ANTIBODY SPECIFICALLY BINDING TO BOVINE PREGNANCY-ASSOCIATED GLYCOPROTEIN 1 AND USE THEREOF

Abstract

The present disclosure relates to an antibody specifically binding to bovine pregnancy-associated glycoprotein 1 (bPAG1) or an antigen-binding fragment thereof. The present disclosure further relates to a hybridoma cell producing the antibody; a composition including the antibody as an effective ingredient for diagnosis of bovine pregnancy; and a kit for diagnosis of bovine pregnancy. The present disclosure also relates to a method of diagnosing bovine pregnancy. Binding specifically to bPAG1, which is a pregnancy-associated plasma protein in bovines, the antibody allows a simple diagnosis of pregnancy in animals such as bovines of which the reproduction is important, so that the antibody may be usefully applied in the livestock industry by increasing reproduction efficiency.

Claims

1. An antibody specifically binding to bovine pregnancy-associated glycoprotein 1 (bPAG1) or an antigen-binding fragment thereof.

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

3. The antibody or the antigen-binding fragment thereof of claim 1, wherein heavy chains of the antibody comprise Immunoglobulin M (IgM).

4. The antibody or the antigen-binding fragment thereof of claim 1, wherein the antibody or the antigen-binding fragment thereof is produced by a hybridoma cell with Accession No: KCLRF-BP-00416.

5. A hybridoma cell (Accession No: KCLRF-BP-00416) producing an antibody specifically binding to bovine pregnancy-associated glycoprotein 1 (bPAG1) or an antigen-binding fragment thereof.

6. A composition for diagnosis of bovine pregnancy, the composition comprising an antibody specifically binding to bovine pregnancy-associated glycoprotein 1 (bPAG1) or an antigen-binding fragment thereof.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0027] FIG. 1 shows the molecular information of bovine pregnancy-associated glycoprotein 1 (bPAG1).

[0028] FIG. 2 shows the results of the isotype screening test.

[0029] FIG. 3 shows the results confirmed by Bradford assay after separating immunoglobulin M. In FIG. 3, FIG. 3A shows a quantitative graph of separated 3B1 clones, FIG. 3B shows a quantitative graph of separated 3B3 clones, and FIG. 3C is a photograph showing the degree of activity of the separated-purified antibody on a membrane.

[0030] FIG. 4 shows the analysis results of epitope for a monoclonal antibody against the bPAG1.

[0031] FIG. 5 is a schematic diagram showing a method of diagnosing bovine pregnancy.

[0032] FIG. 5 is a photograph showing the results of diagnosis of bovine pregnancy by using a simplified kit.

DETAILED DESCRIPTION OF EMBODIMENTS

[0033] Hereinafter, to promote understanding of the present disclosure, preferable Examples are provided. However, Examples below are only provided to more easily understand the present disclosure more easily, and the contents of the present disclosure are not limited by Examples below.

Preparation Example

[0034] As an antigen peptide for preparing an antibody, 40 amino acid sequences were selected from the 150.sup.th to the 200.sup.th amino acid sequences and the 350.sup.th to the 400.sup.th amino acid sequences of bovine pregnancy-associated glycoprotein 1 (bPAG1). Four types of the antigen peptide were used depending on the presence of Keyhole limpet hemocyanin (KLH). The amino acid sequence of the bPAG1 fragment may include sequences of SEQ ID NO: 5 (PAG1_100) and SEQ ID NO: 6 (PAG1_300).

EXAMPLES

Example 1. Induction of Immune Response Using Antigen

[0035] 1-1. bPAG1_100

[0036] 100 l of the antigen (bPAG1_100) of Preparation Example and a complete adjuvant of the same amount were used to prepare an antigen emulsion. Then, an 8-week-old female mouse (BALB/C) was administered subcutaneously with the antigen emulsion, so as to induce an immune response. After the first administration, the mouse was administered subcutaneously with 100 l of the same antigen and an incomplete adjuvant of the same amount every two weeks. Matters to consider regarding the antigen administration schedule or the like are shown in Table 1 below.

[0037] 1-2. bPAG1_100-KLH

[0038] The same process as Example 1-1 was performed, except that the bPAG1_100-KLH antigen was used.

[0039] 1-3. bPAG1_300

[0040] The same process as Example 1-1 was performed, except that the bPAG1_300 antigen was used.

[0041] 1-4. bPAG1_300-KLH

[0042] The same process as Example 1-1 was performed, except that the bPAG1_300-KLH was used.

TABLE-US-00001 TABLE 1 Concentration Dose Order Antigen (mg/ml) (g) First Example 1-1 0.5 75 +Complete adjuvant First Example 1-2 0.3125 46.875 (v/v 1:1 mixture) Second Example 1-1 0.5 75 +Incomplete adjuvant Second Example 1-2 0.3125 46.875 (v/v 1:1 mixture) First Example 1-3 0.5 75 +Complete adjuvant First Example 1-4 0.3125 46.875 (v/v 1:1 mixture) Third Example 1-1 0.5 75 +Complete adjuvant Third Example 1-2 0.3125 46.875 (v/v 1:1 mixture) Second Example 1-3 0.5 75 +Incomplete adjuvant Second Example 1-4 0.3125 46.875 (v/v 1:1 mixture) Third Example 1-3 0.5 75 +Complete adjuvant Third Example 1-4 0.3125 46.875 (v/v 1:1 mixture)

Example 2. Confirmation of Antibody Titer

[0043] 2-1. bPAG1_100

[0044] After the antigen administration of Example 1-1 was performed three times, a small amount of serum was collected from the tail vein of the mouse, so as to confirm the titer of polyclonal antibodies in the serum through ELISA. In detail, a reaction was allowed at a temperature of 4 C. for 18 hours by using 100 l of the PAG1_100 antigen (2 g/ml) per well, and a coating process was performed thereon. On the following day, a reaction blocking process was performed thereon at room temperature for 1 hour by using a blocking buffer. Afterwards, the serum collected from the mouse blood was diluted according to a magnification ratio, and 100 l of the diluted serum was dispensed into each well for a reaction at room temperature for 1 hour. The well plate where the reaction was completed was washed with PBST, and goat anti-mouse IgG-HRP in which HRP was conjugated was diluted at a ratio of 1:2,000, and 100 l of the diluted goat anti-mouse IgG-HRP was dispensed into each well for a reaction at room temperature for 1 hour. Afterwards, the well plate was washed again with PBST, and a TMB solution was dispensed thereinto for a reaction for 15 minutes. Then, a reaction stopping solution was used to terminate the reaction. Titers in the serum were confirmed by measuring absorbance values at 450/620 nm, and the results are shown in Tables 2 and 3 below.

2-2. bPAG1_100-KLH

[0045] The same process as Example 2-1 was performed, except that the bPAG1_100-KLH antigen was used.

2-3. bPAG1_300

[0046] The same process as Example 2-1 was performed, except that the bPAG1_300 antigen was used.

2-4. bPAG1_300-KLH

[0047] The same process as Example 2-1 was performed, except that the bPAG1_300-KLH antigen was used.

TABLE-US-00002 TABLE 2 PAG1-100 coating Serum Example Example Example Example concentration Control NC1 NC2 NC3 2-1 2-2 2-3 2-4 100 A 0.203 0.087 0.145 0.111 2.393 2.483 0.195 0.151 200 B 0.084 0.037 0.062 0.045 2.176 2.265 0.087 0.069 400 C 0.03 0.014 0.025 0.018 2.018 1.916 0.035 0.029 800 D 0.013 0.009 0.011 0.01 1.547 1.518 0.015 0.013 1600 E 0.009 0.006 0.008 0.006 1.029 0.998 0.016 0.019 3200 F 0.006 0.006 0.006 0.007 0.562 0.554 0.014 0.002 6400 G 0.005 0.005 0.011 0.011 0.288 0.263 0.005 0.005 12800 H 0.005 0.005 0.005 0.005 0.134 0.127 0.005 0.005

TABLE-US-00003 TABLE 3 PAG1-300 coating Serum Example Example Example Example concentration Control NC1 NC2 NC3 2-1 2-2 2-3 2-4 100 A 0.15 0.075 0.125 0.082 0.159 0.012 2.558 2.583 200 B 0.059 0.027 0.048 0.033 0.073 0.048 2.317 2.34 400 C 0.021 0.012 0.018 0.015 0.03 0.019 2.114 2.164 800 D 0.01 0.007 0.009 0.008 0.013 0.01 1.828 1.921 1600 E 0.007 0.006 0.006 0.007 0.007 0.005 1.434 1.446 3200 F 0.005 0.005 0.006 0.005 0.006 0.006 0.869 0.887 6400 G 0.005 0.004 0.005 0.005 0.005 0.004 0.385 0.454 12800 H 0.005 0.004 0.005 0.005 0.005 0.005 0.224 0.246

[0048] As a result, as shown in Tables 2 and 3, it was confirmed through the analysis of antigenicity of the bPAG1 that the synthesized PAG1-100 peptide and the synthesized PAG1-300 peptide mass-produced antibodies against the bPAG1.

Example 3. Preparation of Hybridoma Cell Producing Monoclonal Antibody Against bPAG1 and Separation of Monoclonal Antibody

3-1. Preparation of Hybridoma Cell

[0049] Once the production of antibodies, which have high titers based on the titers in the mouse serum confirmed by the antibody titer confirmation test of Example 2, was confirmed, lymphocytes of the mouse were separated and subjected to cell fusion. In detail, the spleen of the mouse in which the immune response was introduced in Examples 1-1 to 1-4 was extracted without damage, washed with Dulbecco/Vogt modified Eagle's minimal essential medium (DMEM). Then, lymphocytes were separated from the extracted spleen and placed on a 60 mm dish containing the DMEM to be separated as single cells. Afterwards, the red blood cells mixed in the lymphocytes were removed by using an RBC lysis buffer, and washed with fresh DMEM. Then, prepared myeloma cells (SP2/0 Ag 14ATCC # CRL-1581) and the treated lymphocytes were hybridized at a ratio of 1:5 based on the number of the myeloma cells. Then, 1.7 ml of PEG1500 was added to the hybridized cells (i.e., hybridoma cells) to induce cell fusion, and 200 l of the resulting hybridoma cells was dispensed into each well of a 96-well plate for incubation in a CO.sub.2 incubator. After 2 days of the cell fusion, 50% of each well was replaced with hypoxanthine-aminopterinthymidine (HAT) medium. After 12 days of the cell fusion, the production of colonies was confirmed to determine whether or not the cells reacted with a bPAG1 peptide (i.e., an antigen to be confirmed by ELISA) in the same manner as ELISA (Asb. 450 nm) of Example 2. Referring to the ELISA results, when the hybridoma cells had the O.D value of 1.0 or more, the hybridoma cells were regarded as positive cells and selected as parent cells for producing monoclonal antibodies. The results are shown in Table 4 below.

TABLE-US-00004 TABLE 4 bPAG100 bPAG300 1A1 1B3 2A6 2C2 3A6 N 4A1 1.877 0.97 2.49 1.475 0.704 0.02 3.036 1A2 1B4 2B1 2C3 3B1 4A2 1.689 1.776 1.797 1.382 1.558 1.966 1A3 1B5 2B2 2C4 3B2 4A3 2.651 0.251 0.985 3.283 2.766 0.389 1A4 2A1 2B3 3A1 3B3 4A4 2.146 2.649 1.11 1.504 2.996 0.181 1A5 2A2 2B4 3A2 3B4 4A5 0.68 2.631 0.157 0.478 1.053 0.146 1A6 2A3 2B5 3A3 3B5 N 1.431 3.164 3.106 0.904 1.058 0.125 1B1 2A4 2B6 3A4 3B6 2.522 2.847 3.16 0.076 0.745 1B2 2A5 2C1 3A5 3C1 0.872 0.108 2.929 1.133 2.246

3-2. Screening of Hybridoma Cell Line Producing Monoclonal Antibody

[0050] Cells specifically reacting only to the bPAG1 among the hybridoma cell group prepared in Example 3-1 were selected. Then, in order to confirm whether the antibodies were produced, screening was performed on the selected from cells according to enzyme immunoassay.

[0051] After 12 days of the cell fusion, the medium was replaced with fresh medium, and the original medium which is regarded as a primary antibody was subjected to ELISA. Following the ELISA, wells showing positive results for the corresponding antigen were selected and transferred to a 24-well plate for culture. A hybridoma cell line cultured in the 24-well plate was subjected again to ELISA in the same manner as described above, so as to confirm antibody titers. At the same time, the antibody-producing cell line was subjected to the second screening. The absorbance (expressed as O.D value) of the hybridoma cells grown in the 24-well plate was confirmed by ELISA. Only the hybridoma cells having the absorbance value of 1 or more were selected and transferred to a 6-well culture flask. After the hybridoma cells were cultured and centrifuged, the supernatant was obtained, confirmed by ELISA again, and subjected to the third screening. The hybridoma cells selected based on the third screening were transferred again to a 75T/C culture flask, and then cultured. The absorbance of the hybridoma cells was confirmed by ELISA, and only the hybridoma cells having the absorbance value of 1 or more were selected. The cell lines selected through the process above are shown in Table 5 below.

TABLE-US-00005 TABLE 5 bPAG1_100 1A6, 2A6, 2B6, 2C4 bPAG1_100-KLH 3A6, 3B1, 3B3, 3B5 bPAG1_300 4A1, 4A3

[0052] As shown in Table 5, 1A6, 2A6, 2B6, 2C4, 3A6, 3B1, 3B3, and 3B5 cell lines were finally selected for producing antibodies against bPAG1-100 as a binding site, and 4A1 and 4A3 cell lines were finally selected from for producing antibodies against bPAG-300 as a binding site. Among these cell lines, the 3B1 cell line was deposited with the KCLRF on Dec. 5, 2017 (Accession No: KCLRF-BP-00416).

Example 4. Production and Purification of Monoclonal Antibody Against bPAG1

[0053] In order to mass-produce monoclonal antibodies against the bPAG1 from the finally selected from hybridoma cells of Example 3-2, 0.5 ml of an incomplete adjuvant was administered to the abdominal cavity of a 6-week-old mouse (BALB/C). After 3 days of the administration, among the finally selected from hybridoma cells of Example 3-1, the cells confirmed to have high O.D values were each administered at 100 l (110.sup.6 cells) per mouse. After 6 to 10 days of the administration, ascite was collected from the abdominal cavity of the mouse.

Example 5. Separation of Monoclonal Antibody Against bPAG1

[0054] In order to separate the antibodies specifically binding to the bPAG1 from the selected hybridoma cells of Example 3, the collected ascite was injected into a dialysis pack by using a syringe. Afterwards, 2 L of 2 mM phosphate buffer (PB) was added to a beaker, and a dialysis solution containing a magnetic bar and a sample was added thereto, followed by dialysis at 4 C. for 3 days to 4 days. Here, the buffer was replaced with fresh buffer once a day. After the dialysis was completed, the liquid present in the dialysis solution was collected and centrifuged. The supernatant was then collected therefrom to quantify proteins, and the antigen-antibody response on a membrane was also confirmed.

[0055] As a result, as shown in FIGS. 3A and 3B, in the case of the 3B1 hybridoma cell line, about 1.5 mg of the antibody was produced per 1 ml of the ascite, and in the case of the 3B3 hybridoma cell line, about 2 mg of the antibody was produced per 1 ml of the ascite. In order to confirm the activity of the antibody separated from the process above on the membrane, a small amount of serum of a pregnant bovine was loaded onto the membrane. The membrane was then dried, and each antibody conjugated with a gold particle was mixed with a developing solution and flowed along the membrane. As a result, as shown in FIG. 3C, the 2C4, 3B1, 3B3, and 4A1 antibodies each showed a clear spot indicating the binding to the antigen (bPAG1).

Example 6. Analysis of Epitope for Monoclonal Antibody Against bPAG1

[0056] Epitope mapping was performed by using the peptides used in the antibody production. As the peptides used in the mapping, only some peptides, e.g., the 100-sized peptides and the 300-sized peptides, of the PAG1 protein used in the production of the existing antigen were used. As shown in Table 6 below, the mapping proceeded with four types of the peptides.

TABLE-US-00006 TABLE6 Peptide1 ASSDLWVPSDFCTSPACSTH SEQIDNO:1 Peptide2 CVRFRHLQSSTFRLTNKTFRI SEQIDNO:2 Peptide3 GAIPRGSEHYVPCSEVNTLP SEQIDNO:3 Peptide4 CSIVFTINGINYPVPGRAYIL SEQIDNO:4

[0057] In detail, each of the antigen bPAG1_100 and the antigen bPAG1_300 was dispensed at 25 l, 50 l, 100 l, and 200 l per well, and a coating process was performed thereon. Then, a reaction was allowed at room temperature for 2 hours. Afterwards, a blocking process was performed thereon at room temperature for 1 hour by using a blocking buffer, and the purified sample was dispensed at 100 l per well. Then, a reaction was allowed at room temperature for 1 hour. Next, the well plate where the reaction was completed was washed with PBST, and goat anti-mouse IgM conjugated with HRP was diluted at a ratio of 1:50,000 and dispensed into each well at 100 l per well. Then, a reaction was allowed for 1 hour. After the reaction was completed, the well plate was washed again with PBST, and a TMB solution was dispensed thereinto for a reaction for 15 minutes. Then, a reaction stopping solution was used to terminate the reaction, and the absorbance values at 450/620 nm were measured, so as to confirm the antibody titers in the sample.

[0058] As a result, as shown in FIG. 4, it was confirmed that the amount of the antibody specifically binding to the antigen increased depending on the concentration of the antigen. In particular, in the case of Peptide 1, the amount of Peptide 1 specifically binding to the antigen was remarkably high compared to the amounts of Peptides 2 to 4 at the same concentration of the antigen, and accordingly, it was confirmed that Peptide 1 had the amino acid sequence of the epitope for the antibody of the present disclosure.

Example 7. Diagnosis of Bovine Pregnancy by Using Separated and Purified Antibody

[0059] By using the antibody selected in Example 3, the bPAG1 which is known to be present in the blood of pregnant bovines was detected. In detail, the antibody was dispensed into a membrane, and an antibody with an antigen-binding site, which is different from the antibody of the present disclosure, to which gold particles are bound was used for artificial insemination. Then, the blood was collected from a 6 weeks-old bovine, a 7 weeks-old bovine, and a 8 weeks-old bovine, and then centrifuged to separate serum. 0.05 ml of the separated serum was loaded onto a sample-loading section of a simplified kit, and after 15 minutes, the results were decided.

[0060] As a result, as shown in FIG. 6, it was confirmed that a band was produced on an inspection line after 6 weeks of the fertilization. That is, the bovine pregnancy was able to be diagnosed by confirming the presence of the bPAG1 in the bovine blood by using the antibody.