阅读说明：本技术 与牛妊娠相关糖蛋白1特异性结合的抗体及其用途 (Antibody specifically binding to bovine pregnancy-associated glycoprotein 1 and use thereof ) 是由 金奉辉 河英柱 金世泳 金志娜 于 2018-11-08 设计创作，主要内容包括：本发明涉及一种与牛妊娠相关糖蛋白1(bovine Pregnancy-associated glycoprotein 1,bPAG1)特异性结合的抗体或其抗原结合片段；产生所述抗体的杂交瘤细胞；包括所述抗体作为有效成分的用于诊断牛妊娠的组合物；用于诊断牛妊娠的试剂盒；以及诊断牛妊娠的方法。由于所述抗体与牛妊娠血浆蛋白bPAG1特异性结合,因此可以容易地诊断对繁殖很重要的牛等动物的妊娠,从而可以通过提高繁殖效率而有效地应用于畜牧业。(The present invention relates to an antibody or an antigen-binding fragment thereof that specifically binds to bovine Pregnancy-associated glycoprotein 1(bPAG 1); a hybridoma cell producing the antibody; a composition for diagnosing bovine pregnancy comprising the antibody as an effective ingredient; a kit for diagnosing pregnancy in cattle; and a method of diagnosing pregnancy in cattle. Since the antibody specifically binds to bovine pregnancy plasma protein bPAG1, pregnancy of animals such as cattle, which are important for breeding, can be easily diagnosed, and thus, the antibody can be effectively applied to animal husbandry by improving breeding efficiency.)

1. An antibody or antigen-binding fragment thereof that specifically binds bovine pregnancy-associated glycoprotein 1(bPAG 1).

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody specifically binds to an epitope polypeptide having the amino acid sequence of SEQ ID NO. 1.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain of said antibody is immunoglobulin m (igm).

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof is produced by hybridoma cell deposited as KCLRF-BP-00416.

5. A hybridoma cell producing an antibody or antigen-binding fragment thereof that specifically binds to bPAG1 (accession number KCLRF-BP-00416).

6. A composition for diagnosing pregnancy in a bovine comprising an antibody or antigen-binding fragment thereof that specifically binds to pag 1.

7. A kit for diagnosing pregnancy in a bovine comprising the antibody or antigen-binding fragment thereof of claim 6.

8. A method of diagnosing pregnancy in a bovine comprising: contacting a test sample with the antibody or antigen-binding fragment thereof of claim 1; and detecting bPAG1 bound to the antibody or antigen binding fragment.

9. The method of claim 8, wherein the detection method comprises performing any one selected from the group consisting of enzyme-linked immunosorbent assay, immunoblotting, immunofluorescence, immunohistochemical staining, flow cytometry, immunocytochemistry, radioimmunoassay, immunoprecipitation assay, immunodiffusion assay, complement fixation assay, and protein chip.

Technical Field

This application claims priority and benefit from korean patent application No. 10-2017-0152257, filed on 15/11/2017, which is hereby incorporated by reference for all purposes as if fully set forth herein.

The present invention relates to an antibody or an antigen-binding fragment thereof that specifically binds to bovine Pregnancy-associated glycoprotein 1(bPAG 1).

Background

In the rearing of cattle as industrial animals, farmer income is mainly realized by the production of calves. On the other hand, in breeding cattle, it is important to perform conception, but if it is diagnosed early whether the fertilized cattle is pregnant, non-pregnant animals can be managed individually at an early stage and the nonpregnant period can be reduced, so that the farmer cost can also be reduced. Currently, most bovine pregnancies are diagnosed by fetal membrane palpation by veterinarians or artificial inseminaters between 30 and 42 days of pregnancy. In addition, there is a method of detecting progesterone in milk using Radioimmunoassay (RIA). According to this method, progesterone is a steroid hormone required for the normal progression and maintenance of pregnancy, and since the serum titer of progesterone is increased in the early stages of pregnancy, the degree of increase of said hormone in cow's milk can be confirmed. However, the radioimmunoassay has problems in that not only is there a risk due to the use of radioactive substances, but also a complicated experimental facility is required, and since the progesterone content per cow is different and the standard is unclear, at least two experiments are required to perform accurate diagnosis. Therefore, in addition to solving the existing problems, there is a need to develop a new method for accurately diagnosing pregnancy of cattle at an early stage with greater ease and rapidity.

Disclosure of Invention

Technical problem

In one aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to bovine Pregnancy-associated glycoprotein 1(bPAG 1).

In another aspect, the present invention provides a hybridoma cell (deposited as KCLRF-BP-00416) producing an antibody or an antigen-binding fragment thereof that specifically binds to bPAG 1.

Another aspect of the invention provides a composition comprising an antibody or antigen-binding fragment thereof that specifically binds to pag 1.

In another aspect, the present invention provides a kit for diagnosing pregnancy in cattle, comprising the above composition.

In another aspect, the present invention provides a method for diagnosing pregnancy in a bovine, comprising: contacting a test sample with an antibody or antigen-binding fragment thereof that specifically binds to bPAG 1; and detecting bPAG1 bound to the antibody or antigen binding fragment.

Technical scheme

The present inventors completed the present invention by preparing hybridoma cells producing a novel monoclonal antibody specifically binding to bovine pregnancy plasma protein bPAG1, isolating and purifying the large amount of antibody from the cells, and confirming whether a bovine is pregnant using a relatively simple method.

In the present specification, the term "bovine Pregnancy-associated glycoprotein 1(bPAG 1)" is a bovine Pregnancy plasma protein, which collectively refers to a blood flow protein that appears or increases with Pregnancy. The bPAG1 includes the native form of the protein, variants thereof, and functional equivalents thereof. The bPAG1 can be isolated from natural cells or recombinant cells, or can be synthesized artificially.

In the present specification, the term "antibody" refers to an antibody that specifically binds to a single antigenic site. Unless otherwise indicated, the antibody may be a molecule or antigen-binding fragment thereof that includes an antigen-binding specific site formed by a heavy chain polypeptide and a light chain polypeptide as known in the art. The antibody may be produced by plasma cells (plasma cells) or hybridoma cells. The antibody may be a monoclonal antibody (monoclonal antibody). Specifically, it may be a monoclonal antibody specifically binding to bPAG1 produced by hybridoma cells deposited as KCLRF-BP-00416. In the present specification, the term "antigen-antibody complex" refers to a conjugate of bPAG1 and an antibody recognizing it. The complex can be used for detecting bPAG1 in a sample. The complex can be used, for example, for detecting bPAG1 in a biological sample (e.g., bovine urine, serum, or plasma).

One aspect of the invention provides an antibody or antigen-binding fragment thereof that specifically binds to bPAG 1. Specifically, the antibody according to the present invention may include an antigen-binding fragment as long as the antibody can selectively recognize bovine pregnancy plasma protein bPAG1, and the antigen-binding fragment may include F (ab')2, Fab, Fv fragments, and the like.

In one example, by analyzing the result of the epitope (epitope) specifically binding to the monoclonal antibody against bPAG1, it can be confirmed that the amount of antibody specifically binding to the antigen increases according to the concentration of the antigen. In particular, the antibody specifically binds to the polypeptide having the amino acid sequence of SEQ ID NO. 1 in a significantly higher amount than the polypeptide having the amino acid sequence of SEQ ID NO. 2 to 4 at the same antigen concentration. Thus, an antibody that specifically binds to the bPAG1 can bind to an epitope polypeptide having the amino acid sequence of seq id No. 1. In addition, the intact form of the antibody is a structure having two full-length light chains and two full-length heavy chains, and each light chain and heavy chain may be linked by a disulfide bond. The heavy chain can be a full-length heavy chain comprising a variable region domain VH and three constant region domains CH1, CH2, and CH3, wherein the variable region domain comprises an amino acid sequence having a variable region sequence sufficient to confer antigen specificity, and fragments thereof. The light chain may refer to a full-length light chain including a variable region domain VL including an amino acid sequence having a variable region sequence sufficient to impart antigen specificity and a constant region domain CL, and fragments thereof. At this time, the heavy chain includes constant regions of gamma (γ), muir (μ), alpha (α), delta () and epxolone (), and the light chain includes constant regions of kappa (κ) and lambada (λ) types. The antibody may for example be immunoglobulin m (igm).

In another aspect, the present invention provides a hybridoma cell (deposited as KCLRF-BP-00416) producing an antibody or an antigen-binding fragment thereof that specifically binds to bPAG 1. The specific contents of the antibody or antigen-binding fragment thereof specifically binding to bPAG1 are as described above. Specifically, in one embodiment of the present invention, only hybridoma cells that identify an antibody that specifically binds to bovine pregnancy plasma protein bPAG1 are selected after obtaining mouse spleen cells and culturing in fusion with mouse myeloma cells. Then, the hybridoma cells were deposited in the Korean Cell Line Research Foundation (KCLRF) and obtained in 22 days 12 months in 2017 under the accession number KCLRF-BP-00416.

The hybridoma cells producing the antibody can be used to culture them in large quantities in vitro or in vivo. The antibody produced by the hybridoma cells may be used without purification, but may be used by purification with a high purity, for example, 95% or more, according to a known method in order to obtain the best result. The antibody can be isolated from the medium or ascites fluid (ascites fluid) using purification methods such as dialysis, salt precipitation, chromatography, and the like.

In another aspect, the present invention provides a composition for diagnosing pregnancy in cattle, comprising an antibody or an antigen-binding fragment thereof specifically binding to bPAG 1. The specific contents of the antibody or antigen-binding fragment thereof specifically binding to bPAG1 are as described above. The composition may include a carrier for the antibody, such as diluents, buffers, stabilizers, and the like. The composition may include a detection reagent for detecting an antigen-antibody complex.

In another aspect, the present invention provides a kit for diagnosing pregnancy in cattle, comprising the antibody or antigen-binding fragment. The specific contents of the antibody or antigen-binding fragment thereof specifically binding to bPAG1 are as described above. Specifically, the kit for diagnosing bovine pregnancy according to the present invention may include an antibody or an antigen-binding fragment thereof specifically recognizing bPAG1 and a tool or a reagent for immunological analysis. The antibody can specifically bind to an epitope polypeptide having an amino acid sequence of sequence No. 1. At this time, the tool or reagent for immunological analysis may include a suitable carrier, a labeling substance that can generate a detectable signal, a solubilizing agent, a detergent, and the like. When the labeling substance is an enzyme, the kit may comprise a substrate that can measure the activity of the enzyme, a reaction terminating reagent, and the like.

The support is a soluble support such as a physiologically acceptable buffer known in the art, e.g., Phosphate Buffered Saline (PBS), an insoluble support such as polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesin, cross-linked dextran, polysaccharide, polymer such as magnetic fine particles plated with metal in latex, other paper, glass, metal, agarose, and combinations thereof.

In another aspect, the present invention provides a method for diagnosing pregnancy in a bovine, comprising: contacting a test sample with an antibody or antigen-binding fragment thereof that specifically binds to bPAG 1; and detecting bPAG1 bound to the antibody or antigen binding fragment. The specific contents of the antibody or antigen-binding fragment thereof specifically binding to bPAG1 are as described above.

A method of diagnosing pregnancy in a bovine according to one embodiment includes the step of contacting an antibody or an antigen-binding fragment thereof specifically binding to pag1 with a subject. The sample may be a biological sample isolated from a bovine. Specifically, the sample may be milk, tissue, cell, blood, serum, plasma, saliva or urine of a cow for which pregnancy is to be confirmed. The step of contacting may be incubating the antibody or antigen-binding fragment thereof and a sample in a liquid medium. The incubation may be performed under temperature, pH and stirring conditions suitable for antigen-antibody binding between the antigen and the antibody in the sample. The incubation may be performed at 2 ℃ to 37 ℃. The incubation may be performed at pH6 to pH 8.

A method of diagnosing pregnancy in a bovine according to one embodiment includes the step of detecting the antibody or antigen binding complex. The step of detecting may be performed by directly isolating the antigen-antibody complex to confirm its presence, or by performing a method such as biochemical or immunochemical analysis with or without isolation. The detection method may be any one selected from enzyme-linked immunosorbent Assay (ELISA), immunoblotting (stereo Blotting), Immunofluorescence (Immunofluorescence), immunohistochemical staining (Immunohistochemistry), Flow cytometry (Flow cytometry), immunocytochemistry, Radioimmunoassay (RIA), Immunoprecipitation Assay (Immunoprecipitation Assay), Immunodiffusion Assay (Immunodiffusion Assay), Complement Fixation Assay (complementary hybridization Assay), and Protein Chip (Protein Chip).

In the method, the antigen-antibody complex may be labeled with a detectable label (detectable label) or a substance that can produce a detectable label, which may be selected from the group consisting of an enzyme, a fluorescent substance, a ligand, a luminescent substance, a microparticle (microparticulate), a redox molecule, and a radioisotope, which may be, for example, β -glucuronidase, β -D-glucosidase, β -D-galactosidase, urease, peroxidase, alkaline phosphatase, acetylcholinesterase, glucose oxidase, hexokinase, and PaGDse, RNase, glucose oxidase and luciferase, phosphofructokinase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphoenolpyruvate decarboxylase, β -lactamase, or a combination thereof, the fluorescent substance includes, but is not limited to, fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, fluorescamine, etc., the ligand includes a biotin derivative, etc., the luminescent substance includes acridinium ester, fluorescein, luciferase, etc., the luminescent substance includes, ruthenium, the microparticle may be, a ruthenium complex, a reduced ion, a colloidal ion, a quinone ion, a ligand including, a ruthenium complex, a quinone ion, a ligand including, a quinone, a ligand including, a ruthenium complex, a ligand including₄W(CN)₈、[Os(bpy)₃]²⁺、[RU(bpy)₃]²⁺Or [ MO (CN)₈]^4-And the like. The radioisotope may be³H、¹⁴C、³²P、³⁵S、³⁶Cl、⁵¹Cr、⁵⁷Co、⁵⁸Co、⁵⁹Fe、⁹⁰Y、¹²⁵I、¹³¹I or¹⁸⁶Re, and the like.

As described above, the antibody or antigen-binding fragment thereof specifically binding to pag1 according to the present invention forms an antigen-antibody complex by specifically binding to plasma proteins that appear or increase according to whether a cow is pregnant, and through analysis of the antigen-antibody complex, it is possible to diagnose whether a cow is pregnant using a relatively simple method, so it is possible to effectively apply to animal husbandry by improving the reproductive efficiency of cows.

Advantageous effects

An aspect of the present invention relates to an antibody or an antigen-binding fragment thereof specifically binding to pag1, wherein the antibody specifically binds to bovine pregnancy plasma protein pag1, so that pregnancy of animals such as cattle, which are important for breeding, can be easily diagnosed, and thus can be effectively applied to animal husbandry by improving breeding efficiency.

Drawings

FIG. 1 shows the molecular information of bPAG1 according to the present invention.

Figure 2 shows the results of the isotype screen test.

Fig. 3 shows the results confirmed by Bradford assay after isolation of immunoglobulin M. In fig. 3, fig. 3a shows a quantitative icon of the isolated 3B1 clone, fig. 3B shows a quantitative icon of the isolated 3B3 clone, and fig. 3c shows a photograph of the activity level of the isolated and purified antibody on the membrane.

Fig. 4 shows epitope analysis results of monoclonal antibodies against pag 1.

Fig. 5 shows a schematic diagram of a diagnostic method for bovine pregnancy.

Fig. 6 shows a photograph of the result of diagnosing bovine pregnancy by using a simply prepared kit.

Detailed Description

The following preferred embodiments will be provided to aid in understanding the present invention. However, the following examples are provided only for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[ PREPARATION EXAMPLES ]

The antigen peptide used for antibody production was selected from 40 of amino acid sequences of 150 to 200 and 350 to 400 of bPAG1, and was classified into 4 types according to the presence of Keyhole Limpet Hemocyanin (KLH). The amino acid sequence of the bPAG1 fragment has the sequence of SEQ ID NO. 5(PAG 1-100) and SEQ ID NO. 6(PAG 1-300).