An antibody or antigen-binding fragment thereof against bovine pregnancy-associated glycoprotein 1 and use thereof

Abstract

The application belongs to the technical field of antibodies, and discloses an antibody or antigen binding fragment thereof against bovine pregnancy-associated glycoprotein 1; the antibody or antigen binding fragment thereof comprises a heavy chain and a light chain; the heavy chain of the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3; and the light chain of the antibody or antigen binding fragment thereof comprises a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3. The antibody or antigen binding fragment thereof can be used for detecting the presence or level of bovine pregnancy-associated glycoprotein 1 in a sample, and provides a quick, simple and cost-effective solution for early pregnancy diagnosis of dairy cows.

Description

Technical Field

[0001] This invention belongs to the field of antibody technology, specifically relating to an antibody against bovine pregnancy-associated glycoprotein 1 or its antigen-binding fragment and its applications. Background Technology

[0002] With the rapid development of dairy farming towards large-scale and intensive operations, efficient reproductive management has become a key aspect of improving farm economic benefits. Optimizing reproductive efficiency can not only effectively shorten the non-pregnant period and calving interval of dairy cows, but also significantly increase individual milk production, thereby creating greater economic value for the farm. In this process, early and accurate pregnancy diagnosis is particularly important; it is a crucial technical guarantee for achieving efficient reproductive management and provides a scientific basis for subsequent feeding management and production planning.

[0003] In the field of early pregnancy diagnosis in dairy cows, two main diagnostic methods are currently employed: direct and indirect. As a traditional diagnostic method, rectal palpation has long dominated dairy farm practice due to its economy and convenience. However, this method has significant technical limitations: firstly, reliable results are difficult to obtain during the critical diagnostic window of 29-31 days post-mating; secondly, its diagnostic accuracy is highly dependent on the operator's clinical experience and skill level. While modern ultrasound diagnostic technology can effectively compensate for the shortcomings of rectal palpation and achieve earlier and more accurate diagnosis, its widespread application in grassroots dairy farms faces challenges due to high equipment investment and the need for specialized personnel. In contrast, indirect diagnostic methods based on body fluid testing demonstrate significant advantages. By detecting dynamic changes in specific biomarkers such as progesterone, early pregnancy factor (EPF), and pregnancy-associated glycoproteins (PAG), combined with antigen-antibody immunoassay techniques, it achieves simple, rapid, and non-invasive pregnancy diagnosis, providing a more valuable technical solution for efficient reproductive management in large-scale dairy farms.

[0004] Bovine pregnancy-associated glycoproteins (bPAGs) are a family of aspartic protease glycoproteins specifically synthesized and secreted by placental trophoblast cells. As important biomarkers of pregnancy, they can be detected in the peripheral blood of cows from days 15 to 22 of gestation and persist throughout the entire pregnancy. Genomic studies have identified 21 PAG genes and 20 PAG-like genes, which can be divided into ancient and modern groups based on phylogenetic analysis. The modern group (including bPAG1, 3-9, and 14-22) is synthesized by binuclear cells of the placental trophoblast and has a molecular weight range of 37-90 kDa. As the first PAG member isolated from bovine placenta, bPAG1 has a molecular weight of 67 kDa and four N-glycosylation sites. Its serum concentration continuously increases with the progression of pregnancy. Radioimmunoassay (RIA) shows that a pregnancy detection rate of 95%-100% can be achieved 30 days after fertilization, reaching a peak at 80 days and remaining at a high level for up to 220 days. Based on the molecular characteristics of bPAG1 and its specific expression pattern in early pregnancy, immunoassay technology developed using bPAG1 as a target provides a rapid, simple, and cost-effective solution for early pregnancy diagnosis in dairy cows. Summary of the Invention

[0005] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, the present invention proposes an antibody against bovine pregnancy-associated glycoprotein 1 or its antigen-binding fragment, which can be used to detect the level of bovine pregnancy-associated glycoprotein 1 in a sample, providing a rapid and accurate detection tool for the diagnosis of early pregnancy in dairy cows.

[0006] This invention also proposes a recombinant protein.

[0007] The present invention also proposes a biomaterial related to an antibody against bovine pregnancy-associated glycoprotein 1 as described in the first aspect of the present invention or an antigen-binding fragment thereof, or a recombinant protein as described in the second aspect of the present invention.

[0008] The present invention also proposes a coupling agent.

[0009] The present invention also proposes a solid-phase support.

[0010] The present invention also proposes an application.

[0011] The present invention also proposes a product.

[0012] The present invention also provides a method for preparing antibodies or antigen-binding fragments thereof as described in the first aspect of the invention or recombinant proteins as described in the second aspect of the invention.

[0013] According to a first aspect of the present invention, an antibody or antigen-binding fragment thereof against bovine pregnancy-associated glycoprotein 1 is provided, said antibody or antigen-binding fragment comprising a heavy chain and a light chain:

[0014] The heavy chain of the antibody or its antigen-binding fragment comprises:

[0015] Heavy chain variable region, wherein the heavy chain variable region includes CDR-H1, CDR-H2 and CDR-H3;

[0016] The light chain of the antibody or its antigen-binding fragment comprises:

[0017] Light chain variable region, wherein the light chain variable region includes CDR-L1, CDR-L2 and CDR-L3;

[0018] The amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, and CDR-L3 of the antibody or its antigen-binding fragment are shown in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 16, and SEQ ID NO: 17, respectively. The amino acid sequence of CDR-L2 is YAS. The CDR is defined according to the IMGT definition scheme.

[0019] The amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the antibody or its antigen-binding fragment are shown in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 17, respectively, wherein the CDR is defined according to the Kabat scheme; or

[0020] The amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the antibody or its antigen-binding fragment are shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 17, respectively, wherein the CDR is defined according to the Chothia scheme; or

[0021] The amino acid sequences of the antibody or its antigen-binding fragments CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are shown in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively, wherein the CDR is defined using the Contact definition scheme.

[0022] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment comprises:

[0023] a1)SEQ ID NO: 2; or

[0024] a2) An amino acid sequence of SEQ ID NO: 2 that has undergone substitution and / or deletion and / or addition of one or more amino acids and has the same function as the protein shown in SEQ ID NO: 2; or

[0025] a3) has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 70%, 60%, 50%, 40%, or 30% homology with SEQ ID NO: 2 and has the same function as the protein shown in SEQ ID NO: 2.

[0026] In some embodiments of the present invention, the amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment comprises:

[0027] b1)SEQ ID NO: 4; or

[0028] b2) A sequence of amino acids in SEQ ID NO: 4 that has undergone substitution and / or deletion and / or addition of one or more amino acids and has the same function as the protein shown in SEQ ID NO: 4; or

[0029] b3) has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 70%, 60%, 50%, 40%, or 30% homology with SEQ ID NO: 4 and has the same function as the protein shown in SEQ ID NO: 4.

[0030] In some embodiments of the present invention, the antibody or its antigen-binding fragment comprises at least one of a full-length antibody, Fab, Fab', F(ab')2, Fv, scFv, bispecific antibody, and multispecific antibody.

[0031] In some embodiments of the present invention, the heavy chain of the antibody or its antigen-binding fragment further includes a heavy chain constant region.

[0032] In some embodiments of the present invention, the light chain of the antibody or its antigen-binding fragment further includes a light chain constant region.

[0033] According to a second aspect of the present invention, a recombinant protein is provided, comprising: an antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention; and

[0034] Optional tag sequences to assist in expression and / or purification.

[0035] In some embodiments of the present invention, the tag sequence is selected from at least one of the following groups: His tag, GGGS sequence, FLAG tag.

[0036] According to a third aspect of the present invention, biomaterials relating to the antibodies or antigen-binding fragments thereof described in the first aspect of the present invention, or the recombinant proteins described in the second aspect of the present invention, are provided, said biomaterials comprising at least one of h1) to h8):

[0037] h1) A nucleic acid molecule encoding an antibody or antigen-binding fragment thereof of any one of claims 1 to 2, or a recombinant protein of claim 3;

[0038] h2) contains an expression cassette of the nucleic acid molecule described in h1);

[0039] h3) A carrier containing the nucleic acid molecule described in h1);

[0040] h4) A carrier containing the expression box described in h2);

[0041] h5) A transgenic cell line containing the nucleic acid molecules described in h1);

[0042] h6) Transgenic cell lines containing the expression cassette described in h2);

[0043] h7) A transgenic cell line containing the vector described in h3);

[0044] h8) is a transgenic cell line containing the vector described in h4).

[0045] In some embodiments of the present invention, the transgenic cell line does not contain propagation material.

[0046] In some embodiments of the present invention, the nucleic acid molecule encoding the antibody or its antigen-binding fragment of the anti-bovine pregnancy-associated glycoprotein 1 as described in the first aspect of the present invention comprises a nucleic acid molecule encoding the heavy chain variable region of the anti-bovine pregnancy-associated glycoprotein 1 as described in the first aspect of the present invention and a nucleic acid molecule encoding the light chain variable region of the anti-bovine pregnancy-associated glycoprotein 1 as described in the first aspect of the present invention.

[0047] In some embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region of anti-bovine pregnancy-associated glycoprotein 1 as described in the first aspect of the present invention comprises:

[0048] a211) The nucleotide sequence shown in SEQ ID NO: 1; or

[0049] a212) A nucleotide sequence of SEQ ID NO: 1 that has undergone substitution and / or deletion and / or addition of one or more nucleotides, and has the same function as the nucleic acid molecule shown in SEQ ID NO: 1; or

[0050] a213) has at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 70%, 60%, 50%, 40%, or 30% homology with SEQ ID NO: 1, and has the same function as the nucleic acid molecule shown in SEQ ID NO: 1.

[0051] In some embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule encoding the light chain variable region of anti-bovine pregnancy-associated glycoprotein 1 as described in the first aspect of the present invention comprises:

[0052] a221) The nucleotide sequence shown in SEQ ID NO: 3; or

[0053] a222) A nucleotide sequence of SEQ ID NO: 3 that has undergone substitution and / or deletion and / or addition of one or more nucleotides, and has the same function as the nucleic acid molecule shown in SEQ ID NO: 3; or

[0054] a223) has at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 70%, 60%, 50%, 40%, or 30% homology with SEQ ID NO: 3, and has the same function as the nucleic acid molecule shown in SEQ ID NO: 3.

[0055] According to a fourth aspect of the present invention, a conjugate is provided, comprising at least one of the antibody or antigen-binding fragment thereof described in the first aspect of the present invention and the recombinant protein described in the second aspect of the present invention;

[0056] And a coupling portion comprising at least one of a detectable marker, drug, toxin, electron-dense marker, biotin / avidin, spin marker, antibody, antibody Fc fragment, antibody scFv fragment, radionuclide, enzyme, gold nanoparticles / nanorobars, magnetic nanoparticles, and viral capsid proteins.

[0057] In some embodiments of the present invention, the detectable marker is a fluorescent or luminescent marker.

[0058] In some preferred embodiments of the present invention, the detectable marker is selected from any one of acridine ester, acridine sulfonamide, luminol, isoluminol, horseradish peroxidase, and alkaline phosphatase.

[0059] In some embodiments of the present invention, the radioactive isotope is selected from at least one of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, and Re-188.

[0060] According to a fifth aspect of the present invention, a solid-phase support is provided, the surface of which is coupled with an antibody against bovine pregnancy-associated glycoprotein 1 as described in the first aspect of the present invention or an antigen-binding fragment thereof and / or a recombinant protein as described in the second aspect of the present invention.

[0061] According to a sixth aspect of the invention, the use of an antibody against bovine pregnancy-associated glycoprotein 1 or an antigen-binding fragment thereof as described in the first aspect of the invention, a recombinant protein as described in the second aspect, a biomaterial as described in the third aspect, a conjugate as described in the fourth aspect, and / or a solid-phase support as described in the fifth aspect in the preparation of a product is proposed.

[0062] In some embodiments of the present invention, the product comprises at least one of a drug, a reagent, a detection plate, a reagent kit, and a detection chip.

[0063] In some embodiments of the present invention, the reagent, detection plate, detection chip, or reagent kit has at least one function among j1) to j2):

[0064] j1) Detect the presence or level of bovine pregnancy-associated glycoprotein 1 in the sample;

[0065] j2) Diagnose early pregnancy in cattle.

[0066] According to a seventh aspect of the present invention, a product is provided, the product comprising at least one of k1) to k4):

[0067] k1) The antibody or antigen-binding fragment thereof as described in the first aspect of the present invention;

[0068] k2) The recombinant protein described in the second aspect of the present invention;

[0069] k3) The coupling described in the fourth aspect of the present invention;

[0070] k4) The solid support described in the fifth aspect of the present invention.

[0071] In some embodiments of the present invention, the product comprises at least one of a drug, a reagent, a detection plate, a reagent kit, and a detection chip.

[0072] In some embodiments of the present invention, the reagent, detection plate, detection chip, or reagent kit has at least one function among j1) to j2):

[0073] j1) Detect the presence or level of bovine pregnancy-associated glycoprotein 1 in the sample;

[0074] j2) Diagnose early pregnancy in cattle.

[0075] According to an eighth aspect of the present invention, a method for preparing an antibody against bovine pregnancy-associated glycoprotein 1 or an antigen-binding fragment thereof as described in the first aspect of the present invention, or a recombinant protein as described in the second aspect of the present invention, is provided, obtained by culturing the transgenic cell line described in the third aspect of the present invention.

[0076] The present invention has at least the following beneficial effects:

[0077] This invention provides an antibody against bovine pregnancy-associated glycoprotein 1 or its antigen-binding fragment thereof. The antibody or its antigen-binding fragment has good specificity for bovine pregnancy-associated glycoprotein 1 and can be used to detect the presence or level of bovine pregnancy-associated glycoprotein 1 in a sample, providing a rapid, simple, economical and efficient solution for early pregnancy diagnosis in dairy cows. Attached Figure Description

[0078] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0079] Figure 1 The image shows the SDS-PAGE results of imidazole eluted samples collected at different times in Example 1; where M is the protein molecular weight standard (Marker) and 1-4 are eluted samples collected at different times with a concentration of 250mM imidazole.

[0080] Figure 2 This is a graph showing the results of serum titer detection in mice after immunization in Example 2;

[0081] Figure 3 This is a graph showing the results of ascites titer detection in mice after injection of cell line PAG1-1 in Example 3;

[0082] Figure 4 This is the result of SDS-PAGE analysis of the purified PAG1-1 monoclonal antibody in Example 3; where M is the protein molecular weight standard (Marker) and 1 is the purified PAG1-1 monoclonal antibody.

[0083] Figure 5 This is a graph showing the results of the PAG1-1 monoclonal antibody specificity detection in Example 3. Detailed Implementation

[0084] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0085] Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in these examples are commercially available.

[0086] Example 1: Obtaining recombinant bovine pregnancy-associated glycoprotein 1 (bPAG1) protein

[0087] In this embodiment, recombinant bPAG1 protein was prepared. The specific preparation method includes the following steps:

[0088] 1. Construction of recombinant expression vectors

[0089] Based on the sequence information (NM_174411.2) retrieved from NCBI, Anshengda Biotechnology Co., Ltd. was commissioned to synthesize the full-length gene sequence of bPAG1 and construct it into the pET28a vector to obtain the recombinant expression vector pET28a-bPAG1.

[0090] 2. Expression and purification of bPAG1 protein

[0091] After confirming the correct BL21(DE3) clone containing the recombinant expression vector pET28a-bPAG1, plasmids were extracted and transformed using standard methods. Single colonies were then picked and cultured in LB medium containing ampicillin resistance until the bacterial concentration reached A... 600When the concentration was 0.5, expression was induced for 18 h with 0.1 mM IPTG at 200 rpm and 16 °C. The cells were collected by centrifugation at 4 °C and 4000 rpm for 20 min, resuspended and washed with 20 mL PBS, and then sonicated on ice for 3 min with Binding buffer at 30 W. The sonication was repeated for 2 seconds with a 2-second interval until the bacterial solution was relatively clear. The supernatant was collected by centrifugation at 4 °C and 12000 rpm for 20 min for purification.

[0092] The target protein (bPAG1 recombinant protein) was purified by nickel-column affinity chromatography: A 50% NI-NTA column was packed, washed with 4 mL of deionized water, and then equilibrated with 5 mL of binding buffer. The sample was loaded, and the supernatant containing the E recombinant protein was added to the column. The permeate was collected and loaded again. Unbound proteins were washed away with binding buffer, followed by 20 mL of washing buffer. Proteins were then eluted with 250 mM imidazole elution buffer. The purity of the eluted sample was determined by SDS-PAGE electrophoresis. The results are shown below. Figure 1 As shown, the bPAG1 recombinant protein obtained by affinity chromatography had high purity. The concentration of the purified bPAG1 recombinant protein was determined by BCA method.

[0093] Example 2: Establishment of a monoclonal antibody cell line for the bPAG1 recombinant protein

[0094] This embodiment prepared a cell line for a recombinant bPAG1 protein monoclonal antibody. The specific preparation method includes the following steps:

[0095] 1. Mouse immunization

[0096] The bPAG1 recombinant protein obtained in Example 1 was used as an immunogen to immunize Balb / c mice. For the first immunization (day 1 of the first immunization), 100 μg of immunogen was emulsified with Freund's complete adjuvant at a 1:1 volume ratio, and injected subcutaneously into five immunization sites on the back and abdomen of each mouse. A second immunization was performed on day 15, using Freund's incomplete adjuvant emulsified with the immunogen at a 1:1 volume ratio, at a dose of 50 μg per mouse, following the same method. A third immunization was performed on day 29, following the same method as the second immunization. On day 36, a small amount of tail blood was collected for ELISA testing. If the antibody titer was greater than 1:10000, antigen pulse immunization and spleen cell fusion could then be performed. If the antibody titer was less than 1:10000, a fourth immunization was performed on day 43, following the same pattern. Three days before cell fusion, an intraperitoneal pulse immunization was performed, with 100 μg of immunogen injected directly into the peritoneum without adjuvant.

[0097] 2. Mouse serum titer detection

[0098] (1) Antigen coating: The coating buffer was adjusted to 1 μg / mL with the bPAG1 recombinant protein obtained in Example 1 as the coating antigen. 100 μL was added to each well of the ELISA plate and incubated overnight at 4°C.

[0099] (2) Washing: On the second day, discard the liquid in the well, pat dry, and wash twice with PBST in a plate washer.

[0100] (3) Sealing: Add 200 μL of sealing solution to each well and incubate at 37°C for 1 h.

[0101] (4) Preparation of serum (primary antibody): Blood was collected from the tail of mice, and serum from unimmunized mice was used as a negative control.

[0102] (5) Add primary antibody: Use blocking buffer to serially dilute the serum samples to be tested. Use non-immunized mouse serum diluted 1:2500 as a negative control and blocking buffer as a blank control. Incubate at 37℃ for 1 hour, discard the liquid in the well, pat dry, wash twice with PBST, and pat dry.

[0103] (6) Add enzyme-labeled secondary antibody: Dilute enzyme-labeled secondary antibody (HRP-goat anti-mouse) 5000 times with blocking buffer, 100 μL / well, incubate at 37℃ for 1 h, discard the liquid in the well, and pat dry.

[0104] (7) Color development and measurement: Add 50 μL of TMB chromogenic solution per well, incubate at 37℃ in the dark for 15 min, then add 100 μL of 2M sulfuric acid per well to terminate the reaction. Measure the A value at 450 nm using a microplate reader. Calculation: The highest antiserum dilution factor when the ratio of the A value of the test well to the A value of the negative control well (P / N) ≥ 2.1 is taken as the serum titer. Mice with a dilution greater than 1:100,000 are prepared for the next fusion step. The results are as follows: Figure 2 As shown, the reaction between recombinant bPAG1 protein and immune serum was identified by indirect ELISA, with a mouse serum titer of 1:32000.

[0105] 3. Cell fusion

[0106] Aseptically, spleen cells from immunized mice were mixed with myeloma cells from SP2 / 0 mice at a ratio of approximately 5:1 in a 50 mL centrifuge tube. After washing twice with culture medium, the supernatant was discarded. Over 50 seconds, 0.9 mL of preheated PEG-1500 was slowly added to disperse the cells as evenly as possible in the PEG. The mixture was allowed to stand for 1 min. Then, 20 mL of preheated serum-free DMEM culture medium at 37°C was slowly added dropwise, 2 mL over the first two minutes and 18 mL over the next two minutes, all within 4 minutes. The mixture was allowed to stand for 3 minutes, then centrifuged at 800 rpm for 5 minutes and the supernatant was discarded. Preheated FBS and HAT culture medium were added, and the mixture was gently pipetted to mix. The mixture was then transferred to 96-well plates at a density of 200 μL per well and incubated in an incubator.

[0107] 4. Screening of positive hybridoma cells

[0108] Ten days after cell fusion, when the fused cells filled more than 50% of the wells, hybridoma cells were screened using an indirect ELISA method.

[0109] 5. Subcloning of positive hybridoma cells

[0110] Subcloning was performed on the positive wells using a limiting dilution method. The number and location of cell clusters in the positive wells were observed under an inverted microscope. Cell clusters were then aspirated using a 200 μL pipette tip in a clean bench, and the cell count was diluted to 1-2 cells per 100 μL. Prepared feeder cells were then added to 100 μL of the diluted cells in a 96-well plate, labeled, and incubated at 37°C for 9 days using a 5% CO2 incubator. After three subcloning cycles until one cell was found per well, the titer of the cell supernatant was detected using an indirect ELISA method. If the positive rate reached 100%, the culture was expanded, and the bPAG1-1 cell line was preserved.

[0111] Example 3b: Preparation and Identification of Monoclonal Antibody for PAG1 Protein

[0112] In this embodiment, a bPAG1 protein monoclonal antibody was prepared and its subclass was identified. The specific steps are as follows:

[0113] 1. Preparation and titer determination of ascites fluid from bPAG1 protein monoclonal antibody.

[0114] 12-16 week old female BALB / c mice were intraperitoneally injected with 0.5 mL of sterile liquid paraffin. Ten days later, each mouse was injected with 0.5 mL of a cell suspension (5 × 10⁻⁶) of the bPAG1-1 cell line preserved in Example 2. 5 Each mouse was injected with cell suspension (day 1 was counted as one cell per mouse); on day 7, after significant abdominal distension, ascites fluid was collected, centrifuged at 3000 rpm for 20 min, adipose tissue was removed, the supernatant was collected, and stored at -20℃ for later use. The titer of the ascites fluid was determined by indirect ELISA, and the results are as follows: Figure 3 As shown: Ascites antibody titer 1:640000.

[0115] 2. Purification of monoclonal antibodies

[0116] Ascites fluid was collected, and bPAG1-1 monoclonal antibody was purified using the caprylic acid-ammonium sulfate precipitation method, as follows: Ascites fluid was collected and centrifuged at 12000 rpm for 5 min at 4°C. The supernatant was collected, and 2 volumes of 0.06 M acetate buffer (pH 4.0) were added to adjust the pH to 4.5. 33 μL of caprylic acid was added per mL of ascites fluid, and the mixture was stirred at room temperature for 30 min. The mixture was then allowed to stand at 4°C for 1 h to allow for complete precipitation of impurities. The mixture was centrifuged at 1000 g for 30 min at 4°C. 0.277 g of ammonium sulfate powder was added per mL of the supernatant. The beaker was placed on a magnetic stirrer and stirred for 1 h. The mixture was then centrifuged at 10000 g for 20 min at 4°C. The supernatant was discarded, and the precipitate was dissolved in PBS. SDS-PAGE was used to identify the purity of the monoclonal antibody. The results are shown below. Figure 4 As shown: High-purity bPAG1-1 monoclonal antibody was obtained.

[0117] 3. Identification of Monoclonal Antibody Types and Subclasses

[0118] The experiment was conducted using the mouse subtype identification kit instructions. The bPAG1-1 monoclonal antibody was identified as IgG1, kappa light chain.

[0119] Meanwhile, Guangzhou Aiji Biotechnology Co., Ltd. was commissioned to sequence the monoclonal antibody produced by the cell line bPAG1-1 preserved in Example 2 (i.e., the bPAG1-1 monoclonal antibody purified in step 2 of Example 3). The results are as follows:

[0120] The nucleotide sequence of the heavy chain variable region of bPAG1-1 monoclonal antibody is: 5'-GAGGTGCAACTGGTGGAGTCT GGGGGAGACTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGTAGCCTCTGGATTCACTTTCAGTAGCTATGGCATGTCTTGGGTTCGCCAGACTCCAGACAAGAGGCTGGAGTGGGTCGCAACCATTAGTAGTGGTGGTTATTACACCTACTATCCAGACAGTGTGAA GGGGCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTGCAAATGAGCAGTCTGAAGTCTGAGGACACAGCCATATATTACTGTGCAAGACATCCAATTGGGACGAGGGAATGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTTTGCA-3'(SEQ ID NO: 1),

[0121] Its corresponding amino acid sequence is: EVQLVESGGDLVKPGGSLKLSCVASGFTFSSYGMSWVR QTPDKRLEWVATISSGGYYTYYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAIYYC ARHPIGTREWFAYWGQGTLVTVFA (SEQ ID NO: 2);

[0122] The nucleotide sequence of the light chain variable region is: 5'-GATATTGTGCTAACTCAGTCTCCAACCACCCTG TCTGTGACTCCAGGAGATAGCGTCAGTCTTTCCTGCAGGGCCAGCCAAAGTATTAGCAACAACCTACACTGGTATCAACAAAAATCACATGAGTCTCCAAGGCTTCTCATCAAGTATGCTTCCCAGTCCATCTCTGGGATCCCCTCC AGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACTCTCAGTATCAACAGTGTGGAGACTGAAGATTTTGGAGTGTATTTCTGTCAACAGAGTAACGGCTGGCCGTACACGTTCGGAGGGGGGACCAAACTGGAAATAAAA-3'(SEQ ID NO: 3),

[0123] The corresponding amino acid sequence is: DIVLTQSPTTLSVTPGDSVSLSCRASQSISNNLHWYQQK SHESPRLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVETEDFGVYFCQQSNGWPYTFG GGTKLEIK (SEQ ID NO: 4).

[0124] The heavy chain variable regions of the bPAG1-1 monoclonal antibody, defined by different schemes as CDR-H1, CDR-H2, and CDR-H3, are shown in Table 1.

[0125] Table 1. CDR-H1, CDR-H2, and CDR-H3 of the heavy chain variable region defined by different schemes.

[0126]

[0127] Table 2 shows the CDR-L1, CDR-L2, and CDR-L3 of the light chain variable region of the bPAG1-1 monoclonal antibody, defined by different definition schemes.

[0128] Table 2. CDR-L1, CDR-L2, and CDR-L3 of the variable region of the light chain defined by different schemes.

[0129]

[0130]

[0131] 4. Specificity detection of monoclonal antibodies

[0132] The antigens used were bovine pregnancy-associated glycoprotein 1 (PAG1), bovine pregnancy-associated glycoprotein 2 (PAG2), bovine pregnancy-associated glycoprotein 5 (PAG5), bovine pregnancy-associated glycoprotein 7 (PAG7), bovine early pregnancy factor (EPF), bovine viral diarrhea virus non-structural protein 3 (NS3), and bovine viral diarrhea virus Erns glycoprotein (Erns). All the antigens used in the tests were obtained by constructing expression vectors in the laboratory and inducing prokaryotic expression.

[0133] The ELISA plate was coated under the same conditions, using the bPAG1-1 monoclonal antibody purified in step 2 of Example 3 as the primary antibody and HRP-labeled goat anti-mouse IgG as the secondary antibody. A values ​​were read on the ELISA reader. 450 The values ​​were calculated by taking the average of three replicates for each sample. The specificity of the monoclonal antibody was detected using an indirect ELISA method (refer to Example 2). The results are as follows: Figure 5 As shown, the monoclonal antibody produced by the cell line bPAG1-1 preserved in Example 2 (i.e., the bPAG1-1 monoclonal antibody purified in step 2 of Example 3) has good specificity and can be used for qualitative or quantitative detection of bovine pregnancy-related glycoprotein 1 in the sample to be tested.

[0134] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. An antibody against bovine pregnancy-associated glycoprotein 1 or its antigen-binding fragment, characterized in that, The antibody or its antigen-binding fragment comprises a heavy chain and a light chain; The heavy chain of the antibody or its antigen-binding fragment comprises: Heavy chain variable region, wherein the heavy chain variable region includes CDR-H1, CDR-H2 and CDR-H3; The light chain of the antibody or its antigen-binding fragment comprises: Light chain variable region, wherein the light chain variable region includes CDR-L1, CDR-L2 and CDR-L3; The amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, and CDR-L3 of the antibody or its antigen-binding fragment are shown in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 16, and SEQ ID NO: 17, respectively. The amino acid sequence of CDR-L2 is YAS. The CDR is defined according to the IMGT definition scheme. The amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the antibody or its antigen-binding fragment are shown in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 17, respectively, wherein the CDR is defined according to the Kabat scheme; or The amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the antibody or its antigen-binding fragment are shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 17, respectively, wherein the CDR is defined according to the Chothia scheme; or The amino acid sequences of the antibody or its antigen-binding fragments CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are shown in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively, wherein the CDR is defined using the Contact definition scheme.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment includes: a1) SEQ ID NO: 2; or a2) An amino acid sequence of SEQ ID NO: 2 that has undergone substitution and / or deletion and / or addition of one or more amino acids and has the same function as the protein shown in SEQ ID NO: 2; The amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment includes: b1) SEQ ID NO: 4; or b2) The amino acid sequence of SEQ ID NO:4 with one or more amino acid substitutions and / or deletions and / or additions that have the same function as the protein shown in SEQ ID NO:

4.

3. The antibody or its antigen-binding fragment according to claim 2, characterized in that, The antibody or its antigen-binding fragment comprises at least one of full-length antibody, Fab, Fab', F(ab')2, Fv, and scFv.

4. The antibody or its antigen-binding fragment according to claim 2, characterized in that, The heavy chain of the antibody or its antigen-binding fragment further includes a heavy chain constant region; and / or The light chain of the antibody or its antigen-binding fragment further includes a light chain constant region.

5. A recombinant protein comprising the antibody or its antigen-binding fragment as described in any one of claims 1 to 4; and Optional tag sequences for expression and / or purification.

6. A biological material relating to the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 4, or the recombinant protein as described in claim 5, wherein the biological material comprises at least one of h1) to h8): h1) A nucleic acid molecule encoding an antibody or antigen-binding fragment thereof of any one of claims 1 to 4, or the recombinant protein of claim 5; h2) An expression cassette containing the nucleic acid molecule described in h1); h3) A carrier containing the nucleic acid molecule described in h1); h4) A carrier containing the expression box described in h2); h5) Transgenic cell lines containing the nucleic acid molecules described in h1); h6) Transgenic cell lines containing the expression cassette described in h2); h7) A transgenic cell line containing the vector described in h3); h8) Transgenic cell lines containing the vector described in h4).

7. A solid-phase support having an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 4, and / or a recombinant protein as described in claim 5, coupled to its surface.

8. The application of at least one of (1) to (4) in the preparation of the product; (1) The antibody or antigen-binding fragment thereof as described in any one of claims 1 to 4; (2) The recombinant protein according to claim 5; (3) The biomaterial as described in claim 6; (4) The solid support as described in claim 7; The product is at least one of reagents, test plates, reagent kits, and test chips.

9. The application according to claim 8, characterized in that, The reagent, detection plate, detection chip, or kit has at least one function of j1) to j2): j1) Detect the presence or level of bovine pregnancy-associated glycoprotein 1 in the sample; j2) Diagnose early pregnancy in cattle.

10. A product comprising at least one of k1) to k3): k1) The antibody or antigen-binding fragment thereof as described in any one of claims 1 to 4; k2) The recombinant protein according to claim 5; k3) The solid support as described in claim 7; The product is at least one of reagents, test plates, reagent kits, and test chips.

11. The product according to claim 10, characterized in that, The reagent, detection plate, detection chip, or kit has at least one function of j1) to j2): j1) Detect the presence or level of bovine pregnancy-associated glycoprotein 1 in the sample; j2) Diagnose early pregnancy in cattle.

12. The method for preparing the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 4 or the recombinant protein as described in claim 5, obtained by culturing the transgenic cell line as described in claim 6.

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