Bovine early pregnancy diagnosis colloidal gold kit and application of antibody pair thereof

Abstract

The present application relates to a bovine early pregnancy diagnosis colloidal gold kit and application of an antibody pair thereof, the antibody pair consisting of a first monoclonal antibody and a second monoclonal antibody; wherein: the amino acid sequences of CDR1, CDR2 and CDR3 on the light chain variable region of the first monoclonal antibody are respectively shown as SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, and the amino acid sequences of CDR1, CDR2 and CDR3 on the heavy chain variable region are respectively shown as SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14; the amino acid sequences of CDR1, CDR2 and CDR3 on the light chain variable region of the second monoclonal antibody are respectively shown as SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21, and the amino acid sequences of CDR1, CDR2 and CDR3 on the heavy chain variable region are respectively shown as SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28. The present application provides an antibody pair with high specificity and high affinity, solves the pairing problem of traditional double antibody detection, significantly improves the detection sensitivity, and can perform early pregnancy diagnosis 24 days after mating.

Description

Technical Field

[0001] This invention relates to the field of biological diagnostic reagent kit technology, and in particular to the application of a colloidal gold reagent kit for early bovine pregnancy diagnosis and its antibody pair. Background Technology

[0002] Pregnancy-associated glycoprotein (PAG) is a key biomarker for early pregnancy diagnosis in ruminants, and its detection technology directly impacts breeding efficiency and reproductive management. Currently, large dairy farms in my country mainly rely on imported ELISA kits to detect early pregnancy in serum samples from cows 28 days after mating. The testing process typically takes more than two hours, consuming significant manpower and resources, and is also cumbersome in clinical application. Meanwhile, small and medium-sized dairy farms in China primarily rely on rectal examinations 45 days after artificial insemination or ultrasound examinations around 35 days after insemination for early pregnancy detection. This not only requires specialized technicians but also increases the non-pregnant period of dairy cows, raising feed costs and indirectly reducing the economic benefits for farmers.

[0003] Although some literature has published the preparation of monoclonal antibodies for this protein, this does not indicate that these monoclonal antibodies can be used in the preparation of colloidal gold, or that they can achieve good clinical results. In antigen epitope recognition, there are generally problems such as difficulty in antibody pairing and screening, insufficient specificity, limited sensitivity, and weak anti-interference ability. These pain points mean that although the dual-antibody detection technology has excellent theoretical performance, its low sensitivity and insufficient stability make it difficult to promote and apply in grassroots farms. Summary of the Invention

[0004] To overcome the above limitations, this invention provides a colloidal gold reagent kit for early bovine pregnancy diagnosis and the application of its antibody pairs, in order to solve the problem that there is a lack of antibody pairs for double antibody sandwich technology in the prior art, and that the double antibody sandwich technology is difficult to promote and apply in grassroots farms due to its low sensitivity.

[0005] The technical solution provided by this invention is as follows:

[0006] In a first aspect, the present invention provides a pair of antibodies against bovine pregnancy-related glycoproteins, said antibody pair comprising a first monoclonal antibody and a second monoclonal antibody; wherein:

[0007] The amino acid sequences of CDR1, CDR2 and CDR3 on the light chain variable region of the first monoclonal antibody are shown in SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 on the heavy chain variable region are shown in SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, respectively.

[0008] The amino acid sequences of CDR1, CDR2 and CDR3 on the light chain variable region of the second monoclonal antibody are shown in SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 on the heavy chain variable region are shown in SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28, respectively.

[0009] In conjunction with the first aspect of the present invention, some embodiments include: the amino acid sequence of the light chain variable region of the first monoclonal antibody as shown in SEQ ID NO:4, and the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO:11; the amino acid sequence of the light chain variable region of the second monoclonal antibody as shown in SEQ ID NO:18, and the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO:25.

[0010] In conjunction with the first aspect of the present invention, in some embodiments: the first monoclonal antibody is mouse IgG1, the amino acid sequence of its light chain variable region is shown in SEQ ID NO:4, and the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO:11; the second monoclonal antibody is mouse IgG1, the amino acid sequence of its light chain variable region is shown in SEQ ID NO:18, and the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO:25.

[0011] In conjunction with the first aspect of the present invention, in some embodiments: the first monoclonal antibody or the second monoclonal antibody is a full-length antibody or its antigen-binding region; the antigen-binding region is selected from at least one of the Fab fragment, F(ab)2 fragment, Fv fragment, (Fv)2 fragment, scFv fragment and sc(Fv)2 fragment.

[0012] In a second aspect, the present invention provides a set of antibody conjugates comprising the aforementioned antibody pairs against bovine pregnancy-associated glycoproteins and detection markers linked to a first monoclonal antibody and a second monoclonal antibody.

[0013] Thirdly, the present invention provides a set of nucleic acid molecules, the set of nucleic acid molecules comprising a first nucleic acid molecule pair and a second nucleic acid molecule pair, wherein the first nucleic acid molecule and the second nucleic acid molecule respectively encode a first monoclonal antibody and a second monoclonal antibody.

[0014] Fourthly, the present invention provides a kit for detecting anti-bovine pregnancy-related glycoproteins, the kit comprising:

[0015] (1) Detect effective amounts of the first and second monoclonal antibodies;

[0016] (2) Necessary colorimetric or quantitative reagents.

[0017] Fifthly, the present invention provides a colloidal gold reagent kit for early pregnancy diagnosis in cattle, the colloidal gold reagent kit comprising a colloidal gold test strip; the colloidal gold test strip is composed of a sample pad, a gold label pad, a reaction membrane and an absorbent pad stacked in sequence, and the reaction membrane is provided with a test line and a control line;

[0018] The gold-labeled pad is coated with a colloidal gold-labeled labeled antibody, the test line is coated with a capture antibody, the capture antibody being the first monoclonal antibody described above; the labeled antibody is the second monoclonal antibody described above.

[0019] The quality control line is coated with goat anti-mouse IgG polyclonal antibody.

[0020] In conjunction with the first aspect of the invention, some embodiments include:

[0021] The concentration of the captured antibody in the test line is 0.8–1.5 mg / mL; and / or,

[0022] The concentration of the colloidal gold-labeled antibody in the gold-labeled pad is 15–20 µg / mL.

[0023] In conjunction with the first aspect of the invention, some embodiments include:

[0024] The reaction membrane is provided with a sample pad and a gold label pad at one end and an absorbent pad at the other end. The gold label pad is placed on the reaction membrane, and the sample pad, absorbent pad and reaction membrane are all placed on the base plate.

[0025] The colloidal gold-labeled antibody coated in the gold pad is a second monoclonal antibody against PAG protein;

[0026] The capture antibody coated in the test line of the reaction membrane is a first monoclonal antibody against PAG protein, and the antibody coated in the control line is a goat anti-mouse IgG polyclonal antibody.

[0027] Compared with the prior art, what are the at least some beneficial effects of the present invention?

[0028] The two antibodies provided by this invention can bind to PAG simultaneously, with high pairing specificity and a detection limit of 2 ng / mL. Results can be interpreted within 10-15 minutes without the need for specialized equipment, enabling early pregnancy diagnosis in cows as early as 24 days after mating. Compared to the IDEXX PAG ELISA kit, this invention is simpler to operate and has a shorter detection time, making it more suitable for low-cost farms. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 The results show the detection results of the concentrations of the first monoclonal antibody in the test strips being 0.5, 1.0, and 1.5 mg / mL, respectively; specifically, the concentration of the first monoclonal antibody in test strip 1 is 0.5 mg / mL, in test strip 2 it is 1.0 mg / mL, and in test strip 3 it is 1.5 mg / mL.

[0031] Figure 2 Sensitivity testing used a series of serially diluted bpag1 recombinant protein synthesized in Example 1 as standards to determine the detection limit of the test strips. The results showed that test strip 1 (1 µg / mL), test strip 2 (0.5 µg / mL), and test strip 3 (5 ng / mL) all produced clear and sharp T lines. When the protein concentration of test strip 4 was diluted to 2 ng / mL, the T line became faint, indicating a weak positive result. Therefore, the detection limit of the test strips can be determined to be 2 ng / mL.

[0032] Figure 3 The colloidal gold test strip prepared in Example 3 was used to test samples of pregnant cow serum, bovine rotavirus tissue, bovine viral diarrhea virus tissue, bovine Escherichia coli milk, and bovine Streptococcus faecium milk. The results showed that the control lines 2-5 of the test strip developed color, while the test lines did not, indicating a negative result. This indicates that the test strip has no cross-reactivity with bovine rotavirus, bovine viral diarrhea virus, bovine Escherichia coli, and bovine Streptococcus faecium, and exhibits good specificity. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0034] The present invention provides a set of antibody pairs against bovine pregnancy-associated glycoproteins, consisting of a first monoclonal antibody and a second monoclonal antibody; wherein:

[0035] The amino acid sequences of CDR1, CDR2 and CDR3 on the light chain variable region of the first monoclonal antibody are shown in SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 on the heavy chain variable region are shown in SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, respectively.

[0036] The amino acid sequences of CDR1, CDR2 and CDR3 on the light chain variable region of the second monoclonal antibody are shown in SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 on the heavy chain variable region are shown in SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28, respectively.

[0037] The CDR sequence (CDR1 / 2 / 3 of the light chain / heavy chain) is the key for antibody recognition of antigens. The CDR sequences of the first monoclonal antibody and the second monoclonal antibody specifically recognize different epitopes of PAG, and there is no steric hindrance effect. They can synergistically form a sandwich structure of "first monoclonal antibody-PAG-second monoclonal antibody". Compared with the detection of PAG by a single anti-PAG antibody, the present invention can significantly reduce false positive results caused by non-specific binding by using the sandwich structure of "first monoclonal antibody-PAG-second monoclonal antibody" to detect PAG, improve the specificity of PAG detection, and improve the sensitivity of detection through signal amplification.

[0038] The framework region (FR, the non-CDR portion of the variable region) stabilizes the spatial conformation of the CDR, determining whether the CDR can correctly fold into an effective binding pocket, thus affecting the antibody's thermostability, pH stability, and expression efficiency. This invention has found that antibodies with the following sequences in their variable regions (containing both CDR and FR) exhibit high affinity and detection sensitivity: the amino acid sequence of the light chain variable region of the first monoclonal antibody is shown in SEQ ID NO:4, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:11; the amino acid sequence of the light chain variable region of the second monoclonal antibody is shown in SEQ ID NO:18, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:25. The affinity of these antibody pairs is as high as 1.2 × 10⁻⁶. -9 M, with a detection limit as low as 2 ng / mL.

[0039] The constant region (especially the Fc segment) provides binding sites for gold nanoparticles. The binding efficiency varies significantly among different constant region subtypes. In some embodiments of this invention, the first monoclonal antibody is mouse IgG1, with the amino acid sequence of its light chain variable region as shown in SEQ ID NO:4 and the amino acid sequence of its heavy chain variable region as shown in SEQ ID NO:11; the second monoclonal antibody is mouse IgG1, with the amino acid sequence of its light chain variable region as shown in SEQ ID NO:18 and the amino acid sequence of its heavy chain variable region as shown in SEQ ID NO:25. These mouse IgG1s can bind efficiently to gold particles. Since the charge distribution of the constant region (e.g., the ratio of acidic to basic amino acids) determines the conformational stability of the antibody at a specific pH, this pair of antibodies is conformationally stable at pH 8.2. When using this pair of antibodies, the pH needs to be controlled at 8.2 ± 0.2. Specifically, the amino acid sequence of the light chain of the first monoclonal antibody is shown in SEQ ID NO:3 and the amino acid sequence of the heavy chain is shown in SEQ ID NO:10; the amino acid sequence of the light chain of the second monoclonal antibody is shown in SEQ ID NO:17 and the amino acid sequence of the heavy chain is shown in SEQ ID NO:24.

[0040] In some embodiments of the present invention, the first monoclonal antibody or the second monoclonal antibody is a full-length antibody (IgG) that works stably at pH 8.2. In order to improve the diffusion rate, a smaller fragment antibody, i.e. an antigen-binding region, can be selected to replace the full-length antibody. The antigen-binding region is selected from at least one of the Fab fragment, F(ab)2 fragment, Fv fragment, (Fv)2 fragment, scFv fragment, and sc(Fv)2 fragment.

[0041] To address the issues of weak signals and insufficient sensitivity in traditional antibody detection methods, this invention conjugates a first monoclonal antibody and a second monoclonal antibody with a detection marker. Through the signal amplification effect of the marker, the detectable signal after antigen-antibody binding is enhanced. Based on this, this invention provides a set of antibody conjugates comprising the aforementioned antibody pairs against bovine pregnancy-related glycoproteins and a detection marker linked to the first and second monoclonal antibodies. The detection marker can be selected from colloidal gold, fluorescent molecules, or horseradish peroxide.

[0042] To address the problems of low yield, loss of antibody genes during long-term passage, and difficulty in large-scale production of antibodies using traditional hybridoma cell antibody preparation methods, this invention provides a set of nucleic acid molecules for recombinant antibody production using genetic engineering technology, ensuring the reproducibility and batch stability of antibody sequences. This set of nucleic acid molecules includes a first nucleic acid molecule pair and a second nucleic acid molecule pair, which encode a first monoclonal antibody and a second monoclonal antibody, respectively.

[0043] In some embodiments of the present invention, the first nucleic acid molecule pair includes the light chain DNA of the first monoclonal antibody and the heavy chain DNA of the first monoclonal antibody, wherein the nucleotide sequence of the variable region of the light chain DNA of the first monoclonal antibody is SEQ ID NO:2, corresponding to the amino acid sequence encoding the variable region of the light chain of the first monoclonal antibody (SEQ ID NO:4); and the nucleotide sequence of the variable region of the heavy chain DNA of the first monoclonal antibody is SEQ ID NO:9, corresponding to the amino acid sequence encoding the variable region of the heavy chain of the first monoclonal antibody (SEQ ID NO:11).

[0044] In some embodiments of the present invention, the light chain DNA nucleotide sequence of the first monoclonal antibody is SEQ ID NO:1, which corresponds to the amino acid sequence encoding the light chain of the first monoclonal antibody (as shown in SEQ ID NO:3); the heavy chain DNA nucleotide sequence of the first monoclonal antibody is SEQ ID NO:8, which corresponds to the amino acid sequence encoding the heavy chain of the first monoclonal antibody (as shown in SEQ ID NO:10).

[0045] In some embodiments of the present invention, the second nucleic acid molecule pair comprises the light chain DNA of the second monoclonal antibody and the heavy chain DNA of the second monoclonal antibody, wherein the nucleotide sequence of the variable region of the light chain DNA of the second monoclonal antibody is SEQ ID NO:16, corresponding to the amino acid sequence encoding the light chain variable region of the second monoclonal antibody (as shown in SEQ ID NO:18); and the nucleotide sequence of the variable region of the heavy chain DNA of the second monoclonal antibody is SEQ ID NO:23, corresponding to the amino acid sequence encoding the heavy chain variable region of the second monoclonal antibody (as shown in SEQ ID NO:25).

[0046] In some embodiments of the present invention, the light chain DNA nucleotide sequence of the second monoclonal antibody is SEQ ID NO:15, which corresponds to the amino acid sequence encoding the light chain of the second monoclonal antibody (as shown in SEQ ID NO:17); the heavy chain DNA nucleotide sequence of the second monoclonal antibody is SEQ ID NO:22, which corresponds to the amino acid sequence encoding the heavy chain of the second monoclonal antibody (as shown in SEQ ID NO:24).

[0047] This invention provides a kit for detecting anti-bovine pregnancy-associated glycoproteins, the kit comprising:

[0048] (1) Detect effective amounts of the first and second monoclonal antibodies;

[0049] (2) Necessary colorimetric or quantitative reagents.

[0050] This kit integrates the detection of effective antibody pairs with chromogenic / quantitative reagents into a complete dual-antibody sandwich detection system. Users do not need to purchase antibodies and markers separately, avoiding the problem of poor reagent compatibility between different suppliers.

[0051] In the kit of the present invention, the effective detection amount of the first monoclonal antibody refers to its concentration in the range of 0.5-2.0 mg / mL, which can ensure a clear T line in a 1 μg / mL PAG sample and a weak positive T line in a 2 ng / mL sample (concordance rate 98.6%).

[0052] In the kit of the present invention, the necessary colorimetric or quantitative reagent for the second monoclonal antibody is a colloidal gold reagent.

[0053] In the kit of the present invention, the pH value of the second monoclonal antibody after mixing with the necessary colorimetric or quantitative reagents is 7.8-9.0.

[0054] Existing double antibody tests require specialized equipment and are cumbersome to operate, making them unsuitable for grassroots farms. This invention provides a colloidal gold reagent kit for early pregnancy diagnosis in cattle. This kit is simple to use, requires no processing of whole blood samples, is stable, low-cost, highly specific, prevents misdiagnosis, and highly sensitive, prevents missed diagnosis. It is suitable for grassroots farming scenarios, allowing non-professionals to accurately conduct early pregnancy detection in cattle.

[0055] Specifically, the colloidal gold reagent kit includes a colloidal gold test strip; the colloidal gold test strip is composed of a sample pad, a gold label pad, a reaction membrane and an absorbent pad stacked in sequence, and the reaction membrane is provided with test lines and control lines;

[0056] The gold-labeled pad is coated with a colloidal gold-labeled labeled antibody, the test line is coated with a capture antibody, the capture antibody being the first monoclonal antibody described above; the labeled antibody is the second monoclonal antibody described above.

[0057] The quality control line is coated with goat anti-mouse IgG polyclonal antibody.

[0058] The colloidal gold test strip consists of a sample pad, a gold-labeled pad, a reaction membrane (including test lines and control lines), and an absorbent pad stacked sequentially. Sample flow is driven by capillary action. The functions of each component are as follows: The sample pad receives the sample, filters impurities, and balances the sample pH to ensure uniform diffusion of the sample to the gold-labeled pad; the gold-labeled pad is pre-coated with a colloidal gold-labeled first monoclonal antibody, with the colloidal gold particles appearing red as a visual signal marker; two lines are fixed on the reaction membrane: the test line, coated with a second monoclonal antibody, specifically binds to bovine pregnancy-associated glycoproteins; and the control line, coated with goat anti-mouse IgG polyclonal antibody, used to verify the effectiveness of the test strip, because the first monoclonal antibody is a mouse-derived monoclonal antibody, and its constant region can be recognized by goat anti-mouse IgG; the absorbent pad adsorbs sample liquid through capillary action, driving the entire chromatography process and absorbing excess liquid to prevent over-wetting of the reaction membrane.

[0059] In some embodiments of the present invention, the reaction membrane is a nitrocellulose membrane.

[0060] In some embodiments of the present invention, the concentration of the capture antibody in the test line is 0.8–1.5 mg / mL; and / or, the concentration of the colloidal gold-labeled antibody in the gold pad is 15–20 µg / mL.

[0061] In some embodiments of the present invention, one end of the reaction membrane is provided with a sample pad and a gold-labeled pad, and the other end is provided with an absorbent pad. The gold-labeled pad is placed on the reaction membrane, and the sample pad, absorbent pad, and reaction membrane are all placed on a base plate. The colloidal gold-labeled antibody coated in the gold-labeled pad is a second monoclonal antibody against PAG protein. The capture antibody coated in the test line of the reaction membrane is a first monoclonal antibody against PAG protein, and the antibody coated in the control line is a goat anti-mouse IgG polyclonal antibody.

[0062] The colloidal gold reagent kit of the present invention also includes a sample diluent for diluting serum samples, with 3 drops of diluent used for 1 drop of serum. In some embodiments, the sample diluent is 0.01 M phosphate buffer, pH=8.2±0.2, containing 0.9wt% sodium chloride and 0.05wt% Tween-20.

[0063] The technical solution of the present invention will be described in detail below through specific embodiments. Unless otherwise specified, the reagents and instruments used in the following embodiments are common reagents and instruments in the art.

[0064] Example 1: Preparation of PAG protein monoclonal antibody

[0065] 1. Synthesis of bPAG1 protein

[0066] Referring to the 1143 bp bPAG1 gene sequence on NCBI, an xhoI restriction site was introduced upstream and a BamhI restriction site was introduced downstream. This plasmid was optimized and synthesized by Sangon Biotech (Shanghai) Co., Ltd., and provided as plasmid pUC57-bPAG1. The correctly constructed plasmid was transfected into HEK-293 cells. Cells were harvested 24 hours later. Western blot results showed that the bPAG1 protein size was approximately 43 kDa, consistent with the expected size.

[0067] 2. Animal immunization and potency determination

[0068] Using bPAG1 protein as an immunogen, three 8-week-old female Balb / c mice were immunized for the first time with 100 μg of PAG protein per mouse after emulsification with Freund's complete adjuvant. Three booster immunizations (100 μg of PAG protein per mouse each time) were administered at 14, 28, and 42 days after the first immunization. The first two booster immunizations used Freund's complete adjuvant, while the final booster immunization (at 42 days) did not use adjuvant. Approximately 7 days after the final booster immunization, serum samples were collected to detect anti-PAG protein antibody levels. Mice with high titers were selected for subsequent cell fusion experiments.

[0069] 3. Cell fusion experiment

[0070] Preparation of feeder cells: Peritoneal macrophages of ICR mice were used as feeder cells, resuspended in DMEM medium containing 15% rabbit serum, and then seeded into 10 96-well cell culture plates and cultured in a 37°C, 5% CO2 incubator for later use.

[0071] Preparation of myeloma cells: SP2 / 0 myeloma cell culture in logarithmic growth phase was centrifuged and the supernatant was discarded. The cells were washed twice with D-Hanks' solution. After washing, the cell pellet was resuspended in an appropriate amount of D-Hanks' solution. A small amount of the cell suspension was stained with trypan blue, and the viable cell concentration was counted for later use.

[0072] Preparation of immunosuppressed spleen cells: Spleens were harvested from mice (samples 1 and 2) 3 days after a booster treatment. Blood was collected from the eyes to separate positive serum for later use. Mice were euthanized and immersed in 75% alcohol for 5 minutes before being transferred to a biosafety cabinet. The spleen was aseptically removed and crushed to fully release the spleen cells, creating a spleen cell suspension. Positive spleen cells were isolated using streptavidin magnetic beads combined with biotinylated antigen, and counted after trypan blue staining.

[0073] Cell fusion: Tumor cells were mixed with magnetically sorted positive spleen cells and centrifuged. Polyethylene glycol was added, and the cells were resuspended in HAT medium. The cells were then seeded into 10 96-well plates containing feeder cells and cultured in a 37°C, 5% CO2 incubator. After fusion, HAT medium was added again, and the medium was changed midway through the process to allow new clonal clusters to grow.

[0074] 4. Screening and cloning of positive hybridomas

[0075] Hybridoma cells were screened approximately 9 days after fusion. Hybridoma wells secreting the desired antibodies were selected from the numerous wells for antibody detection. The ELISA method was used to select the monoclonal antibody with the highest sensitivity. The screening method was consistent with the method used to detect mouse titers. First, a piece of cell plate supernatant was tested to determine the supernatant dilution, establishing a 1:10 sampling concentration for the cell supernatant and a 1:50 sampling concentration for the antigen supernatant. After all plates were tested, the selected positive wells were re-screened on the same ELISA plate using the same procedure to compare the reaction strength of each well. Finally, 39 positive wells were selected for hybridoma cell cloning.

[0076] 5. Hybridoma cell cloning

[0077] Hybridoma cell culture is initially unstable; some cells may lose parts of their chromosomes and thus lose their ability to produce antibodies. To remove these antibody-deficient cells and obtain stable monoclonal hybridoma cell lines, cloning is necessary. The hybridoma cells to be cloned, obtained during retesting, are mixed by pipetting in the culture wells. Positive hybridomas are cloned using the limiting dilution method. Cell suspension is added to 96-well plates already coated with feeder cells. After dilution, the hybridoma cells are incubated at 37°C. After approximately 7–8 days of clonal growth, the cells are closely observed, and wells containing single clonal clusters are identified under a microscope. Approximately 20 single-clonal wells from each cloning plate are selected, recorded, and the clones are detected using the ELISA method used during screening.

[0078] After cloning detection was completed, each cloning plate showed positive results, and the cells were numbered bpag (1~39). Ultimately, only about 1-2 positive clones were selected from each plate, and culture medium was immediately added to the selected clone wells for expansion. After the cells grew to a certain number, the cell supernatant was collected to test their viability. The cells were then resuspended in 10% DMSO and transferred to cryovials, frozen overnight at -80°C, and then transferred to liquid nitrogen for long-term storage.

[0079] 6. Preparation and purification of ascites antibodies

[0080] Two mice were prepared for each clone. The mice were sensitized with paraffin oil about one week in advance. The cells were resuspended in DMEM medium, and the injection volume was 500,000 cells per mouse. The mice's abdomen and survival were closely observed for about 7-10 days. The mice were sacrificed when their viability decreased and their abdominal cavity was as large as possible, and the ascites fluid was collected. The fluid was temporarily stored at 4°C for purification or frozen at -20°C.

[0081] The ascites antibody sample was purified using the caprylic acid-ammonium sulfate method. The specific steps are as follows: Add 4 times the volume of ascites fluid to 60mM pH 4.0 NaAc-HAc buffer to dilute the antibody. Add at least 0.025 times the total volume of caprylic acid and stir at room temperature for 30 min. Centrifuge at 9000 rpm / min for 20 min at 4℃. Collect the supernatant, filter through filter paper, and add at least 0.35 times the total volume of solid (NH4)2SO4 and stir. After complete dissolution, adjust the pH to 8.0 with ammonia and centrifuge at 9000 rpm / min for 20 min at 4℃. Discard the supernatant. Reconstitute the precipitate with 2–4 mL of PBS and dialyze against PBS overnight at 4℃. Change the dialysate the next day, repeating approximately three times until dialysis is complete. The protein concentration after dialyzing was measured using a UV spectrophotometer. The purified antibody was then added to 10% sodium azide.

[0082] A total of 39 hybridoma cell lines resistant to bovine pregnancy-associated glycoproteins were obtained. Monoclonal antibodies were prepared using mouse ascites fluid, and the purified monoclonal antibodies obtained by the caprylic acid-ammonium sulfate method will be used in subsequent antibody pairing experiments using colloidal gold test strips.

[0083] 7. Antibody pairing

[0084] Two monoclonal antibodies were selected from 39 monoclonal antibody strains: one as a capture antibody and the other as a labeling antibody (HRP-labeled). The capture antibody was coated onto an antigen plate. Antigen was added first, and after incubation for 1 hour, unbound antigen was washed away. Then, the labeling antibody was added and incubated for 30 minutes, after which unbound labeling antibody was washed away. Finally, chromogenic buffer was added for color development. After screening using ELISA, the second monoclonal antibody was ultimately selected as the labeling antibody, and the first monoclonal antibody as the capture antibody.

[0085] The amino acid and nucleotide sequences of the first and second monoclonal antibodies are shown in Table 1.

[0086] Table 1

[0087]

[0088] Example 2: Establishment of a colloidal gold reagent method for detecting early pregnancy in ruminants

[0089] 1. Antibody treatment

[0090] The monoclonal antibody and the second monoclonal antibody used for labeling were dialyzed three times with phosphate buffer (10mM, pH 7.2) and the concentration was adjusted to 2mg / mL. Finally, the antibody was centrifuged at 12000rpm for 15min to remove impurities and agglomerates.

[0091] 2. Colloidal gold production

[0092] Add 1 mL of 1% chloroauric acid (purchased from Sigma) to an Erlenmeyer flask containing 99 mL of ultrapure water to prepare a 0.01% chloroauric acid solution. Shake well and heat to boiling using a constant-temperature electromagnetic stirrer. While maintaining high temperature and continuous stirring, add 1.0–1.7 mL of 1 W / v% trisodium citrate (purchased from Sigma). Continue stirring and heating at a constant speed for about 10 minutes. The solution color first changes from yellow to colorless, then to black, and then slowly to a clear wine-red. When the solution color no longer changes, continue the reaction for 10 minutes, then stop heating, continue stirring, and cool. Dilute to 100 mL with ultrapure water and store at 2–8 °C protected from light. The prepared colloidal gold is pure, clear, and free of precipitates and floating matter.

[0093] 2. Determination of the optimal pH for labeled antibodies

[0094] Analysis using Bioedit software revealed the antibody's isoelectric point to be around 8.2. First, pH paper was used for calibration. The pH of the colloidal gold solution was adjusted to near 8.0 using 0.2 mol / L K₂CO₃. Then, the optimal pH for antibody labeling was determined by titration. The titration method is shown in Table 2. 200 µL of pH-adjusted colloidal gold solution was added to the ELISA plate. Then, a certain volume of 0.2 mol / L K₂CO₃ solution was added to each well, followed by excess antibody. After labeling for a certain period, sodium chloride was added. If color change and agglutination occurred, it indicated incomplete labeling due to unsuitable pH. If the solution state in the well remained unchanged, it indicated complete colloidal gold labeling under the specified pH conditions. The pH of the colloidal gold solution was adjusted using the ratio of colloidal gold to K₂CO₃ for each well. The results showed that the colloidal gold-antibody complex exhibited the best binding stability when 4 µL of 0.2 mol / L K₂CO₃ was added. Therefore, pH 8.2 of the colloidal gold solution was the optimal pH for the binding of colloidal gold and the second antibody.

[0095] Table 2

[0096]

[0097] 3. Determination of the optimal dosage of labeled antibody

[0098] The antibody dosage was determined by titration. The colloidal gold solution was adjusted to pH 8.2, and 1 mL was added to each of seven test tubes. Then, 0, 5, 10, 15, 20, 25, and 30 µL of the pre-treated second monoclonal antibody solution (1 mg / mL) were added to each tube, respectively. After mixing, the tubes were allowed to stand for 10 min to allow the second monoclonal antibody labeling to complete. Finally, 100 µL of 10% NaCl was added to each tube, and the tubes were mixed and allowed to stand for 10 min for observation. The optimal antibody labeling concentration was determined when the colloidal gold concentration remained unchanged and no precipitation occurred. Results showed that the colloidal gold-labeled second antibody maintained a stable pink color at concentrations above 20 µg / mL. Therefore, the concentration of monoclonal antibody at 20 µg / mL was selected as the optimal concentration for colloidal gold labeling and used in subsequent operations.

[0099] 4. Colloidal gold labeling and purification of antibodies

[0100] Colloidal gold labeling was performed based on the pH value and amount of the second monoclonal antibody determined in the above experiments. 10 mL of colloidal gold solution was taken, the pH was adjusted with K₂CO₃, and the calculated amount of the second monoclonal antibody solution was added. The mixture was stirred for 10 min, then 0.1 mL of BSA (10%, w / v) was added, and the reaction was continued for another 10 min. The reaction mixture was centrifuged at 12000 rpm for 30 min, the supernatant was carefully removed, and the precipitate was resuspended in Tris buffer (20 mm Tris, 1% BSA, pH 8.0). Then, it was centrifuged at low speed to remove large particles or agglomerates. These two steps were repeated once to thoroughly remove unlabeled second monoclonal antibody and impurities. Finally, the colloidal gold-labeled second monoclonal antibody (hereinafter referred to as gold-labeled antibody) was resuspended in Tris buffer at a concentration of 20 µg / mL, sealed, and stored for later use.

[0101] 5. Optimization of the optimal coating amount for capture antibodies

[0102] The first monoclonal antibody was sprayed onto the test line at three concentration gradients (0.5 mg / mL, 1.0 mg / mL, and 1.5 mg / mL) to determine the optimal coating amount of the test line, which was 1.0 mg / mL.

[0103] Example 3: Preparation of colloidal gold test strips

[0104] In this embodiment, a glass fiber membrane (GF / AVA) is used as the sample pad for the test strip, Accuflow G as the gold-labeled pad, a nitrocellulose membrane (AE99) as the reaction membrane (hereinafter referred to as the NC membrane), and a CF6 filter membrane as the absorbent pad. All components and the backing plate are cut to appropriate sizes according to the number of test strips to be produced. The sample pad needs to be completely wetted with sample pad treatment solution and then dried before use. The gold-labeled pad needs to be completely wetted with gold-labeled pad treatment solution containing gold-labeled antibodies and then dried before use.

[0105] Assemble the gold-labeled test strip: First, fix the backing plate on the lab bench, peel off the wax paper to expose the adhesive surface, use tweezers to pull the NC membrane taut, and place the membrane in the middle of the adhesive surface of the backing plate in a straight, taut state. Gently press with a roller to remove air bubbles. Next, attach the absorbent pad to the top of the backing plate, maintaining a 2mm overlap with the NC membrane, and flatten it with a roller. Place the gold-labeled pad below the NC membrane, using the same placement and rolling method as the absorbent pad, also maintaining a 2mm overlap with the NC membrane. After placing the gold-labeled pad, place the sample pad, with the sample spot below the gold-labeled pad, overlapping it by 2mm, and place it in the same way as above.

[0106] After all components are successfully bonded, place the membrane on the Biodot 3D spraying platform and spray test lines and control lines onto the NC membrane. The test lines contain the first monoclonal antibody, and the control lines contain goat anti-mouse IgG polyclonal antibody (Abcam catalog number: AB97023). After the sprayed test lines and control lines have dried, place them in a strip cutter to cut them into 4mm wide test strips, and then place them in an aluminum foil bag containing desiccant.

[0107] The detection method is as follows:

[0108] 1. Sample addition: For serum or plasma samples, add 1 drop (approximately 30µL) of the sample directly into the sample well using a dropper. For whole blood samples, add 2 drops (approximately 60µL) of the sample into the sample well using a dropper, along with 3 drops of diluent.

[0109] 2. The judgment criteria are as follows:

[0110] Negative: A red band appears at the control line, and the test line is blank, indicating a negative result;

[0111] Positive: Both the control line and the test line show red bands, indicating a positive result.

[0112] Invalid: The control line is blank, regardless of whether there is a red band at the test line, the result is considered invalid.

[0113] Quality testing of colloidal gold paper strips: Based on the results of steps (3) and (5) in Example 2, gold-labeled pads were finally prepared using gold-labeled antibodies at a concentration of 20 µg / mL, and the test line was coated with the first monoclonal antibody as the capture antibody at a concentration of 1.0 mg / mL to develop colloidal gold immunochromatographic test strips for detecting PAG.

[0114] Sensitivity testing was performed using a series of serially diluted bpag1 recombinant protein synthesized in Example 1 as a standard to determine the detection limit of the test strip. The dilution gradients and detection results are shown in the figure. Figure 2 The experimental results show that standards with concentrations greater than 1 µg / mL all produce clear and sharp test lines. When the standard concentration is diluted to 2 ng / mL, the test line becomes faint, indicating a weak positive result. Therefore, the detection limit of the test strip can be determined to be 2 ng / mL.

[0115] Example 4: Clinical Sample Testing

[0116] 1. Blood sample collection

[0117] Sixty-seven cows were randomly selected from four standardized dairy farms. On the 24th day after artificial insemination, 2 mL of blood was collected from the tail vein using a coagulation-promoting vacuum blood collection tube. The blood was immediately placed at 4°C for refrigeration, and the serum was separated. The serum sample was then stored at -20°C for later use.

[0118] 2. Colloidal gold test strip detection

[0119] Equilibrate the test reagents and samples to room temperature. Place the colloidal gold test strip prepared in Example 3 flat on the table, add 1 drop of serum sample to the sample well, and add 3 drops of diluent to the diluent well. Observe the results after 15 minutes.

[0120] Result interpretation: Positive: The appearance of two bands indicates that the animal is pregnant; Negative: The appearance of only one band in the control area (C) and no band in the test area (T) indicates that the animal is not pregnant; Invalid: No band appears in the control area (C), indicating that the operation process was incorrect or the reagent has deteriorated or been damaged.

[0121] 3. Detection using the IDOX PAG ELISA kit

[0122] Before starting the test, bring the IDEXX PAG ELISA kit (Beijing IDEXX Yuanheng Biotechnology Co., Ltd., catalog number: 99-41169 J401) and serum sample to room temperature. Add the serum sample to the sample well and then strictly follow the standardized steps of the kit.

[0123] Result interpretation: An OD value ≥ 0.3 indicates a positive result; an OD value < 0.3 indicates a negative result; invalid detection is when the OD values ​​of both the positive and negative control wells are less than 0.3.

[0124] 4. Ultrasound examination

[0125] Farm technicians used ultrasound to diagnose pregnancy in dairy cows 35 days after mating, and used the ultrasound results on day 35 as a benchmark to calculate the positive concordance rate of two pregnancy diagnostic kits (IDEX PAG ELISA kit and colloidal gold test strip prepared in Example 3).

[0126] 5. Results Analysis

[0127] Table 3. Ultrasound examination results on day 35 post-mating.

[0128]

[0129] Table 4 Comparison of colloidal gold test strips prepared in Example 3 and ultrasound examination results

[0130]

[0131] Note: a represents the number of positive results obtained by the colloidal gold test strip for early pregnancy in dairy cows, b represents the number of false positive results, c represents the number of false negative results, and d represents the number of negative results.

[0132] Table 5 Comparison of IDS PAG ELISA kit and ultrasound examination results

[0133]

[0134] Note: a represents the number of positive results obtained by the colloidal gold test strip for early pregnancy in dairy cows, b represents the number of false positive results, c represents the number of false negative results, and d represents the number of negative results.

[0135] As shown in Tables 4 and 5, the detection results of the colloidal gold test strip prepared in Example 3 were highly consistent with the ultrasound detection results 35 days after mating, with a positive concordance rate of 96.2%, making it a reliable on-site detection tool for early pregnancy in dairy cows. The detection results of the colloidal gold test strip prepared in Example 3 were close to the positive concordance rate of the commercially available IDEXX PAG ELISA kit, and the colloidal gold test strip prepared in Example 3 was simpler to operate and had a shorter detection time, making it more suitable for low-cost farm applications.

[0136] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A pair of antibodies against bovine pregnancy-associated glycoproteins, characterized in that: The antibody pair consists of a first monoclonal antibody and a second monoclonal antibody; wherein: The amino acid sequences of CDR1, CDR2 and CDR3 on the light chain variable region of the first monoclonal antibody are shown in SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 on the heavy chain variable region are shown in SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, respectively. The amino acid sequences of CDR1, CDR2 and CDR3 on the light chain variable region of the second monoclonal antibody are shown in SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 on the heavy chain variable region are shown in SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28, respectively.

2. The antibody pair against bovine pregnancy-associated glycoproteins according to claim 1, characterized in that: The amino acid sequence of the light chain variable region of the first monoclonal antibody is shown in SEQ ID NO:4, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:11; The amino acid sequence of the light chain variable region of the second monoclonal antibody is shown in SEQ ID NO:18, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

25.

3. The antibody pair against bovine pregnancy-associated glycoproteins according to claim 1, characterized in that: The first monoclonal antibody is mouse IgG1, the amino acid sequence of its light chain variable region is shown in SEQ ID NO:4, and the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO:11; The second monoclonal antibody is mouse IgG1, the amino acid sequence of its light chain variable region is shown in SEQ ID NO:18, and the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO:

25.

4. The antibody pair against bovine pregnancy-associated glycoproteins according to claim 1, characterized in that: The first monoclonal antibody or the second monoclonal antibody is a full-length antibody or its antigen-binding region; the antigen-binding region is selected from one of the Fab fragment, F(ab)2 fragment, Fv fragment, (Fv)2 fragment, scFv fragment and sc(Fv)2 fragment.

5. A group of antibody conjugates, characterized in that, It consists of an antibody pair against bovine pregnancy-associated glycoprotein as described in any one of claims 1 to 4, a detection marker linked to a first monoclonal antibody, and a detection marker linked to a second monoclonal antibody.

6. A group of nucleic acid molecules, characterized in that, The group of nucleic acid molecules includes a first nucleic acid molecule pair and a second nucleic acid molecule pair, wherein the first nucleic acid molecule pair and the second nucleic acid molecule pair respectively encode the first monoclonal antibody and the second monoclonal antibody as described in any one of claims 1 to 4.

7. A kit for detecting anti-bovine pregnancy-associated glycoproteins, characterized in that, The kit contains: (1) Detecting an effective amount of the first monoclonal antibody and the second monoclonal antibody as described in claim 1; (2) Necessary colorimetric or quantitative reagents.

8. A colloidal gold reagent kit for early pregnancy diagnosis in cattle, characterized in that, The product includes colloidal gold test strips; the colloidal gold test strips are composed of a sample pad, a gold label pad, a reaction membrane and an absorbent pad stacked in sequence, and the reaction membrane is provided with test lines and control lines; The gold-labeled pad is coated with a colloidal gold-labeled labeled antibody, and the test line is coated with a capture antibody, wherein the capture antibody is the first monoclonal antibody according to any one of claims 1 to 4; the labeled antibody is the second monoclonal antibody according to any one of claims 1 to 4; The quality control line is coated with goat anti-mouse IgG polyclonal antibody.

9. The colloidal gold reagent kit for early bovine pregnancy diagnosis according to claim 8, characterized in that: The concentration of the captured antibody in the test line is 0.8–1.5 mg / mL; and / or, The concentration of the colloidal gold-labeled antibody in the gold-labeled pad is 15–20 µg / mL.

10. The colloidal gold reagent kit for early bovine pregnancy diagnosis according to claim 8, characterized in that: The reaction membrane is provided with a sample pad and a gold label pad at one end and an absorbent pad at the other end. The gold label pad is placed on the reaction membrane, and the sample pad, absorbent pad and reaction membrane are all placed on the base plate. The colloidal gold-labeled antibody coated in the gold pad is a second monoclonal antibody against PAG protein; The capture antibody coated in the test line of the reaction membrane is a first monoclonal antibody against PAG protein, and the antibody coated in the control line is a goat anti-mouse IgG polyclonal antibody.

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