a flow cytometry kit, and the kit further comprises a fluorescence-coupled anti-rabbit IgG secondary antibody.

The invention will be further illustrated with reference to specific examples. The experimental methods in which specific conditions are not specified in the following examples are generally conducted under conventional conditions, for example, those described in the molecular cloning Experimental guidelines (fourth edition) published in Cold spring harbor laboratory, or are generally conducted under conditions recommended by the manufacturer.

Example 1 preparation of Rabbit-derived antibodies against human TRAIL receptor 3 (TRAIL-R3, CD 263)

The CD263 protein is immunized into white rabbits, then B cells capable of recognizing target antigens are enriched and separated from spleen of the rabbits based on single B cell marking and sorting technology, the separated B cells are cultured in a single cell form to obtain monoclonal antibody 1D9, finally, natural paired antibody light chain (VL) and heavy chain variable region (VH) genes are obtained from the B cells secreting the monoclonal antibody through PCR amplification by a genetic engineering recombinant expression technology, the genes are inserted into an expression vector in series with the light Chain (CL) and the heavy chain constant region (CH), and the monoclonal antibody 1D9 is produced in a large quantity through recombinant expression of the antibody genes. The sequencing work was done by Jin Kairui Biotechnology Inc., the Amino Acid (AA) and nucleotide (DNA) sequences of the antibodies are shown in Table 1, where LCDR1-3 represent CDRs 1-3 on the light chain and HCDR1-3 represent CDRs 1-3 on the heavy chain, respectively.

TABLE 1 sequence information of rabbit monoclonal antibody 1D9 of this example

1.1, Antigen preparation, wherein the adopted immunogen is a human TRAIL-R3/CD263 protein 26-236aa polypeptide fragment obtained by 293F expression and purification, the amino acid sequence of the whole length of the CD263 protein is shown in Uniprot No. O14798 or NCBI accession number NP-003832.3, and the gene sequence is shown in NCBI accession number NM-003841.5. The gene sequence corresponding to the 26-236aa polypeptide fragment is constructed into a pBR322 vector, 293F cells are transformed, high-quality recombinant Human CD263 mature protein with biological activity is expressed, and the purity of the obtained protein is more than 90%.

1.2, Animal immunization, wherein 2 New Zealand white rabbits are immunized by recombinant human TRAIL-R3/CD263 protein according to 200 mug, antigen is mixed with equal amount of complete Freund's adjuvant (purchased from Sigma company) to prepare an emulsifying agent before the first immunization, the emulsifying agent is subcutaneously injected into the abdomen and the back of the rabbits, 100 mug of immunogen is mixed with equal amount of incomplete Freund's adjuvant (purchased from Sigma company) every 3 weeks after the first immunization to prepare the emulsifying agent, and the emulsifying agent is subcutaneously injected into the abdomen and the back of the rabbits for two times. Rabbit serum is collected after six immunizations and diluted according to a ratio of 1:243000, titer of the rabbit serum against antigen protein is measured by an enzyme-linked immunosorbent assay (ELISA), rabbit with OD450nm more than 0.2 is taken, the rabbit is subjected to subcutaneous multipoint injection with 200 mug immunogen for boosting once, spleen is taken after three to four days, and affinity and specific reaction of the rabbit serum with a cell sample to be tested are measured by a Flow Cytometry (FC) after final immune serum dilution.

ELISA method for determining immune serum titer comprises (1) adding neutral avidin (Neutravidin) protein with final concentration of 2 μg/mL into ELISA plate at 25 μL/hole, coating at 4deg.C overnight, (2) adding biotinylated TRAIL-R3 polypeptide antigen with final concentration of 1 μg/mL into ELISA plate at 25 μL/hole, incubating at room temperature for 1h, (3) adding washing buffer (PBS containing 0.05% (v/v) Tween-20) at 75 μL/hole for 5 times, then adding blocking buffer (PBS containing 1% BSA, 0.5% gelatin and 5% sucrose) at 50 μL/hole, incubating at room temperature for 1h, (4) repeating (3) washing plate process for washing plate, incubating at 25 μL/hole for 4 ℃ overnight, starting with 1:1000 of serum to be tested, performing triple gradient dilution with dilution buffer (PBS containing 1% BSA), incubating for 8 gradients, (5) repeating (3) plate washing process for washing at 75 μL/hole, adding HRP (PBS containing 0.05% (v/v) Tween-20) for 5% of room temperature, adding 5% of anti-enzyme-coupling buffer (PBS containing 5% BSA at 25 μL/hole for 5nm, and stopping the ELISA plate at 37 ℃ for 1:37 ℃ for 1.37 nm (light-dark contrast), repeating the incubation for 1 ℃ for 1.5 ℃ for 1 nm (5 nm, and performing coupling reaction), the positive immune serum is obtained by taking the ratio of the measured value to the control value not less than 2.1.

The FC determination of immune serum specificity comprises the steps of (1) irradiating an ultraviolet sterilization super clean bench for 15-20min, opening a fan for 5min, preparing sterile work, (2) collecting and washing cells, determining total number of cells, checking that cell viability is 90% -95%, measuring cell samples including human chorionic tumor cells (BEWO) and human leukemia chronic myelogenous cells (K562), (3) resuspending the cells to about 3×10 ⁶-5×10⁶ cells/mL by using PBS buffer, dispensing the cells into 96-well V-shaped plates according to 100 mu L/well, washing once by using 1×PBS, (4) diluting L/D staining solution (Live/DEAD STAINING solution) of a biological Zombie NIR Fixable Viability Kit kit (product number 423105) according to a ratio of 1:1500, dispensing the diluted L/D staining solution into an orifice plate according to a ratio of 100 mu L/well, reacting for 15min at room temperature, (5) centrifuging for 5min, discarding the supernatant by using FACS buffer for 2 times, (6) dispensing 1× IntracelluLar Fixation buffer cells into a 96-well V-shaped plates according to 100 mu L/well, dispensing the diluted L/DEAD STAINING solution into a hole for 1×5min, centrifuging for 5min, dispensing the diluted L/DEAD STAINING solution into a hole for each time (centrifuging for 5 min) and performing a reaction of 5×498 m, dispensing the diluted L/37 min into a well plate according to a ratio of 100 mu L/well, washing 2 times with 1X Permeablization buffer, (10) dispensing fluorescent secondary antibody (Fluorescein (FITC) AffiniPure F (ab') 2Fragment Goat Anti-Rabbit IgG, available from jackson, cat. No. 111-096-046) diluted with 1X Permeablization buffer (dilution ratio 1:200) into well plates at 100. Mu.L/well, resuspending cells in the wells for 30min at room temperature, (11) washing 2 times with 1X Permeablization buffer, followed by resuspension of cells in each well with 200. Mu.LFACS buffer, preserving in the dark, AND (12) analyzing by Beckman cytoflex flow analyser using AND maintenance SOP-105-AND-CA-008 procedure. Rabbit IgG isotype control was used as isotype control.

The serum titer test results are shown in FIG. 1, wherein WA-64580D is the project number, and N18742 and N18743 are the rabbit numbers. The results of FC detection of immune serum and positive cells BEWO expressing CD263 protein and negative cells K562 not expressing CD263 are shown in FIGS. 2-3, respectively, wherein the abscissa represents relative fluorescence intensity, the ordinate represents relative cell number, the red curve is a blank control, the blue curve is a isotype control, the yellow curve is serum to be tested, and the dilutions of serum from left to right are 1:500 and 1:2000, respectively. From FIGS. 1-3, it can be seen that a stronger immune response is generated in rabbits during the sixth immunization and the seventh boost, immune serum has specific binding on positive cells, better fluorescence transition, no specific binding on negative cells, no fluorescence transition, and it is determined that high-specificity and high-affinity antibodies which can be used for flow detection are generated in rabbits, and the stage of separating spleen cells can be entered.

1.3 Isolation of B cells from spleen and sorting of antigen-specific B cells related methods see published patent "methods for efficient isolation of individual antigen-specific B lymphocytes from spleen cells" (publication No. CN110016462A, publication No. 2019-07-16) "and patent" an in vitro B lymphocyte culture system and use (publication No. CN111518765A, publication No. 2020-08-11) ".

Cloning of Gene encoding Rabbit-derived monoclonal antibody Positive clones were identified by antigen-coated ELISA of the cultured B cell supernatant. Cells of positive clones were collected and lysed, and RNA was extracted using Quick-RNA ^TM Micro Prep kit (available from ZYMO, cat. No. R1051) and reverse transcribed into cDNA. The natural paired rabbit antibody light chain variable region (VL) and heavy chain variable region (VH) genes were obtained by PCR amplification using cDNA as template and sequenced. The PCR system consisted of 4. Mu.L of cDNA, 1. Mu.L of forward primer (10 mM), 1. Mu.L of reverse primer (10 mM), 12.5. Mu.L of 2X Gloria HiFi (from ABclonal, cat. RK 20717) and 6.5. Mu. L H ₂ O, the PCR procedure consisting of 98℃for 30s followed by 40 cycles at 98℃for 10s,64℃for 30s,72℃for 30s and finally 72℃for 5min, the reaction was stored at 4 ℃. Primer sequences (5 '-3') for amplifying the VL and VH genes are shown below, and F and R represent forward and reverse primers, respectively.

VL-F TGAATTCGAGCTCGGTACCCATGGACACGAGGGCCCCCAC (see SEQ ID NO. 15);

VL-R CACACACACGATGGTGACTGTTCCAGTTGCCACCTGATCAG (see SEQ ID NO. 16);

VH-F TGAATTCGAGCTCGGTACCCATGGAGACTGGGCTGCGCTG (see SEQ ID NO. 17);

VH-R GTAGCCTTTGACCAGGCAGCCCAGGGTCACCGTGGAGCTG (see SEQ ID NO. 18).

Sequencing the amplified DNA product to obtain VL sequence shown as SEQ ID No.2 and VH sequence shown as SEQ ID No.7, and searching the IMGT online database (www.imgt.org) to obtain constant region sequence to obtain the complete light chain (FL) shown as SEQ ID No.1 and complete heavy chain (FH) shown as SEQ ID No.6 as antibody 1D9.

1.5, Expression and scale production of rabbit antibody 1D9, namely, respectively loading the obtained antibody heavy chain and light chain genes on an expression vector. In this example, the light chain constant region (CL) and heavy chain constant region gene (CH) were inserted into the mammalian expression vector pBR322 in advance, and the expression patterns thereof are shown in fig. 4, in which pBR322origin and f1origin are replication promoters, AMPCILLIN is a resistance gene, CMVpromoter is a transcription promoter, SV40PAterminator is a tailing signal, LIGHT CHAIN constant is the nucleic acid sequence of CL (left panel), and HEAVY CHAIN constant is the nucleic acid sequence of CH (right panel). And then connecting VL and VH genes with signal peptides at the upstream with expression vectors pBR322 carrying CL and CH genes respectively linearized by XbaI (955 bp) and NheI (949 bp) restriction endonucleases in a homologous recombination mode to obtain complete light chain (FL) and heavy chain (FH) gene expression vectors, and verifying that the vectors are successfully constructed through sequencing.

In order to more conveniently purify the antibody, secretory expression of the antibody is realized by adding a signal peptide upstream of VL and VH genes, the signal peptide is expressed by the antibody commonly used in the field, such as a rabbit monoclonal antibody of the patent 'anti-Human interferon alpha 2' and application thereof (publication number: CN116063487A, publication date: 2023-05-05) ', a high affinity Human IL-5 rabbit monoclonal antibody of the patent' high affinity Human IL-5 'and application thereof (publication number: CN115819578A, publication date: 2023-03-21)', a signal peptide 'MDTRAPTQLLGLLLLWLPGATF (the coding gene of the embodiment is atggacacgagggcccccact cagctgctgggtctgctgcttctgtggctgcctggcgctaccttt)', and a signal peptide 'METGLRWLLLVA VLKGVQC (the coding gene of the embodiment is atggagactgggctgcgctggcttctcctggtggcagttctgaaaggcgtgcagtg t)'. Of course, the person skilled in the art may also replace other signal peptide after obtaining the antibody sequence of the present invention to perform antibody expression, and thus the signal peptide sequence is not represented in the antibody sequences of table 1 in this example.

And co-transfecting the FL and FH expression vectors successfully constructed into 293F cells, and culturing for 72-96 hours after transfection to obtain the antibody for recognizing the human TRAIL-R3 protein in culture supernatant. The antibody of interest was purified from the culture supernatant using a proteona affinity gel resin (purchased from heaven and earth, cat No. SA 015100). The purity of the antibody was confirmed to be not less than 95% by 12% polyacrylamide gel electrophoresis (SDS-PAGE). The purified antibody is packaged and stored at low temperature of-20 ℃ for standby.

Example 2 establishment and Effect evaluation of an immunoassay method based on anti-human TRAIL-R3 rabbit antibody 1D9

In the embodiment, a flow analysis, immunoblotting and immunohistochemical detection system is established aiming at the rabbit monoclonal antibody 1D9, and the application performance of the antibody is evaluated.

1. Establishment of Flow Cytometry (FC) detection system

The FC assay was identical to example 1, except that the primary antibody was antibody 1D9 and the final concentration of primary antibody was 2. Mu.g/mL.

The results of FC detection of antibody 1D9 on positive cells BEWO expressing TRAIL-R3/CD263 protein (left panel) and on negative cells K562 not expressing TRAIL-R3/CD263 protein (right panel) are shown in FIG. 5, wherein the abscissa indicates the relative fluorescence intensity, the ordinate indicates the relative cell number, the red curve is a negative control, the blue curve is a isotype control, and the yellow curve is antibody 1D9. The figure shows that the antibody 1D9 has obvious fluorescence signal transition difference between a positive sample and a negative sample, namely, has specific binding on positive cells and no specific binding on negative cells MCF7, and can be proved to be capable of specifically recognizing TRAIL-R3/CD263 expressed by cells, effectively resisting interference of cell components, has no cross reaction with other complex antigen components on the cells, and has good specificity and accuracy for detecting TRAIL-R3/CD263 protein.

2. Establishment of immunoblotting (WB) detection system

The cell sample of WB is positive cell BEWO, and the detection steps comprise (1) electrophoresis, namely, cell lysis, protein lysate, electrophoresis by using Tris-Glycine-SDS-Buffer conventional electrophoresis solution (purchased from Monad) and 6% SDS-PAGE separation gel, (2) transfer of gel protein bands onto NC membrane in an electrotransfer system according to conventional method, (3) sealing, namely, placing NC membrane in TBST sealing solution containing 3% of skimmed milk powder for 30min at room temperature, (4) primary antibody incubation, namely, adding the antibody prepared in example 1 (the final concentration of the primary antibody is 0.5 mu g/mL), and incubating for 1h at room temperature, (5) secondary antibody incubation, namely, washing membrane 3-4 times by using TBST, adding secondary antibody working solution (from Abclonal, product number AS014, secondary antibody dilution ratio of 1:5000), room temperature for 1h, (5) color development, namely, washing membrane 3-4 times by using TBST, adding ECL hypersensitive color development solution, and developing.

The WB assay results of antibody 1D9 on positive cells BEWO are shown in FIG. 6. The antibody 1D9 can be seen to detect a single band of about 65kDa in BEWO cell lysates, has a strong band signal, accords with the TRAIL-R3/CD263 protein size, presents a positive signal, and shows that the antibody has good recognition specificity and sensitivity to TRAIL-R3/CD 263.

3. Construction of an immunohistochemical (Immunohistochemistry, IHC) detection System

The slice samples comprise human spleen tissue slices and human brain tissue slices, wherein the human spleen tissue slices are positive samples, and the human brain tissue slices are negative samples. Baking slices, namely immersing paraffin tissue slices which are baked at the constant temperature of 56 ℃ for 30min into dewaxing liquid 1, taking out the slices after 5min, immersing the paraffin slices in sequence of dewaxing liquid 2, dewaxing liquid 3, absolute ethyl alcohol 1, absolute ethyl alcohol 2 and absolute ethyl alcohol 3, placing the slices for 5min when the slices are placed in dewaxing liquid, and placing the slices for 3min when the slices are placed in absolute ethyl alcohol; washing the slice with running water for 3min, dewaxing 1-3 from the Santa Classification of Xiushi river, high pressure heat repair of 0.01M Tris-EDTA repair solution (pH 9.0), inactivation of endogenous peroxidase, soaking in PBS buffer solution for 3 times and 3min each time to remove buffer solution on the slice, immersing the slice in 3% hydrogen peroxide solution, incubating at room temperature for 10min, (4) sealing, soaking in PBS buffer solution for 3 times and 3min each time, removing buffer solution, dropping PBS sealing solution in the region to be detected, incubating at room temperature for 30min, (5) primary antibody incubation, dropping antibody 1D9 dilution (primary antibody final concentration is 5 mu g/mL), incubating for 60min, removing antibody working solution, quick rinsing in PBS buffer solution for 1 time and 3 times each time, soaking and washing for 3min each time, (6) secondary antibody incubation, namely, dropping secondary antibody working solution (purchased from Note, SD, incubating for 3100 min), removing buffer solution, dropping PBS sealing solution in the region to be detected, incubating at room temperature for 30min, dropping antibody 1D9 dilution (primary antibody final concentration is 5 mu g/mL), incubating at room temperature, incubating for 60min, dropping antibody working solution, rinsing the working solution is removed, quick rinsing the working solution is quickly, the color is changed when the color is required to be washed, and the color is changed under the condition of a microscope is obtained when the test is changed when the test is in a proper condition of the test condition of high, washing with inflow water for 10min, (8) counterstaining, immersing the slightly drained tissue slice in Mayer's hematoxylin for 1min, washing with running water for 3min, (9) returning blue, immersing the slightly drained tissue slice in saturated aqueous solution of lithium carbonate for bluing for 3s, washing with running water for 3min, (10) dewatering, immersing the tissue slice in absolute ethyl alcohol for 2 times, lifting up and down for several times during immersing, taking out after 10s, drying the tissue slice at high temperature (54-58 ℃), and (11) sealing the tissue slice, wherein a proper amount of neutral gum is dripped into the center of the tissue slice, and covering the tissue slice with a cover glass, wherein the tissue slice is completely covered after the cover glass is covered with a proper amount of gum, and the glue cannot overflow, and finally performing the scanning of the tissue slice.

IHC detection results of antibody 1D9 on positive tissue human spleen (left panel) and negative tissue human brain (right panel) are shown in FIG. 7.TRAIL-R3/CD263 protein is highly expressed in spleen, bone marrow, lung, pancreas, etc., and is not expressed in human brain tissue. The staining of the human spleen samples of the positive tissue of the left graph in FIG. 7 shows a pronounced tan reaction, with the darker the color and the greater the extent of the reaction. The negative human brain tissue sample has no obvious and clear brown color reaction, and the identification of the TRAIL-R3/CD263 protein by the antibody 1D9 in the tissue detection has good specificity and is not influenced by the tissue complexity, so that the detection accuracy and reliability can be improved when the antibody is used for detecting pathological tissue samples.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.

Claims (10)

1. The rabbit antibody for resisting the human TRAIL-R3 protein is characterized by comprising a light chain variable region and a heavy chain variable region, wherein the amino acid sequences of CDR1, CDR2 and CDR3 on the light chain variable region are respectively shown as SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, and the amino acid sequences of CDR1, CDR2 and CDR3 on the heavy chain variable region are respectively shown as SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO. 10.

2. The rabbit antibody against human TRAIL-R3 protein according to claim 1, wherein the amino acid sequence of the light chain variable region is shown in SEQ ID No.2 and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 7.

3. The rabbit source antibody of anti-human TRAIL-R3 protein according to claim 2, wherein the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO. 6.

4. The rabbit antibody against human TRAIL-R3 protein according to claim 1, wherein the antibody is a full-length antibody or an antigen-binding region thereof, and wherein the antigen-binding region is at least one selected from the group consisting of a Fab fragment, a F (ab) ₂ fragment, an Fv fragment, (Fv) ₂ fragment, an scFv fragment and an sc (Fv) ₂ fragment.

5. A nucleic acid molecule encoding a rabbit-derived antibody against a human TRAIL-R3 protein according to any one of claims 1-4.

6. The nucleic acid molecule of claim 5, wherein the light chain variable region of the antibody has a nucleic acid sequence as shown in or complementary to SEQ ID No.12 and the heavy chain variable region has a nucleic acid sequence as shown in or complementary to SEQ ID No. 14.

7. The nucleic acid molecule of claim 6, wherein the light chain of said antibody has a nucleic acid sequence as shown in or complementary to SEQ ID No.11 and the heavy chain has a nucleic acid sequence as shown in or complementary to SEQ ID No. 13.

8. Use of a rabbit-derived antibody against human TRAIL-R3 protein or an antibody conjugate thereof according to any one of claims 1-4 in the manufacture of a human TRAIL-R3 protein immunoassay kit, wherein the antibody conjugate is formed by the antibody ligation detection label.

9. A human TRAIL-R3 protein immunoassay kit comprising a rabbit-derived antibody against a human TRAIL-R3 protein, or an antibody conjugate thereof, according to any one of claims 1-4, formed from said antibody linked to a detection label.

10. The human TRAIL-R3 protein immunoassay kit of claim 9, which is an immunoblotting kit, an immunohistochemical kit, or a flow cytometry kit, further comprising a fluorescence-coupled anti-rabbit IgG secondary antibody.