Alliin monoclonal antibody and its preparation method and application

Abstract

The present invention provides an alliin monoclonal antibody, a preparation method, and an application thereof. Specifically, the present invention provides an anti-alliin antibody or an antigen-binding fragment thereof, which has excellent specificity and can be used to effectively detect alliin in a sample.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological preparations, and in particular relates to an alliin monoclonal antibody and a preparation method and application thereof. Background Art

[0002] Alliin is the signature sulfur-containing amino acid of garlic, containing carboxyl and amino groups, with a molecular formula of C6H 11 NO3S has a relative molecular mass of 177.22 and is stable. Domestic and international studies have shown that alliin has antibacterial, non-alcoholic fatty liver disease improvement, myocardial protection, and immune regulation effects.

[0003] Immunoassays rely on antigen-antibody specific reactions to rapidly detect small molecules. Antibody preparation is a hot topic and a challenge. Because alliin is a small molecule and non-immunogenic, it must be coupled to a carrier protein to form a complete antigen. Whether this conjugate is immunogenic and can stimulate antibody production is crucial to the preparation of alliin antibodies.

[0004] Therefore, developing an antibody against a complete antigen including the hapten alliin for the effective detection of alliin in garlic, garlic-related medicines, health foods and health products (disinfection products, food), and biological samples (blood, tissue samples) in the body after taking the above products is of great value to the basic research on garlic and the clinical research on garlic preparations. Summary of the Invention

[0005] The present invention provides an alliin monoclonal antibody and a preparation method and application thereof.

[0006] In a first aspect of the present invention, an anti-alliin antibody or antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the complementarity determining region (CDR) of the heavy chain variable region and the complementarity determining region (CDR) of the light chain variable region are selected from the following group:

[0007] (1) HCDR1 with the amino acid sequence shown in SEQ ID NO: 3,

[0008] The amino acid sequence of HCDR2 is shown in SEQ ID NO: 4,

[0009] The amino acid sequence of HCDR3 is shown in SEQ ID NO: 5,

[0010] LCDR1 having an amino acid sequence as shown in SEQ ID NO: 10,

[0011] LCDR2 having an amino acid sequence as shown in SEQ ID NO: 11, and

[0012] LCDR3 having an amino acid sequence as shown in SEQ ID NO: 12;

[0013] (2) HCDR1 with an amino acid sequence as shown in SEQ ID NO: 17,

[0014] The amino acid sequence of HCDR2 is shown in SEQ ID NO: 18,

[0015] The amino acid sequence of HCDR3 is shown in SEQ ID NO: 19,

[0016] LCDR1 having an amino acid sequence as shown in SEQ ID NO: 24,

[0017] LCDR2 having an amino acid sequence as shown in SEQ ID NO: 25, and

[0018] LCDR3 having an amino acid sequence as shown in SEQ ID NO: 12;

[0019] (3) HCDR1 with an amino acid sequence as shown in SEQ ID NO: 30,

[0020] The amino acid sequence of HCDR2 is shown in SEQ ID NO: 18,

[0021] The amino acid sequence of HCDR3 is shown in SEQ ID NO: 31,

[0022] LCDR1 having an amino acid sequence as shown in SEQ ID NO: 36,

[0023] LCDR2 having an amino acid sequence as shown in SEQ ID NO: 11, and

[0024] The amino acid sequence of LCDR3 is shown in SEQ ID NO: 37.

[0025] In another preferred embodiment, the heavy chain variable region and light chain variable region of the antibody or antigen-binding fragment thereof are selected from the following groups:

[0026] (1) a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 1, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 8;

[0027] (2) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 15, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 22;

[0028] (3) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 28, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 34.

[0029] In another preferred example, the heavy chain variable region includes the three complementary determining regions HCDR and the framework region of the heavy chain variable region for connecting the heavy chain variable region HCDR; the light chain variable region includes the three light chain complementary determining regions LCDR and the framework region of the light chain variable region for connecting the light chain complementary determining regions LCDR.

[0030] In another preferred embodiment, the anti-alliin antibody or antigen-binding fragment thereof is selected from the following group: Fab fragment, Fab' fragment, F(ab)'2 fragment, F(ab)'3 fragment, Fv, single-chain Fv antibody ("scFv"), double scFv, (scFv)2.

[0031] In another preferred embodiment, the light chain of the antibody further includes a light chain constant region.

[0032] In another preferred embodiment, the light chain constant region is of rabbit origin.

[0033] In another preferred embodiment, the heavy chain of the antibody further includes a heavy chain constant region.

[0034] In another preferred embodiment, the heavy chain constant region is of rabbit origin.

[0035] In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.

[0036] In another preferred embodiment, the antibody is a monoclonal antibody.

[0037] In another preferred embodiment, the antibody includes a monospecific, bispecific, trispecific antibody or a multispecific antibody.

[0038] In a second aspect of the present invention, a recombinant protein is provided, wherein the recombinant protein has:

[0039] (i) the anti-alliin antibody or antigen-binding fragment thereof according to the first aspect of the present invention;

[0040] and (ii) optionally a tag sequence to facilitate expression and / or purification.

[0041] In another preferred embodiment, the tag sequence includes a 6His tag.

[0042] In another preferred embodiment, the recombinant protein (or polypeptide) includes a fusion protein.

[0043] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a multimer.

[0044] In a third aspect of the present invention, a nucleotide molecule is provided, wherein the nucleotide molecule encodes the anti-alliin antibody or antigen-binding fragment thereof according to the first aspect of the present invention.

[0045] In another preferred embodiment, the nucleotide molecule comprises a nucleotide sequence encoding the heavy chain variable region as shown in SEQ ID NO: 2, and a nucleotide sequence encoding the light chain variable region as shown in SEQ ID NO: 9.

[0046] In another preferred embodiment, the nucleotide molecule comprises a nucleotide sequence encoding the heavy chain variable region as shown in SEQ ID NO: 16, and a nucleotide sequence encoding the light chain variable region as shown in SEQ ID NO: 23.

[0047] In another preferred embodiment, the nucleotide molecule includes a nucleotide sequence encoding the heavy chain variable region as shown in SEQ ID NO: 29, and a nucleotide sequence encoding the light chain variable region as shown in SEQ ID NO: 35.

[0048] In the fourth aspect of the present invention, a vector is provided, wherein the vector contains the nucleotide molecule described in the first aspect of the present invention.

[0049] In the fifth aspect of the present invention, a host cell is provided, which contains the vector described in the fourth aspect of the present invention, or has the nucleotide molecule described in the third aspect of the present invention integrated into its genome, or expresses the anti-alliin antibody or antigen-binding fragment thereof as described in the first aspect of the present invention.

[0050] In a sixth aspect of the present invention, a method for detecting alliin in a sample is provided, the method comprising the steps of:

[0051] (1) contacting a sample with the anti-alliin antibody or antigen-binding fragment thereof according to the first aspect of the present invention;

[0052] (2) Detecting whether a hapten-antibody complex is formed, wherein the formation of the complex indicates the presence of alliin in the sample.

[0053] In a seventh aspect of the present invention, a method for preparing the anti-alliin antibody or antigen-binding fragment thereof according to the first aspect of the present invention is provided, the method comprising the following steps:

[0054] (a) culturing the host cell according to the fifth aspect of the present invention under expression conditions to express the anti-alliin antibody or antigen-binding fragment thereof;

[0055] (b) isolating and purifying the anti-alliin antibody or antigen-binding fragment thereof described in (a).

[0056] In an eighth aspect of the present invention, there is provided a use of the anti-alliin antibody or antigen-binding fragment thereof according to the first aspect of the present invention, or the recombinant protein according to the second aspect of the present invention, for preparing a reagent, a detection plate, or a kit;

[0057] The reagent, detection plate or test kit is used for detecting whether a sample contains alliin, or for capturing alliin.

[0058] In the ninth aspect of the present invention, a detection plate is provided, comprising a substrate (support sheet) and a test strip, wherein the test strip contains the anti-alliin antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, or the recombinant protein as described in the second aspect of the present invention.

[0059] In another preferred embodiment, the test strip further comprises an antigen spotting area.

[0060] In another preferred embodiment, the antigen is a hapten or a complete antigen.

[0061] In another preferred embodiment, the test strip is composed of filter paper, chromatography material, nitrocellulose membrane and absorbent paper overlapped in sequence.

[0062] In the tenth aspect of the present invention, a kit is provided, comprising a container and the anti-alliin antibody or antigen-binding fragment thereof according to the first aspect of the present invention located in the container, or the kit comprises the detection plate and instructions for use according to the ninth aspect of the present invention.

[0063] In another aspect of the present invention, a kit is provided, comprising:

[0064] (1) a first container containing the anti-alliin antibody or antigen-binding fragment thereof according to the first aspect of the present invention; and / or

[0065] (2) a second container containing a second antibody directed against the anti-alliin antibody or antigen-binding fragment thereof according to the first aspect of the present invention; and / or

[0066] (3) a third container containing a cell lysis reagent;

[0067] or,

[0068] The kit contains the detection plate as described in the ninth aspect of the present invention.

[0069] In another preferred embodiment, the antibody in the first container is detectably labeled.

[0070] In another preferred embodiment, the antibody in the second container is detectably labeled.

[0071] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 AC shows the expression and purification test results of 1A10, 1F3 and 3E12 cloned recombinant antibodies (Coomassie Brilliant Blue staining), respectively: M: protein marker, IN: original sample, FT: flow-through, W: washing solution, E: eluate.

[0073] Figure 2 The titer curve of the antibody 1A10 of the present invention is shown.

[0074] Figure 3 The titer curve of the antibody 1F3 of the present invention is shown.

[0075] Figure 4 The potency curve of the antibody 3E12 of the present invention is shown. DETAILED DESCRIPTION

[0076] After extensive and in-depth research, the inventors have developed for the first time a complete antigen based on the alliin hapten and hemocyanin (KLH). Through extensive screening, they have developed and obtained specific preferred antibodies (1A10, 1F3, and 3E12) based on this antigen. The antiserum titer of the antibodies of the present invention can reach 1:32K, and the antibodies of the present invention have good affinity. The antibodies of the present invention can be used for the effective detection of alliin in garlic, garlic-related medicines, health foods, and health products (disinfectants, foods), as well as in biological samples (blood, tissue samples) in the body after taking the above products. The present invention was completed on this basis.

[0077] the term

[0078] In order to make the present invention easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used herein have the meanings generally understood by those of ordinary skill in the art to which the present invention belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can change. It should also be understood that the terms used herein are intended only to describe specific embodiments and are not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0079] As used herein, when used in reference to a specific recited value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0080] As used herein, the terms "comprise," "include," and "contain" are used interchangeably to include not only closed definitions but also semi-closed and open definitions. In other words, the terms include "consisting of," "consisting essentially of."

[0081] Hapten

[0082] Alliin is the signature sulfur-containing amino acid of garlic, containing carboxyl and amino groups, with a molecular formula of C6H 11 NO3S has a relative molecular mass of 177.22 and is stable. Domestic and international studies have shown that alliin has antibacterial, non-alcoholic fatty liver disease improvement, myocardial protection, and immune regulation effects.

[0083] Alliin has a very small molecular weight and is a hapten. It only has immunoreactivity but no immunogenicity, and cannot be used directly to immunize animals to produce antibodies. Therefore, the present invention uses alliin coupled to the carrier protein KLH (hemocyanin) to form an immunogenic complex, thereby inducing an immune response and producing the corresponding antibodies.

[0084] As used herein, the "hapten" of the present invention refers to alliin.

[0085] Complete antigen

[0086] Usually, haptens need to be covalently coupled to macromolecules such as KLH (hemocyanin) or BSA (bovine serum albumin) to become complete antigens that are both immunoreactive and immunogenic.

[0087] As used herein, the "complete antigen" of the present invention refers to the product of combining the hapten of the present invention with an appropriate protein carrier.

[0088] As used herein, the "protein carrier" in the present invention refers to any immunologically acceptable protein used to form a complete antigen, which may be, for example, hemocyanin, bovine serum albumin, etc.

[0089] The complete antigen prepared by the present invention, alliin-KLH, has excellent immunogenicity and can stimulate New Zealand white rabbits to produce a strong immune response. After one basic immunization and three booster immunizations, the antiserum titer can reach 1:32K; the complete antigen alliin-KLH well retains the immunoreactivity of alliin.

[0090] Antibody

[0091] In the present invention, the terms "antibody (Ab)" and "immunoglobulin G (IgG)" are heterotetrameric glycoproteins with the same structural characteristics, which are composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is connected to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes varies. Each heavy chain and light chain also has regularly spaced intrachain disulfide bonds. One end of each heavy chain has a variable region (VH), followed by a constant region. The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. One end of each light chain has a variable region (VL) and the other end has a constant region. The light chain constant region includes a domain CL; the constant region of the light chain pairs with the CH1 domain of the heavy chain constant region, and the variable region of the light chain pairs with the variable region of the heavy chain. Constant regions are not directly involved in antibody-antigen binding, but they exhibit various effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC). Heavy chain constant regions include IgG1, IgG2, IgG3, and IgG4 subtypes; light chain constant regions include κ (Kappa) or λ (Lambda). The heavy and light chains of an antibody are covalently linked by disulfide bonds between the CH1 domain of the heavy chain and the CL domain of the light chain. The two heavy chains of an antibody are covalently linked by an inter-polypeptide disulfide bond formed between the hinge region.

[0092] As used herein, the terms "Fab" and "Fc" refer to the fact that papain can cleave antibodies into two identical Fab fragments and one Fc fragment. The Fab fragment is composed of the VH and CH1 domains of the antibody's heavy chain and the VL and CL domains of the light chain. The Fc fragment (Fc) is composed of the CH2 and CH3 domains of the antibody. The Fc fragment has no antigen-binding activity and is the site of interaction between the antibody and effector molecules or cells.

[0093] In the present invention, the term "scFv" refers to a single-chain antibody fragment (scFv), which is composed of the antibody heavy chain variable region and the light chain variable region connected by a short linker of usually 15 to 25 amino acids.

[0094] As used herein, the term "variable" refers to the fact that certain portions of the variable region of an antibody differ in sequence, which contributes to the binding and specificity of each particular antibody for its specific antigen. However, this variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three segments within the heavy and light chain variable regions, known as the complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable region are known as the framework regions (FRs). The variable regions of native heavy and light chains each contain four FRs, which generally have a β-pleated sheet configuration and are connected by three CDRs that form a connecting loop, which in some cases may form a partial β-pleated sheet structure. The CDRs in each chain are closely aligned by the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. 1, pp. 647-669 (1991)).

[0095] As used herein, the term "framework region" (FR) refers to the amino acid sequence inserted between CDRs, that is, it refers to those parts of the light chain and heavy chain variable regions of immunoglobulins that are relatively conserved among different immunoglobulins in a single species. The light chain and heavy chain of immunoglobulins each have four FRs, respectively referred to as L-FR1, L-FR2, L-FR3, L-FR4 and H-FR1, H-FR2, H-FR3, H-FR4. Accordingly, the light chain variable domain can be referred to as:

[0096] (L-FR1)-(L-CDR1)-(L-FR2)-(L-CDR2)-(L-FR3)-(L-CDR3)-(L-FR4), and the heavy chain variable domain can be represented as (H-FR1)-(H-CDR1)-(H-FR2)-(H-CDR2)-(H-FR3)-(H-CDR3)-(H-FR4). Preferably, the FR of the present invention is a human antibody FR or a derivative thereof, wherein the derivative of the human antibody FR is substantially identical to the naturally occurring human antibody FR, i.e., the sequence identity reaches 85%, 90%, 95%, 96%, 97%, 98% or 99%.

[0097] Knowing the amino acid sequence of the CDRs, one skilled in the art can easily determine the framework regions L-FR1, L-FR2, L-FR3, L-FR4 and / or H-FR1, H-FR2, H-FR3, H-FR4.

[0098] As used herein, the term "human framework region" is a framework region that is substantially identical (about 85% or more, specifically 90%, 95%, 97%, 99% or 100%) to the framework region of a naturally occurring human antibody.

[0099] As used herein, the term "linker" refers to one or more amino acid residues inserted into an immunoglobulin domain that provide sufficient mobility for the light and heavy chain domains to fold into an exchange dual variable region immunoglobulin. In the present invention, preferred linkers are Linker1 and Linker2, wherein Linker1 connects the VH and VL of a single-chain antibody (scFv), and Linker2 is used to connect the scFv to the heavy chain of another antibody.

[0100] Examples of suitable linkers include a single glycine (Gly), or serine (Ser) residue. The identity and sequence of the amino acid residues in the linker may vary depending on the type of secondary structural element desired to be achieved in the linker.

[0101] In the present invention, the antibodies of the present invention also include conservative variants thereof, which refer to polypeptides in which up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids are replaced with amino acids having similar or similar properties compared to the amino acid sequence of the bispecific antibody of the present invention. These conservative variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0102] Table A

[0103]

[0104]

[0105] In the present invention, the terms "anti-", "binding", "specific binding" refer to the non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen to which it is directed. Typically, an antibody binds to the antigen with an equilibrium dissociation constant (KD) of less than about 10-7M, for example, less than about 10-8M, 10-9M, 10-10M, 10-11M or less. In the present invention, the term "KD" refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen. For example, surface plasmon resonance (SPR) is used to measure the binding affinity of an antibody to an antigen in a BIACORE instrument or the relative affinity of an antibody to an antigen is measured using ELISA.

[0106] In the present invention, the term "epitope" refers to a polypeptide determinant that specifically binds to an antibody. The epitope of the present invention is a region of an antigen that is bound by an antibody.

[0107] The present invention also provides polynucleotide molecules encoding the above-mentioned antibodies, fragments thereof, or fusion proteins thereof. The polynucleotides of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand.

[0108] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0109] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0110] Antibody preparation

[0111] The sequence of the DNA molecule of the antibody of the present invention or its fragment can be obtained by conventional techniques, such as PCR amplification or genomic library screening. In addition, the coding sequences of the light chain and heavy chain can be fused together to form a single-chain antibody.

[0112] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0113] In addition, the sequences can also be synthesized by artificial synthesis, especially when the fragment length is shorter. Usually, a long fragment can be obtained by synthesizing multiple small fragments and then connecting them.

[0114] Currently, DNA sequences encoding the antibodies (or fragments thereof, or derivatives thereof) of the present invention can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the present invention through chemical synthesis.

[0115] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0116] The host cell can be a prokaryotic cell, such as a bacterial cell, a lower eukaryotic cell, such as a yeast cell, or a higher eukaryotic cell, such as a mammalian cell. Preferred animal cells include (but are not limited to): CHO-S and HEK-293 cells.

[0117] Typically, the transformed host cells are cultured under conditions suitable for expression of the antibodies of the present invention. The antibodies of the present invention are then purified using conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography, among other conventional separation and purification methods well known to those skilled in the art.

[0118] The resulting monoclonal antibodies can be characterized by conventional means. For example, the binding specificity of the monoclonal antibodies can be determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of the monoclonal antibodies can be determined, for example, by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0119] The antibodies of the present invention can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be separated and purified by various separation methods utilizing its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic shock, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods.

[0120] Detection Uses and Kits

[0121] The antibodies of the present invention can be used in detection applications, for example, for detecting samples, thereby providing detection information.

[0122] In the present invention, the samples used include garlic, garlic-related medicines, health foods and health products (disinfection products, foods), cells, and tissue samples.

[0123] The samples used in the present invention include fixed or preserved garlic, garlic-related medicines, health foods and health products (disinfection products, foods), cells or tissue samples.

[0124] The present invention also provides a kit containing the antibody (or fragment thereof) of the present invention. In a preferred embodiment of the present invention, the kit further comprises a container, instructions for use, a buffer, etc. In a preferred embodiment, the antibody of the present invention can be fixed to a detection plate.

[0125] The main advantages of the present invention include:

[0126] (a) The complete antigen of the present invention has high immunogenicity and retains the immunoreactivity of alliin.

[0127] (b) The anti-alliin monoclonal antibodies 1A10, 1F3, and 3E12 of the present invention have excellent specificity and high titer, reaching 1:32K.

[0128] (c) In the animal immunization of the present invention, New Zealand white rabbits are selected as experimental animals. Rabbit monoclonal antibodies have the advantages of high specificity, high diversity, high stability and high affinity.

[0129] (d) The anti-alliin monoclonal antibodies 1A10, 1F3 and 3E12 of the present invention have good affinity.

[0130] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0131] Example 1 Preparation of Alliin-KLH Complete Antigen

[0132] The antigen preparation method of the present invention is as follows: the immune antigen is prepared by coupling the natural alliin small molecule hapten with the carrier protein KLH (hemocyanin). Specifically, it includes the following steps:

[0133] (1) 1 mg of protein was dialyzed / dissolved in 1 ml of 25 mM 2-(N-morpholino) (MES) ethanesulfonic acid, and 1 mg of alliin was added and mixed to dissolve.

[0134] (2) Add 0.5 ml of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) solution to 1 ml of the antigen-alliin small molecule mixture;

[0135] (3) Add 0.16 ml of 25 mM MES (pH = 5) to the mixture in (2);

[0136] (4) Mix and incubate at 4°C for 2 h;

[0137] (5) Dialyze at 4°C for 3 times, 2 h each time, to remove the unbound alliin molecules in the free state.

[0138] Example 2 Preparation of alliin monoclonal antibody

[0139] 2.1 Animal immunization

[0140] Eight healthy female New Zealand white rabbits (A, B, C, D, E, F, G, H) aged 4 months and weighing 2.1 kg were selected. The specific immunization schedule is shown in Table 1.

[0141] Table 1 Immunization schedule

[0142]

[0143] Final release: Whole blood was collected from the carotid artery, kept at 4°C overnight, and serum was cryopreserved. Spleen cells were collected and cryopreserved in liquid nitrogen.

[0144] After the second and fourth immunizations, the serum titer was tested by indirect ELISA. The specific steps are as follows:

[0145] (1) Coating Antigen: Dilute the BSA-alliin screening agent to 2 μg / ml with 0.05 mol / L carbonate (pH = 9.6), add 100 μl / well, and incubate overnight at 4°C. Pour off the solution in the plate and wash three times with 0.05% Tween-20 (PBST), each wash for 3 minutes.

[0146] (2) Blocking: Add 150 μl of 5% skim milk powder to each well and block at 37°C for 60 min.

[0147] (3) Washing the plate: Take out the plate and wash it three times with PBST, 3 minutes each time.

[0148] (4) Add primary antibody: dilute the antiserum starting at 1:1000 and incubate at 37°C for 1 h. Pour off the solution in the plate and wash three times with PBST, 3 min each time.

[0149] (5) Add secondary antibody: horseradish peroxidase-conjugated goat anti-rabbit IgG (H+L), dilute 1:5000, and incubate at 37°C for 45 minutes. Pour off the solution in the plate and wash five times with 0.05% Tween-20 (PBST), 3 minutes each time.

[0150] (6) Color development: Add TMB color development solution (100 μl / well), react for 10 minutes, and add 100 μl of 2 mol / L sulfuric acid to terminate the reaction.

[0151] (7) Determination: OD value was measured at a wavelength of 450 nm using an enzyme-labeled instrument.

[0152] The results of indirect ELISA test on serum after the second immunization are shown in Table 2. The results of indirect ELISA test on serum after the fourth immunization are shown in Table 3.

[0153] Table 2 Results of indirect ELISA test on serum after the second immunization

[0154]

[0155]

[0156] Table 3 Results of indirect ELISA test on serum after four immunizations

[0157] A Rabbit B Rabbit C Rabbit D Rabbit E Rabbit F Rabbit G Rabbit H Rabbit Blank control 0.045 0.055 0.051 0.062 0.047 0.052 0.059 0.051 2K 2.296 0.924 2.151 2.231 1.833 2.26 1.759 1.156 4K 1.984 0.594 1.967 1.908 1.686 2.011 1.407 0.686 8K 1.879 0.344 1.682 1.694 1.194 1.846 1.084 0.502 16K 1.679 0.206 1.407 1.385 0.835 1.476 0.819 0.341 32K 1.209 0.158 1.023 1.103 0.572 1.079 0.502 0.298 64K 0.999 0.140 0.554 0.591 0.386 0.954 0.324 0.151 128K 0.629 0.134 0.389 0.344 0.288 0.620 0.285 0.144

[0158] In Tables 2 and 3, the higher the OD value, the stronger the antiserum titer. Finally, the antisera of rabbits A, C, D, and F were selected for competitive ELISA testing. The specific steps of competitive ELISA testing are as follows:

[0159] (1) Coating Antigen: Dilute the BSA-alliin screening agent to 2 μg / ml with 0.05 mol / L carbonate (pH = 9.6), add 100 μl / well, and incubate overnight at 4°C. Pour off the solution in the plate and wash three times with 0.05% Tween-20 (PBST), each wash for 3 minutes.

[0160] (2) Blocking: Add 150 μl of 5% skim milk powder to each well and block at 37°C for 60 min.

[0161] (3) Washing the plate: Take out the plate and wash it three times with PBST, 3 minutes each time.

[0162] (4) Adding the primary antibody: dilute the antiserum at 1:32,000 and mix with 100 μg / well of the competitor alliin (in a 2-fold serial dilution) and incubate at 37°C for 1 h. Pour off the solution in the plate and wash three times with PBST, 3 min each time.

[0163] (5) Add secondary antibody: horseradish enzyme-conjugated goat anti-rabbit IgG (H+L), dilute 1:5000, and incubate at 37°C for 45 min. Pour off the solution in the plate and wash five times with PBST, 3 min each time.

[0164] (6) Color development: Add TMB color development solution (100 μl / well), react for 10 min, and add 100 μl 2 mol / L sulfuric acid to terminate the reaction.

[0165] (7) Determination: OD value was measured at a wavelength of 450 nm using an enzyme-labeled instrument.

[0166] The results of the competitive ELISA test of serum after four immunizations are shown in Table 4.

[0167] Table 4 Results of serum competition ELISA test after four immunizations

[0168]

[0169]

[0170] 2.2B cell collection

[0171] Peripheral blood B cells were collected and washed twice with 1× PBS buffer, 50 ml per wash, at 1500 rpm / min, for centrifugation for 5 min, and the supernatant was discarded to collect the B cells.

[0172] 2.3 Single B cell screening

[0173] Positive B cells were screened by flow cytometry and antigen specificity.

[0174] 2.4 Cloning

[0175] Positive B cells were counted and plated in limiting dilution in 96-well plates. Cultured in M40105 medium for 15 days, the supernatant was collected for indirect ELISA. Sixty-five clones with high positive values ​​(OD values) were selected for subsequent competitive ELISA. The four clones with the highest difference between the competitive and control groups were selected for further testing. Ultimately, four clones, 1A10, 1F3, 2F3, and 3E12, were selected.

[0176] Among them, the results of indirect ELISA detection of B cell culture supernatant are shown in Table 5, and the results of competitive ELISA detection of B cell culture supernatant are shown in Table 6.

[0177] Table 5B Indirect ELISA test results of cell culture supernatant

[0178]

[0179]

[0180] Note: The bold black parts in the table are candidate positive clones.

[0181] Table 6B Competitive ELISA test results of cell culture supernatant

[0182]

[0183]

[0184] Note: The bold black part in the table is the candidate competitive positive clone; the primary antibody of the control group is 50μl PBS + 50μl 96-well positive supernatant screened in the first round; the primary antibody of the competition group is 50μl (concentration 20μg / ml, i.e. 1μg per well) alliin and 50μl 96-well positive supernatant screened in the first round.

[0185] 2.5 Single B Cell Sequencing

[0186] RNA was extracted from single B cells of four clones, 1A10, 1F3, 2F3, and 3E12, and reverse transcribed to amplify the heavy and light chain sequences, which were then sequenced. The sequencing results for antibodies 1A10, 1F3, and 3E12 are shown in Table B of the present invention.

[0187] Example 3 Expression of recombinant alliin antibody

[0188] 3.1 Expression and purification test

[0189] Based on the sequencing results in Example 2, the target genes (VH and VL coding sequences) of four monoclonal antibodies were transfected (transiently transfected) into HEK293 cells (human embryonic kidney 239 cells) for protein expression and purification tests.

[0190] The specific transfection experimental process is as follows:

[0191] (1) According to 0.5×10 6 Cells were inoculated into 300 ml of culture medium in a 1 L shake flask at an inoculum volume of cells / ml.

[0192] (2) Incubate in a shaking incubator at 37°C, 120 rpm, and 5% carbon dioxide for 24 h until the cell density reaches 1×10 6 cells / ml.

[0193] (3) Pipette 300 μg of DNA (sterilized by filtration) and add it to 30 ml of PBS, then vortex for 3 seconds to mix thoroughly.

[0194] (4) Add 1.2 ml of filter-sterilized Lip2000 solution to the PBS / DNA mixture.

[0195] (5) The Lip2000-DNA mixture was allowed to stand at room temperature for 20 minutes.

[0196] (6) Add the DNA / Lip2000 mixture to the cells. The cell density must reach 1.5-2.0×10 6 cells / ml.

[0197] (7) After transfection, the cells were cultured in a shaking incubator at 37°C, 120 rpm, and 5% carbon dioxide for 6 days.

[0198] (8) Centrifuge at 3000 × g for 5 min to collect the culture supernatant and cell pellet, and store at -80°C or use for subsequent purification.

[0199] 3.2 Expression Conditions

[0200] HEK293 cell density: 1.5-2.0 × 10 6 cells / ml; culture temperature: 37°C; culture speed: 130 rpm; culture sampling time: Day 6.

[0201] 3.3 Purification scheme

[0202] The specific steps are as follows:

[0203] (1) The resin was washed three times with 100 mM potassium acetate and 50 mM Tris (pH 7.5), and then equilibrated with PBS (pH 7.4).

[0204] (2) Use 1.25 ml of ProA column material per liter of culture. Incubate the culture supernatant with the affinity column material and place it in one or more 50 ml centrifuge tubes at 4°C with gentle rotation for 30-120 minutes.

[0205] (3) Centrifuge at 3000×g for 1 min at 4°C and discard the supernatant.

[0206] (4) Wash with 45 ml of pre-cooled PBS buffer.

[0207] (5) Add 10 ml of eluent (0.1 M glycine, pH 3.0) to the filler in sequence, adding 1 ml each time, and collect the antibody sample.

[0208] (6) SDS-PAGE detection of antibodies.

[0209] The results of expression and purification of 1A10, 1F3 and 3E12 cloned recombinant antibodies are as follows Figure 1 As shown in AC.

[0210] 3.4 Detection of positive recombinant antibody expression supernatant

[0211] The results of indirect ELISA detection of the recombinant antibody expression supernatant are shown in Table 7.

[0212] Table 7 Indirect ELISA test results of recombinant antibody expression supernatant

[0213] Sample Information OD value A 1A10 1.724 B 1F3 1.529 C 2F3 0.455 D 3E12 1.589 E Blank control 0.074 F Blank control 0.076 G Blank control 0.095 H Positive control 1.688

[0214] According to the test results, the antibody titer of clone 2F3 was too low after recombinant expression, so it was discarded. 1A10, 1F3 and 3E12 were selected to use the competition method to detect the specificity of the antibody and alliin small molecule. The results are shown in Table 8.

[0215] Table 8 Competitive ELISA test results of recombinant antibody expression supernatant

[0216]

[0217]

[0218] Note: The primary antibody in the negative control group was 200 μl PBS; the primary antibody in the positive control group was 50 μl of recombinant expression supernatant; the primary antibody in the competition group was 50 μl (concentration 20 μg / ml, i.e. 1 μg per well) alliin and 50 μl of recombinant expression supernatant.

[0219] Example 4

[0220] 4.1 Reagents and Materials

[0221] Alliin antibodies 1A10, 1F3, and 3E12 (experimental group), negative control (negative antibody), BSA-alliin, coating buffer (0.05 mol / L carbonate buffer, pH 9.6), washing buffer (PBS-T, PBS containing 0.05% Tween 20), blocking buffer (PBS containing 5% BSA), antibody diluent (PBS containing 1% BSA), enzyme conjugate (HRP-labeled goat anti-rabbit secondary antibody), color development reagent (TMB solution), stop solution (2 M H2SO4), 96-well ELISA plate.

[0222] Experimental procedures

[0223] 4.2.1 Board coating

[0224] (1) Add 100 μL of coating buffer containing BSA-alliin small molecules to each ELISA plate well at a concentration of 2 μg / mL.

[0225] (2) Incubate at 4°C overnight.

[0226] (3) Discard the coating solution and wash the plate three times with 300 μL of washing buffer each time.

[0227] 4.2.2 Board Closure

[0228] (1) Add 200 μL of blocking buffer to each well and incubate at room temperature for 1 hour.

[0229] (2) Pour off the blocking solution and wash the plate three times with 300 μL of washing buffer each time.

[0230] 4.2.3 Antibody incubation

[0231] (1) Different alliin antibodies (1 mg / ml) were diluted in antibody diluent according to different concentration gradients (1:500, 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000; i.e., 1:0.5K, 1:1K, 1:2K, 1:4K, 1:8K, 1:16K, 1:32K, 1:64K, 1:128K).

[0232] (2) Add 100 μL of diluted antibody solution to each well, including the experimental group and the negative control group.

[0233] (3) Incubate at 37°C for 1 hour.

[0234] (4) Pour off the antibody solution and wash the plate five times with 300 μL of wash buffer each time.

[0235] 4.2.4 Secondary Antibody Incubation

[0236] (1) Dilute HRP-labeled goat anti-rabbit secondary antibody (dilution ratio, 1:5000).

[0237] (2) Add 100 μL of diluted secondary antibody solution to each well.

[0238] (3) Incubate at 37°C for 1 hour.

[0239] (4) Pour off the secondary antibody solution and wash the plate five times with 300 μL wash buffer each time.

[0240] 4.2.5 Color reaction

[0241] (1) Add 100 μL of color development reagent (TMB solution) to each well.

[0242] (2) Incubate at room temperature for 10-20 minutes, protected from light.

[0243] (3) Add 50 μL of stop solution (2 M H2SO4) to each well to terminate the color development reaction.

[0244] 4.2.6 Results Reading

[0245] (1) Use an ELISA plate reader to read the absorbance value (OD450) at a wavelength of 450 nm.

[0246] (2) The potency of different antibodies (1A10, 1F3, and 3E12) against alliin small molecules was evaluated based on the absorbance values. The results are shown in Tables 9, 10, and 11, respectively.

[0247] Table 9

[0248]

[0249] Table 10

[0250]

[0251] Table 11

[0252]

[0253]

[0254] 4.3 Data Analysis

[0255] Experimental groups: OD450 values ​​of alliin antibodies at different dilution concentrations.

[0256] Negative control: OD450 value of control antibody.

[0257] Titer calculation: Compare the absorbance values ​​of the experimental group and the negative control group, draw the antibody titer curve, and determine the titer of the antibody to the alliin small molecule.

[0258] The results are as follows Figure 2-4 As shown. The affinity EC of the anti-alliin small molecule antibody 1A10 was obtained. 50 :180.8ng / ml, anti-alliin small molecule antibody 1A10 antibody affinity EC 50 : 132.1ng / ml, and the affinity EC of anti-alliin small molecule antibody 1A10 50 :136.3ng / ml.

[0259] In summary, the complete antigen alliin-KLH prepared in this invention exhibits excellent immunogenicity and can stimulate a strong immune response in New Zealand white rabbits. After one primary immunization and three booster immunizations, the antiserum titer reached 1:32k. The resulting monoclonal antibodies 1A10, 1F3, and 3E12 against alliin all exhibited good affinity. The antibody sequences are shown in Table B.

[0260] The sequence information of the present invention is shown in Table B.

[0261] Table B

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269] Note: The CDR sequences in the table are determined using the Kabat rule.

[0270] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. An anti-alliin antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, characterized in that: The complementarity determining region CDR (HCDR) of the heavy chain variable region and the complementarity determining region CDR (LCDR) of the light chain variable region are: The amino acid sequence of HCDR1 is shown in SEQ ID NO: 30, The amino acid sequence of HCDR2 is shown in SEQ ID NO: 18, The amino acid sequence of HCDR3 is shown in SEQ ID NO: 31, LCDR1 having an amino acid sequence as shown in SEQ ID NO: 36, LCDR2 having an amino acid sequence as shown in SEQ ID NO: 11, and The amino acid sequence of LCDR3 is shown in SEQ ID NO:

37.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The heavy chain variable region and light chain variable region of the antibody or antigen-binding fragment thereof are: the heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 28, and the light chain variable region with an amino acid sequence as shown in SEQ ID NO:

34.

3. A recombinant protein, characterized in that The recombinant protein has: (i) the anti-alliin antibody or antigen-binding fragment thereof according to claim 1; and (ii) a tag sequence to facilitate expression and / or purification.

4. The recombinant protein according to claim 3, characterized in that The recombinant protein includes a fusion protein.

5. The recombinant protein according to claim 3, characterized in that The tag sequence includes a 6His tag.

6. A polynucleotide molecule, characterized in that The polynucleotide molecule encodes the anti-alliin antibody or antigen-binding fragment thereof according to claim 1.

7. A carrier, characterized in that The vector contains the polynucleotide molecule according to claim 6.

8. A host cell, characterized in that The host cell contains the vector according to claim 7, or has the polynucleotide molecule according to claim 6 integrated into its genome, or expresses the anti-alliin antibody or antigen-binding fragment thereof according to claim 1.

9. A method for detecting alliin in a sample, characterized in that: The method comprises the steps of: (1) contacting a sample with the anti-alliin antibody or antigen-binding fragment thereof according to claim 1; (2) Detecting whether a hapten-antibody complex is formed, wherein the formation of the complex indicates the presence of alliin in the sample.

10. A method for preparing the anti-alliin antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The method comprises the following steps: (a) culturing the host cell of claim 8 under expression conditions to express the anti-alliin antibody or antigen-binding fragment thereof; (b) isolating and purifying the anti-alliin antibody or antigen-binding fragment thereof described in (a).

11. Use of the anti-alliin antibody or antigen-binding fragment thereof according to claim 1 and the recombinant protein according to claim 3, characterized in that: For preparing reagents, test plates or kits; The reagent, detection plate or test kit is used for detecting whether a sample contains alliin, or for capturing alliin.

12. A detection board, characterized in that: The detection plate comprises a substrate and a test strip, wherein the test strip contains the anti-alliin antibody or antigen-binding fragment thereof according to claim 1, or the recombinant protein according to claim 3.

13. The detection board according to claim 12, wherein: The test strip further comprises an antigen spotting area, and the antigen is a hapten or a complete antigen.

14. A kit, characterized in that The kit comprises a container and the anti-alliin antibody or antigen-binding fragment thereof according to claim 1 located in the container, or the kit comprises the detection plate according to claim 12 and instructions for use.

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