An immunochromatographic kit for detecting pregnant mare serum gonadotropin and application thereof

Abstract

This invention relates to an immunochromatographic kit for detecting pregnant mare serum gonadotropins and its application. Specifically, this invention provides an antibody or antigen-binding fragment thereof that specifically binds to pregnant mare serum gonadotropins (PMSG), which can specifically and with high affinity bind to the target antigen. This results in a highly sensitive detection product with 100% positive and negative detection rates. It also offers significant advantages such as ease of operation, rapid detection, and low cost, making it suitable for rapid on-site diagnosis of early pregnancy in equines.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, specifically to an immunochromatographic kit, and more specifically, to an immunochromatographic kit for detecting gonadotropins in pregnant mare serum and its application. Background Technology

[0002] Mare serum gonadotropin (PMSG) is secreted by the endometrium of the uterus of a pregnant mare. This hormone can be detected in the serum of pregnant mares between day 40 and day 120 of gestation, reaching its peak concentration around day 60. Because this hormone can only be detected in pregnant mares, PMSG testing is a highly effective method for detecting pregnancy in mares, and this test can be performed using a PMSG kit. After 150 days of gestation, PMSG becomes undetectable. The PMSG diagnostic test is an effective and simple method for detecting pregnancy. Compared to ultrasound, it determines pregnancy earlier, allowing operators to obtain information about a mare's pregnancy more quickly and economically.

[0003] There is an urgent need in this field to develop a pregnant mare serum gonadotropin detection kit that offers advantages such as rapid detection and low cost, solves problems such as cumbersome operation and the need for professional technicians, and enables rapid batch testing, thereby reducing costs and increasing efficiency for farms and ranches. Summary of the Invention

[0004] To address the shortcomings of current methods for detecting early pregnancy in equines, such as cumbersome procedures, high costs, long testing cycles, and invasive damage to the embryos of breeding horses, a kit for detecting serum gonadotropins in pregnant equines is provided. This kit reduces the number of steps, is convenient and easy to use, and is inexpensive. It can detect pregnant equines up to 40 days after mating, providing ranches with refined management, cost reduction, and efficiency improvement, resulting in higher economic benefits.

[0005] To achieve the objectives of the invention described above, the present invention provides the following technical solution: An antibody or its antigen-binding fragment against pregnant mare serum gonadotropin is provided.

[0006] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region; In some specific embodiments, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the amino acid sequence shown in SEQ ID NO: 12 or 18; In some specific embodiments, the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the amino acid sequence shown in SEQ ID NO: 14 or 20; In some implementations, HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system; in some specific implementations, HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 are defined according to the Kabat numbering system; wherein, the aforementioned system is the IMGT system.

[0007] In some embodiments, the amino acid sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region are shown in SEQ ID NO: 1-3, respectively; and the amino acid sequences of LCDR1, LCDR2 and LCDR3 in the light chain variable region are shown in SEQ ID NO: 7 and 9, respectively, wherein the amino acid sequence of LCDR2 is KVS; In some embodiments, the amino acid sequences of HCDR1 and HCDR3 in the heavy chain variable region are shown in SEQ ID NO: 4-6, respectively; and the amino acid sequences of LCDR1 and LCDR3 in the light chain variable region are shown in SEQ ID NO: 10, 11, respectively, and the amino acid sequence of LCDR2 is KVS.

[0008] In some embodiments, the heavy chain variable region comprises an amino acid sequence as shown in or having at least 80% identity with SEQ ID NO: 12 or 18; in some specific embodiments, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 12 or 18; in some specific embodiments, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 12; in some specific embodiments, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 18. In some embodiments, the light chain variable region comprises an amino acid sequence as shown in or having at least 80% identity with SEQ ID NO: 14 or 20; in some specific embodiments, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 14 or 20; in some specific embodiments, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 14; in some specific embodiments, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 20.

[0009] In some specific embodiments, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 12, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 14; In some specific embodiments, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 12; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 20; In some specific embodiments, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 18, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 14; In some specific embodiments, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 18; and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 20.

[0010] In some embodiments, the antibody or its antigen-binding fragment is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody; in some embodiments, the antibody or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment, wherein: the heavy chain framework region of the humanized antibody or its antigen-binding fragment is derived from IGKV3-21*01 or IGKV4-30-4*01; and / or, the light chain framework region is derived from IGKV1-33*01 or IGKV1-27*01.

[0011] In some embodiments, the antibody or its antigen-binding fragment further includes a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4, and a light chain constant region of human κ or λ chains; in some specific embodiments, the antibody or its antigen-binding fragment further includes a heavy chain constant region of human IgG1, and a light chain constant region of human κ or λ chains; in some specific embodiments, the antibody or its antigen-binding fragment further includes a heavy chain constant region of human IgG1, and a light chain constant region of human κ chains.

[0012] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain having an amino acid sequence as shown in or having at least 80% identity with SEQ ID NO: 16 or 22, and a light chain having an amino acid sequence as shown in or having at least 80% identity with SEQ ID NO: 17 or 8.

[0013] In some specific embodiments, the antibody or its antigen-binding fragment comprises a heavy chain of an amino acid sequence as shown in SEQ ID NO: 16 or 22, and a light chain comprising an amino acid sequence as shown in SEQ ID NO: 17 or 8; In some specific embodiments, the antibody or its antigen-binding fragment comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO: 16, and a light chain with an amino acid sequence as shown in SEQ ID NO: 17; In some specific embodiments, the antibody or its antigen-binding fragment comprises a heavy chain of an amino acid sequence as shown in SEQ ID NO: 16, and a light chain comprising an amino acid sequence as shown in SEQ ID NO: 8; In some specific embodiments, the antibody or its antigen-binding fragment comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO: 22, and a light chain with an amino acid sequence as shown in SEQ ID NO: 17; In some specific embodiments, the antibody or its antigen-binding fragment comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO: 22, and a light chain with an amino acid sequence as shown in SEQ ID NO: 8.

[0014] A reagent strip for detecting gonadotropins in pregnant mare serum is provided.

[0015] In some embodiments, the test strip includes an antibody or antigen-binding fragment thereof that specifically binds to serum gonadotropin (PMSG); in some specific embodiments, the test strip includes an antibody or antigen-binding fragment thereof as described in any of the foregoing.

[0016] The use of the test strips for detecting pregnant mare serum gonadotropins as described in any of the above descriptions in the preparation of a test kit for detecting pregnant mare serum gonadotropins is provided.

[0017] In some embodiments, the test kit includes a test card comprising a plastic casing containing the test strips described in any of the preceding descriptions, as well as the test strips and a desiccant.

[0018] A kit for detecting gonadotropins in pregnant mare serum is provided.

[0019] In some embodiments, the test kit includes a test strip for pregnant mare serum gonadotropins as described in any of the places above.

[0020] In some embodiments, the test strip includes a PVC sheet with a nitrocellulose membrane on it. An absorbent pad is provided at the upper end of the nitrocellulose membrane, a gold label pad is provided at the lower end of the nitrocellulose membrane, and a sample pad is located below the gold label pad.

[0021] In some embodiments, the gold-labeled pad is coated with colloidal gold-labeled anti-PMSG monoclonal antibody, the T-line of the nitrocellulose membrane is coated with anti-PMSG monoclonal antibody, and the C-line is coated with goat anti-mouse IgG.

[0022] In some embodiments, the gold-labeled pad is coated with colloidal gold-labeled anti-PMSG monoclonal antibody 12A5, the T-line of the nitrocellulose membrane is coated with anti-PMSG monoclonal antibody 6D13, and the C-line is coated with goat anti-mouse IgG.

[0023] In some embodiments, the method for preparing the gold-labeled pad includes: labeling PMSG monoclonal antibody 12A5 onto colloidal gold (λ=530±5nm, OD=2) at a rate of 8-15ug / ml, concentrating it to 30 times with colloidal gold reconstitution solution, and spraying it onto the treated conjugation pad at a rate of 1-2ul / cm using an XYZ three-dimensional streak sprayer, and drying it at 45°C for 24h.

[0024] In some embodiments, the T-line of the nitrocellulose membrane is coated with PMSG monoclonal antibody 6D13 at a concentration of 1.0-2.0 mg / ml; the C-line is coated with goat anti-mouse IgG at a concentration of 0.5-1.5 mg / ml.

[0025] In some embodiments, the sample pad is prepared by soaking in a sample pad treatment solution and then drying at 45°C for 24 hours. The sample pad treatment solution is a 1L solution of 10-50mM tris(hydroxymethyl)aminomethane (Tris) containing 1-5g S9 and 5-10g polyvinyl alcohol (PVA) with a pH of 8.5-9.0.

[0026] In some embodiments, the colloidal gold complexation solution is 1L of 20-100mM borate buffer (BBS) containing 1-5g Tween-20, 1-5g bovine serum albumin, 1-5g surfactant S9, and 50-100g sucrose, with a pH of 8.0-9.0.

[0027] In some embodiments, the coating buffer is 1L of 20-100mM PBS solution containing 5-10g of sucrose, with a pH of 7.4-8.0; the diluent is 1L of 10mM-50mM PBS solution containing 1-5ml of Tween-20 and 1-3g of polyvinylpyrrolidone K30 (PVP K30), with a pH of 7.4-7.8.

[0028] Provide the use of antibodies or antigen-binding fragments thereof against anti-pregnant mare serum gonadotropins as described in any of the above descriptions, test strips, and / or test kits in the preparation of serum gonadotropin detection products.

[0029] Provide the use of antibodies or antigen-binding fragments thereof against anti-pregnant equine serum gonadotropins as described in any of the above descriptions, test strips, and / or test kits in the preparation of equine serum gonadotropin detection products.

[0030] Provide the use of antibodies or antigen-binding fragments thereof against pregnant mare serum gonadotropins as described in any of the above descriptions in the preparation of pregnant mare serum gonadotropin detection products.

[0031] Provide the use of antibodies or antigen-binding fragments thereof against anti-pregnant mare serum gonadotropins as described in any of the above descriptions, test strips, test kits, and / or test products in the detection of serum gonadotropins.

[0032] Provide the use of antibodies against equine serum gonadotropins or their antigen-binding fragments as described in any of the above descriptions, test strips, test kits, and / or test products in the detection of pregnant mare serum gonadotropins.

[0033] Provide the use of antibodies or antigen-binding fragments thereof against anti-pregnant mare serum gonadotropins as described in any of the above descriptions, test strips, test kits, and / or test products in the detection of pregnant mare serum gonadotropins.

[0034] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) Compared with conventional ultrasound examination methods, the kit of the present invention is simpler to operate and cheaper, which can greatly reduce the inspection cost of farms and the workload of inspection personnel.

[0035] (2) The monoclonal antibody used in the kit of this invention is a self-developed monoclonal antibody that can bind to the target antigen with high specificity.

[0036] (3) The kit of the present invention detects gonadotropins in whole blood, serum and plasma samples of pregnant mare. The colloidal gold immunochromatography used is the first of its kind in the world. Attached Figure Description

[0037] Figure 1 This is an SDS-PAGE electrophoresis image of the PMSG protein. M represents the protein marker, and channel 1 corresponds to the PMSG sample.

[0038] Figure 2 This is an SDS-PAGE electrophoresis image of PMSG monoclonal antibodies 12A5 / 6D13. M represents the protein marker, and channels 1 and 2 correspond to monoclonal antibodies 12A5 and 6D13, respectively.

[0039] Figure 3 This is a schematic diagram of the test strip structure. In the diagram, 1-1 is the PVC sheet, 1-2 is the nitrocellulose membrane, 1-3 is the absorbent pad, 1-4 is the gold label pad, 1-5 is the sample pad, C represents the control line, and T represents the detection line.

[0040] Figure 4 This is a diagram illustrating the interpretation of the results.

[0041] Figure 5 The results are from the PMSG protein control test. Detailed Implementation

[0042] Exemplary embodiments of the present disclosure will now be described in more detail with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present disclosure and to fully convey its scope to those skilled in the art. It should be noted that, unless otherwise specified, embodiments and features described herein can be combined with each other. The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0043] Unless otherwise specified, all conditions in the following examples were performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0044] Example 1: Preparation of Antigen Pregnant mare serum gonadotropin (PMSG) protein was prepared for antibody screening.

[0045] Blood was collected from pregnant equines during their first 1-3 months of gestation at the horse farm. Serum was collected and stored at -20°C for later use. 200 ml of the frozen pregnant mare serum was thawed at 4°C and centrifuged at 10,000 rpm for 10 min at 4°C. The supernatant was collected, and the pH was adjusted to 3.5 with 1M metaphosphoric acid. The supernatant was then centrifuged again. The supernatant was placed in an ice box, and pre-chilled 95% ethanol was added dropwise with magnetic stirring to a final alcohol concentration of 35%. The mixture was allowed to stand for 2 hours, then centrifuged at 10,000 rpm for 30 min at 4°C, and the supernatant was collected. Solid sodium chloride was added to the supernatant to a concentration of 0.05M. 95% ethanol was continued to be added dropwise with stirring in an ice bath to a final concentration of 50%. The mixture was allowed to stand overnight at low temperature, then centrifuged at 10,000 rpm for 40 min at 4°C, and the precipitate was collected. The precipitate was carefully washed once with pre-chilled 50% ethanol; this is the crude extract of equine gonadotropin. The precipitate was dissolved in PB solution (pH 7.2) and dialyzed into this solution. Filter using a 0.45µm filter membrane. Take 5ml of a pre-packed DEAE chromatography column, equilibrate with PB at pH 7.2, load the dialysis sample, and after equilibration, elute with a gradient of equilibration buffer containing 1M sodium chloride. The target protein is in the 0.15M sodium chloride fraction. Dialyze the collected target fraction to PBS at pH 7.4, concentrate, and perform gel electrophoresis to obtain 8mg of the target protein with a purity of 90% (see...). Figure 1 ).

[0046] Example 2: Preparation of anti-equine gonadotropin (PMSG) mouse monoclonal antibody 1. Animal immunization Five 6-8 week old female Balb / c mice were immunized. Purified equine gonadotropin (PMSG) protein was mixed with Freund's complete adjuvant at a dose of 50 μg / mouse, completely emulsified, and administered in 200 μL per mouse per immunization. The mice were immunized for the first time at multiple sites on the back, groin, and axilla. Two weeks later, a second immunization was administered at the same dose and route, but with an incomplete adjuvant. Fifteen days later, a third immunization was administered at the same dose, with Freund's incomplete adjuvant added, via intraperitoneal injection. One week later, blood was collected from the tails of all immunized mice, and serum antibody titers were detected by ELISA. The results are shown in Table 1. Mice with serum titers of 100,000 or higher were selected for fusion. Three days before fusion, mice were immunized intraperitoneally at a dose of 100 μg / mouse without adjuvant.

[0047] Table 1. PMSG serum antibody titers

[0048] 2. Construction of B cells 2.1 Culture and preparation of myeloma cell lines This invention uses the SP / 20 myeloma cell line, which exhibits excellent growth and fusion efficiency, with a doubling time of 10-12 hours. Cells in the logarithmic growth phase, with good cell morphology and viability, are selected for fusion. Myeloma cells should undergo adaptive culture before fusion to allow them to grow to their optimal state (i.e., logarithmic growth phase). On the day of fusion, myeloma cells are gently blown off the flask wall using a bent-tip pipette and collected in a 50mL centrifuge tube or fusion tube. The cells are centrifuged at 1000 rpm for 5-10 minutes, the supernatant is discarded, and 30mL of culture medium is added to the precipitate. After washing once, the cells are centrifuged again for 5-10 minutes, the supernatant is discarded, and the precipitate is mixed with 20mL of culture medium for later use.

[0049] 2.2 Preparation of spleen cells BALB / c mice that had already undergone animal immunization were selected. Blood was collected by enucleation, and the serum was separated using a centrifuge to serve as a positive control serum for antibody detection. Mice were euthanized by cervical dislocation. The mice were then immersed in 75% alcohol for 5 minutes. The spleen was aseptically removed in a laminar flow hood and placed in a petri dish containing 10 mL of culture medium. The spleen was gently washed, and the surrounding connective tissue was removed. The spleen was punctured with a sterile syringe until it turned white. The spleen cells were collected from the petri dish into a 50 mL centrifuge tube and centrifuged at 1000 rpm for 10 minutes. The supernatant was discarded, and 10 mL of culture medium was added. A small amount of the medium was then used for 10-fold dilution and counting.

[0050] 2.3 Cell Fusion Three days prior to fusion, positive mice were boosted with immunization. Mouse spleen cells and myeloma cells sp2 / 0 were mixed at a ratio of 10:1, fused with PEG, and then cultured in HAT selective medium. Ten days later, the hybridoma cell supernatant was screened by ELISA. The selected positive hybridoma cells were cloned using limiting dilution. After five rounds of screening, six cell lines were finally identified as positive.

[0051] 2.4 Screening of hybridoma cells ELISA Method: Horse gonadotropin (PMSG) protein (10 μg / mL, 50 μL / well) was coated onto a 96-well plate and incubated overnight at 4°C. The next morning, the liquid in the wells was discarded, and the plates were washed three times with washing buffer and patted dry. 100 μL of blocking buffer was added to each well, and the plates were blocked at 37°C for 2 hours, washed twice, and patted dry. 100 μL of culture supernatant from six hybridoma cell lines was added to each well, along with positive, negative, and blank controls. The plates were incubated at 37°C for 0.5 hours, washed four times, and patted dry. Horseradish enzyme-labeled goat anti-mouse IgG (1:10000) was added to each well at 100 μL, and the plates were incubated at 37°C for 30 minutes, washed four times, and patted dry. Finally, 100 μL of TMB substrate chromogenic solution was added to each well, and the reaction was terminated with 50 μL of 2 mol / L dilute hydrochloric acid after 15 minutes of development.

[0052] As shown in Table 2, the OD values ​​of each well were measured at dual wavelengths of 450 nm and 630 nm. The blank well value was below 0.02, and the negative well value was below 0.1, showing a clear distinction between positive and negative values. This indicates that hybridoma cells resistant to equine gonadotropin (PMSG) secrete antibodies that specifically recognize PMSG protein. The same method was used to measure the titer in mouse ocular blood, achieving a ratio of 1:100,000, which is suitable for cell fusion.

[0053] Table 2. PMSG hybridoma cell screening

[0054] 3. Production and purification of monoclonal antibodies 3.1 Ascites preparation Two hybridoma cell lines with relatively strong hybridoma cell lines were selected for ascites production. Balb / c mice weighing approximately 25 grams were selected, and each mouse was injected intraperitoneally with 500 μL of liquid paraffin. Two weeks later, the mice were injected intraperitoneally with anti-equine gonadotropin (PMSG) hybridoma cells at a dose of 10⁶ cells / mouse. Ten days later, the mice were continuously observed. After the abdomens were significantly distended, the ascites was collected, centrifuged at 10,000 rpm for 10 minutes, and the supernatant was collected, impurities were removed, and the supernatant was stored at -20°C.

[0055] 3.2 Ascites purification The ascites fluid was removed from the -20°C freezer, thawed, and centrifuged at 10,000 rpm. The supernatant was filtered through a 0.22 μm filter. Affinity purification was performed according to the instructions for the protein-A column from Tiandi Renhe. The purified antibody was collected and dialyzed against 20 Mm pH 7.4 PBS. After 72 h, the antibody was collected, filtered through a 0.22 μm filter, aliquoted, and stored at -20°C for subsequent detection and validation. The sequences of the two obtained anti-PMSG antibodies, 12A5 and 6D13, are shown in Table 3 below. Table 3. Antibody CDR Sequences ; >12A5 Heavy Chain Variable Region SVQLQESGGGFVKPGGSLKLSCAASGFTFSYYAMSWVRQTPEKRLEWVASISRGGKTFYSDRVQGRFTISRRDNARDILYLQMSSLRSEDTAMYYCVRGTTEIADFWGQGTTLTVSS (SEQ ID NO:12) >12A5 heavy chain variable region gene sequence: AGTGTgCAACTGCAGGAGTCAGGGGGAGGCTTTGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTTACTATGCCATGTCTTGGGTTCGCCAGACTCCAGAGAAGAGGCTGGAGTGGGTCGCATCCATTAGTCGTGGCGGTAAAACCTTC TATTCAGACAGAGTACAGGGCCGATTCACCATCTCCAGAGATAATGCCAGGGACATCCTGTACCTGCAAATGAGCAGTCTGAGGTCTGAGGACACGGCCATGTATTACTGTGTAAGAGGTACTACGGAAATAGCTGACTTCTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO:13) >12A5 Light Chain Variable Zone DIVMTQSPLSLPVSLGDQVSISCRSSQSLVYSNGNTYFYWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQGTHVPWTFGGGTKLEIK (SEQ ID NO:14) >12A5 Light chain variable region gene sequence: gatattgtgatgacccaatctccactctccctgcctgtcagtcttggagatcaagtctccatctcttgcagatctagtcagagccttgtatacagtaatggaaacacctatttttattggtacctgcagaagccaggccagtctccaaagctcctgatctacaaagtttccaaccgattttctggggtcccagacaggttcagtggcagtggatcagggacagatttcacactcaagatcagcagagtggaggctgaggatctgggagtttatttctgctctcaaggtacacatgttccgtggacgttcggtggaggcaccaagctggaaatcaaa (SEQ ID NO:15) >12A5 Heavy chain SVQLQESGGGFVKPGGSLKLSCAASGFTFSYYAMSWVRQTPEKRLEWVASISRGGKTFYSDRVQGRFTISRDNARDILYLQMSSLRSEDTAMYYCVRGTTEIADFWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO:16) >12A5 Light chain DIVMTQSPLSLPVSLGDQVSISCRSSQSLVYSNGNTYFYWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQGTHVPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO:17) >6D13 Heavy Chain Variable Region DVQLQESGPDLVKPSQSLSLTCTVTGYSITSGYAWHWFRQFPGNKLEWMGYIHYRGDTSYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCARDYAGWGQGTLVTVSA (SEQ ID NO:18) >6D13 Heavy Chain Variable Region Gene Sequence GATGTGCAGCTTCAGGAGTCAGGACCTGACCTGGTGAAACCTTCTCAGTCACTTTCACTCACCTGCACTGTCACTGGTTACTCCATCACCAGTGGTTATGCCTGGCACTGGTTCCGGCAGTTTCCAGGAAACAAACTGGAATGGATGGGCTACATTCACTACAGAGGTGACACTAGCTACAACCCATCTCTCAAAAGTCGAATCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCTTGCAATTGAATTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCAAGAGATTACGCGGGCTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO:19) >6D13 Light Chain Variable Region DIVMTQSPLSRPVSLGDQASISCGSSQSLVHSNGNTYLQWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAADLGVYFCSQSTHIPLTFGAGTKLELK (SEQ ID NO:20) >6D13 Light chain variable region gene sequence gatattgtgatgacacaatctccactctcccggcctgtcagtcttggagatcaagcctccatctcctgcggatctagtcagagccttgttcacagtaatggaaacacctatttacagtggtacctgcagaagccaggccagtctccaaagctcctgatctacaaagtttccaaccgattttctggggtcccagacaggttcagtggcagtggatcagggacagatttcacactcaagatcagcagagtggaggctgcggatctgggagtttatttctgctctcaaagtacacatattccgctcacgttcggtgctgggaccaagctggagctgaaa (SEQ ID NO:21) >6D13 Heavy chain DVQLQESGPDLVKPSQSLSLTCTVTGYSITSGYAWHWFRQFPGNKLEWMGYIHYRGDTSYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCARDYAGWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO:22) >6D13 Light chain DIVMTQSPLSRPVSLGDQASISCGSSQSLVHSNGNTYLQWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAADLGVYFCSQSTHIPLTFGAGTKL ELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO:8) Note: The constant regions of the monoclonal antibodies described in this invention are all κ chains, and the constant regions of the heavy chains are all IgG1 type.

[0056] 4. Characterization of monoclonal antibodies Antibody concentration determination: The absorbance values ​​(A280) and (A260) of the monoclonal antibody at 280 nm and 260 nm were determined by ultraviolet spectrophotometry. Protein content was calculated using the following formula: Protein content (mg / mL) = (A280 × dilution factor) / 1.35. The resulting protein contents were PMSG-12A5 (3.24 mg / mL) and PMSG-6D13 (2.65 mg / mL).

[0057] Antibody molecular weight determination: Monoclonal antibodies were measured using SDS-PAGE, such as... Figure 2 As shown, the heavy chain of the monoclonal antibody is approximately 46 kDa, and the light chain is approximately 25 kDa.

[0058] ELISA titer determination: The purified ascites monoclonal antibody was determined by indirect ELISA, as shown in Table 4. The results showed that the purified titer was greater than 1:100000.

[0059] Table 4. PMSG Monoclonal Antibody Titer Detection

[0060] Example 3: Preparation of a kit for detecting gonadotropins in pregnant mare serum The test kit contains a test card with a test strip. The test strip (e.g.) Figure 3 The sample includes a PVC sheet 1-1, a nitrocellulose membrane 1-2 pasted on the top of the PVC sheet 1-1, an absorbent pad 1-3 at the top of the nitrocellulose membrane, a gold label pad 1-4 at the bottom, and a sample pad 1-5 overlapping below the gold label pad 1-4.

[0061] The test strips are prepared as follows: (1) Preparation of colloidal gold solution: Add 1 ml of 2% HAuCl4 to 100 ml of purified water, heat to boiling, then add 1 ml of 2% trisodium citrate, heat until the color turns wine red, and continue heating for 10 minutes. After natural cooling, measure the maximum absorption wavelength and OD value at 400-600 nm using a spectrophotometer.

[0062] (2) Colloidal gold labeling: Take 60 ml of colloidal gold solution (λ=530±5 nm, OD=2), adjust the pH to 8.0 with 0.1 M K2CO3; after thorough mixing, add 600 μg of PMSG monoclonal antibody 12A5 and react for 45 min; then add 600 μl of 10% BSA solution to block for 30 min; centrifuge at 9000 rpm at 4℃ for 30 min, take the supernatant, reconstitute the precipitate with 2 ml of gold reconstitution solution, and store at 2-8℃ in the dark for later use.

[0063] (3) Preparation of PMSG gold-labeled pads: Take 2 ml of the gold marker prepared in step (2), concentrate it 30 times, and spray it onto the treated conjugate pad at 2 μl / cm using an XYZ three-dimensional gold spraying instrument. Dry it at 45°C for 24 hours. Then cut it into single strips using a strip cutter, seal and dry it for later use.

[0064] (4) Preparation of PMSG-NC membrane: The PMSG monoclonal antibody 6D13 was diluted to 1.5 mg / ml with coating buffer to prepare the T-line working solution; the goat anti-mouse IgG was diluted to 1.5 mg / ml with coating buffer to prepare the C-line working solution; the T-line and C-line working solutions were applied to the corresponding positions on the nitrocellulose membrane using a membrane scribing instrument, dried at 45°C for 24 hours, sealed and stored for later use, which is the PMSG membrane.

[0065] (5) PMSG sample pad preparation: Weigh 6.06g of Tris, 5g of S9 and 10g of PVA using an electronic analytical balance. Mix thoroughly with 1L of purified water. Adjust the pH to 9.0 with 6M HCl solution to obtain the PMSG sample pad treatment solution. Take a 20*30cm glass fiber, apply PMSG sample pad treatment solution at a rate of 40ml / sheet onto the glass fiber, and dry it at 45℃ for 24h. Cut it into 2cm pieces using a strip cutter, seal and store for later use; this is the PMSG sample pad.

[0066] (6) PMSG reagent assembly: Assemble the PMSG-NC membrane, gold label pad, sample pad, and absorbent paper according to... Figure 3 The reagent is assembled, cut to a certain width using a strip cutter, then placed into a plastic casing, and finally sealed in an aluminum foil bag to obtain the PMSG reagent.

[0067] Example 4: Instructions for use of the test kit The qualitative detection of pregnant mare serum gonadotropin (PMSG) in equine blood using colloidal gold assay reagents is performed as follows: (1) Tear open along the cut of the aluminum foil bag and take out the test reagent card.

[0068] (2) Place the test card on a flat table, use a disposable dropper to draw whole blood, serum or plasma, add 1 drop into the sample well of the test card, and then add 1 drop of sample diluent into the sample well of the test card.

[0069] (3) If the test line T and control line C appear in the observation area within 20 minutes, it indicates pregnancy, and the test is completed. Results displayed 30 minutes after sample addition are invalid.

[0070] (4) Interpretation of the interpretation results (see) Figure 4 ): Positive: Colored bands appear at both the test line (T) and the control line (C), indicating pregnancy.

[0071] Negative: Only the control line C showed a colored band, indicating that the patient was not pregnant.

[0072] Invalid: If there is no colored band at position C of the quality control line, the test is invalid regardless of whether a colored band appears at position T of the test line.

[0073] Example 5: Evaluation of reagent kit sensitivity The test method includes the following steps: (1) Use a pipette to take 50 μl of the quality control sample into 50 μl of the sample dilution solution and mix thoroughly; (2) Take out the PMSG test reagent card and place it flat on a horizontal table; (3) Use a pipette to take 80 μL of the diluted quality control sample mixture, time for 10 min, and wait for the result to be read.

[0074] The extracted PMSG protein was serially diluted with serum samples from non-pregnant horses to create quality control samples of 500 ng / ml, 50 ng / ml, 10 ng / ml, 5 ng / ml, 1 ng / ml, and 0 ng / ml. The test results were obtained according to the prescribed method. Figure 5 As shown, PMSG protein can be detected down to 1 ng / ml, indicating that the PMSG sensitivity of this invention is high.

[0075] Example 6: Investigation of the positive concordance rate of whole blood samples from pregnant mares The test method includes the following steps: (1) Use a pipette to draw 50 μl of whole blood sample from pregnant mare into 50 μl of sample diluent and mix thoroughly; (2) Take out the PMSG test reagent card and place it flat on a horizontal table; (3) Use a pipette to draw 80 μl of the diluted sample mixture, time for 20 min, and wait for the result to be read.

[0076] Fifty whole blood samples from pregnant mares confirmed by ultrasound were collected and tested according to the procedure. The test results are shown in Table 5. The results showed that all 50 whole blood samples from pregnant mares were positive, which was completely consistent with the ultrasound test results, with a positive concordance rate of 100%.

[0077] Table 5. Test results of 50 positive whole blood samples from pregnant malts ; Example 7: Investigation of the negative concordance rate of mare samples The test method includes the following steps: (1) Use a pipette to draw 50 μl of mare whole blood sample into 50 μl of sample diluent and mix thoroughly; (2) Take out the PMSG test reagent card and place it flat on a horizontal table; (3) Use a pipette to draw 80 μl of the diluted sample mixture, time for 20 min, and wait for the result to be read.

[0078] Fifty whole blood samples from individuals who were confirmed not to be pregnant by ultrasound (B-scan) were collected and tested according to the procedure. The test results are shown in Table 6. The results showed that all 50 whole blood samples from individuals who were not pregnant were negative, which was completely consistent with the B-scan test results, with a negative concordance rate of 100%.

[0079] Table 6. Test results of negative samples from 50 non-pregnant mares ; The above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the protection scope of the claims of the present invention.

Claims

1. An antibody or its antigen-binding fragment against pregnant mare serum gonadotropin, characterized in that, It contains antibody heavy chain variable regions and light chain variable regions selected from any of the following groups: (1) The amino acid sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region are shown in SEQ ID NO: 1-3, and the amino acid sequences of LCDR1 and LCDR3 in the light chain variable region are shown in SEQ ID NO: 7 and 9, respectively. The amino acid sequence of LCDR2 is KVS. or, (2) The amino acid sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region are shown in SEQ ID NO: 4-6, and the amino acid sequences of LCDR1 and LCDR3 in the light chain variable region are shown in SEQ ID NO: 10 and 11, respectively. The amino acid sequence of LCDR2 is KVS.

2. The antibody against pregnant mare serum gonadotropin or its antigen-binding fragment as described in claim 1, characterized in that, It contains antibody heavy chain variable regions and light chain variable regions selected from any of the following groups: (1) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 12, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 14; or, (2) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

20.

3. The antibody against pregnant mare serum gonadotropin or its antigen-binding fragment as described in claim 1 or 2, characterized in that, The antibody or its antigen-binding fragment further includes the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4, and the light chain constant region of human κ or λ chains.

4. A test strip for detecting gonadotropins in pregnant mare serum, characterized in that, The test strip includes an antibody or antigen-binding fragment thereof that specifically binds to serum gonadotropin (PMSG), as described in any one of claims 1-3.

5. The application of the test strip for detecting pregnant mare serum gonadotropins as described in claim 4 in the preparation of a test kit for detecting pregnant mare serum gonadotropins.

6. A detection kit for pregnant mare serum gonadotropins, characterized in that, The test kit includes the test strip for detecting pregnant mare serum gonadotropins as described in claim 4.

7. The detection kit as described in claim 6, characterized in that, The test strip includes a PVC sheet with a nitrocellulose membrane on it. An absorbent pad is located at the upper end of the nitrocellulose membrane, and a gold label pad is located at the lower end of the nitrocellulose membrane. A sample pad is located below the gold label pad.

8. The detection kit as described in claim 7, characterized in that, The gold-labeled pad is coated with colloidal gold-labeled anti-PMSG monoclonal antibody 12A5, the nitrocellulose membrane has T-line coated with anti-PMSG monoclonal antibody 6D13 and C-line coated with goat anti-mouse IgG. The heavy chain variable region and light chain variable region of the monoclonal antibody 12A5 are as described in group (1) of claim 1 or 2; The heavy chain variable region and light chain variable region of the monoclonal antibody 6D13 are as described in group (2) of claim 1 or 2.

9. The detection kit as described in claim 8, characterized in that, The method for preparing the gold-labeled pad includes: labeling PMSG monoclonal antibody 12A5 onto colloidal gold at a rate of 8-15 ug / ml, concentrating it to 30 times with colloidal gold reconstitution solution, and spraying it onto the treated conjugation pad at a rate of 1-2 μl / cm using an XYZ three-dimensional streak sprayer, and drying it at 45°C for 24 hours.

10. The detection kit as described in claim 9, characterized in that, The nitrocellulose membrane is coated with PMSG monoclonal antibody 6D13 on the T line at a concentration of 1.0-2.0 mg / ml; and coated with goat anti-mouse IgG on the C line at a concentration of 0.5-1.5 mg / ml.

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