A goat milk adulterated with cow milk rapid detection colloidal gold kit based on mouse anti-cow IgG monoclonal antibody and application thereof

Abstract

The application discloses a goat milk adulterated with cow milk rapid detection colloidal gold kit based on mouse anti-cow IgG monoclonal antibody and application thereof, and belongs to the technical field of animal husbandry and veterinary medicine. The goat milk adulterated with cow milk rapid detection colloidal gold kit based on mouse anti-cow IgG monoclonal antibody contains mouse anti-cow IgG monoclonal antibody. The kit can rapidly and conveniently detect the adulterated cow milk in goat milk, and has the advantages of higher sensitivity, simpler operation, independence from laboratory conditions, convenience in application in production and the like compared with the prior art.

Description

Technical Field

[0001] This invention relates to the field of animal husbandry and veterinary technology, and more specifically to a colloidal gold reagent kit for rapid detection of adulterated cow's milk based on mouse anti-bovine IgG monoclonal antibody and its application. Background Technology

[0002] Goat milk generally has a higher nutritional composition than cow milk and is closer to human milk, possessing broad market prospects and huge development potential. However, the limited number of dairy goats, along with their short lactation period and low milk yield, results in a market price for goat milk that is significantly higher than that of cow milk. Therefore, driven by profit, the adulteration of goat milk with cow milk is widespread, severely disrupting market order and harming the industry's health. Current conventional methods for detecting adulteration in goat milk (high-performance liquid chromatography, mass spectrometry, enzyme-linked immunosorbent assay, and PCR, etc.) suffer from drawbacks such as long detection cycles, high costs, and cumbersome operations, failing to quickly and accurately identify whether goat milk has been adulterated with cow milk. Therefore, it is particularly necessary to develop an efficient and rapid method for identifying adulteration in goat milk during the fresh milk warehousing inspection process.

[0003] Therefore, providing a colloidal gold reagent kit for rapid detection of adulterated cow's milk based on mouse anti-bovine IgG monoclonal antibody and its application is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0004] In view of this, the present invention provides a colloidal gold rapid detection kit for adulterated cow's milk based on mouse anti-bovine IgG monoclonal antibody and its application. Addressing the problems of cumbersome detection methods, slow detection time, and difficulty in application at the grassroots level, the present invention employs colloidal gold rapid detection technology, selects IgG protein in cow's milk as the antigen, obtains highly specific monoclonal antibodies through immunological methods and hybridoma cell culture technology, and uses a double-antibody sandwich method to fabricate a highly sensitive colloidal gold rapid test strip. Through comparative optimization, a test strip with high sensitivity and strong specificity was selected.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A hybridoma cell line, produced by fusing bovine IgG-immunized mouse lymphocytes with SP2 / 0 cells, can proliferate indefinitely and secrete mouse anti-bovine IgG monoclonal antibodies. Its accession number is CCTCC NO: C2024137. It has been deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, on November 29, 2024, and is classified as hybridoma cell line 2-D7.

[0007] Furthermore, a mouse anti-bovine IgG monoclonal antibody, which can specifically bind to the species-specific region of bovine IgG, is produced by the hybridoma cell line described above.

[0008] Furthermore, the application of the aforementioned mouse anti-bovine IgG monoclonal antibody in the specific detection of bovine IgG.

[0009] Furthermore, the application of the aforementioned mouse anti-bovine IgG monoclonal antibody in the detection of adulterated cow's milk in goat's milk.

[0010] Furthermore, a colloidal gold reagent kit for rapid detection of adulterated cow's milk in goat's milk based on mouse anti-bovine IgG monoclonal antibody contains the aforementioned mouse anti-bovine IgG monoclonal antibody.

[0011] Furthermore, the kit is used in the detection of adulterated goat milk and cow milk.

[0012] As can be seen from the above technical solution, compared with the prior art, this invention discloses a colloidal gold rapid detection kit for adulterated cow's milk in goat milk based on mouse anti-bovine IgG monoclonal antibody and its application. This invention selects the economical, practical, rapid, and convenient colloidal gold rapid detection technology, selects IgG in cow's milk as the target antigen, and combines hybridoma cell technology to produce a hybridoma cell line that can generate highly specific monoclonal antibodies. Through ascites proliferation and affinity column purification, mouse anti-bovine IgG monoclonal antibody is obtained. Further, gold-labeled mouse anti-bovine IgG monoclonal antibody is prepared using immunogold labeling technology. Using this as the core, rabbit anti-bovine IgG polyclonal antibody as the detection line, and purified bovine IgG as the quality control line, through comparative optimization, a double-antibody sandwich method "colloidal gold rapid diagnostic kit for adulterated cow's milk in goat milk" is assembled. When the proportion of milk adulteration is 10% or higher, this kit can accurately and rapidly identify "milk adulteration positive". In summary, this invention achieves the goal of rapidly and conveniently detecting adulterated cow's milk in goat milk. Compared with the prior art, it has advantages such as higher sensitivity, simpler operation, no dependence on laboratory conditions, and ease of application in production. Attached Figure Description

[0013] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0014] Figure 1 The attached figure is a technical roadmap of the present invention;

[0015] Figure 2 The attached figure shows the electrophoretic identification of the purified immunogen-bovine IgG protein of this invention;

[0016] Where M is the protein standard marker; both lanes contain purified bovine IgG.

[0017] Figure 3 The attached figure shows the change in serum antibody titer of New Zealand rabbits after immunizing them with bovine IgG protein according to this invention.

[0018] Figure 4 The attached figure shows the indirect ELISA specificity detection of rabbit anti-bovine IgG polyclonal antibody according to the present invention;

[0019] Figure 5 The attached figure shows the changes in serum antibody titers in mice after immunization with bovine IgG according to the present invention.

[0020] Figure 6 The attached figure shows images of the monoclonal hybridoma cell line (left) and the expanded hybridoma cells (right) observed under an inverted microscope.

[0021] Figure 7 The attached figure shows the specificity of the ELISA method of the present invention for the detection of monoclonal antibodies in the prepared hybridoma cell supernatant;

[0022] Figure 8 The attached figure shows the results of the identification of antibody subtypes in the supernatant of hybridoma cell lines according to the present invention;

[0023] Figure 9 The attached figure shows the results of the karyotype analysis of hybridoma cells in this invention (left: Giemsa staining 100×, right: chromosome number statistics);

[0024] Figure 10 The attached figure illustrates the specificity detection of clinical samples using the sandwich ELISA method assembled from the prepared monoclonal and polyclonal antibodies of this invention.

[0025] Figure 11 The attached figure shows the results of the optimal pH screening for colloidal gold-labeled monoclonal antibodies according to this invention;

[0026] Figure 12 The attached figure shows the screening of the optimal concentration of colloidal gold-labeled monoclonal antibodies according to the present invention;

[0027] Figure 13 The attached figure is a schematic diagram of the sandwich colloidal gold test strip structure assembled according to the present invention. Detailed Implementation

[0028] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] A rapid detection method for colloidal gold, the technology roadmap is as follows: Figure 1 As shown, IgG protein from milk was selected as the antigen. Highly specific monoclonal antibodies were obtained through immunological methods and hybridoma cell culture technology. Highly sensitive colloidal gold rapid test strips were fabricated using a double-antibody sandwich method or a competitive method. Through comparative optimization, test strips with high sensitivity and strong specificity were selected.

[0030] (1) Selection of antigens

[0031] By comparing the differences between cow and goat milk, it was found that cow's whey protein contains a relatively high amount of bovine IgG (approximately 0.61 mg / mL). Bovine IgG protein has a species-specific amino acid sequence and different immunoeptopes from goat IgG and other proteins in goat milk, allowing it to be recognized by its specific antibodies. Conversely, goat IgG and other proteins in goat milk cannot be recognized by antibodies targeting the species-specific sequence of bovine IgG. Therefore, this invention selects bovine IgG as the specific antigen and obtains highly specific mouse anti-bovine IgG monoclonal antibodies and rabbit anti-bovine IgG polyclonal antibodies through immunological methods and hybridoma cell culture technology to prepare highly sensitive colloidal gold test strips.

[0032] (2) Preparation of antigen

[0033] To prepare high-purity bovine IgG as an immunogen, this study collected blood from healthy adult female Holstein cows, separated serum, and obtained crude IgG by octanoic acid-saturated ammonium sulfate salting-out and dialysis. The crude IgG was then further purified using a protein G affinity column (Tiandi Renhe, rProtein G Beads gravity column, SA016GC01) to obtain high-purity bovine IgG as an antigen.

[0034] (3) Preparation of polyclonal antibodies

[0035] New Zealand rabbits were immunized with a mixture of Freund's complete adjuvant and purified bovine IgG protein. After multiple immunizations, rabbit blood with high antibody titers was collected to separate serum, which was then purified by octanoic acid-saturated ammonium sulfate salt precipitation, dialysis, and protein G affinity chromatography to obtain purified rabbit anti-bovine IgG polyclonal antibody.

[0036] (4) Preparation of monoclonal antibodies

[0037] Mice were immunized with a mixture of Freund's complete adjuvant and purified bovine IgG protein. B lymphocytes that could produce high-maturity, high-affinity antibodies were obtained through shock immunization. Hybridoma cells were selected to obtain monoclonal cell lines. Subsequently, mouse anti-bovine IgG protein monoclonal antibodies were prepared by ascites proliferation, octanoic acid-saturated ammonium sulfate salting out, dialysis, and protein G affinity chromatography.

[0038] (5) Assembly of colloidal gold immunochromatographic test strips

[0039] Colloidal gold-labeled antibodies were prepared based on the obtained mouse anti-bovine IgG monoclonal antibody. Simultaneously, an immunochromatographic test strip was assembled using the prepared rabbit anti-bovine IgG polyclonal antibody as the detection line and purified bovine IgG as the quality control line. The sensitivity and specificity of the test strip were tested, and the most suitable detection system was selected through comparison and optimization.

[0040] Key Point 1: High Target Specificity;

[0041] Key Point Two: Monoclonal antibodies with high specificity against target antigens have strong sensitivity and specificity;

[0042] Key Point 3: The assembly method using colloidal gold is simpler to operate, has a shorter detection time, and lower costs.

[0043] Example 1: Preparation of Polyclonal Antibodies

[0044] 1. Antigen preparation

[0045] One healthy adult female Holstein cow was selected, and 40 mL of blood was collected via jugular vein puncture using disposable vacuum blood collection tubes (10 mL per tube, 4 tubes in total). After standing at room temperature for 30 min, the blood was centrifuged at 3500 rpm for 10 min to obtain bovine serum. The serum was crudely purified by octanoic acid-saturated ammonium sulfate salting-out and then dialyzed to obtain crude IgG. The bovine IgG in the serum was further purified using a protein G affinity column (Changzhou Tiandi Renhe Biotechnology Co., Ltd., rProtein GBeads gravity column, catalog number: SA016GC01). The purity was determined by SDS. Figure 2 As shown, Coomassie Brilliant Blue staining results revealed only two bands in the purified protein solution, approximately 25 kDa and 55 kDa, indicating a relatively pure purified bovine IgG band. The protein concentration was determined to be 6.2 mg / mL using the Bicinchoninic Acid Assay (BCA) protein concentration assay kit (Jiangsu Kaiji Biotechnology).

[0046] 2. Preparation of polyclonal antibodies

[0047] Using the purified bovine IgG protein obtained in the above steps as the immunogen, healthy adult New Zealand rabbits were immunized according to the procedure in Table 1: For the initial immunization (first immunization), Freund's complete adjuvant (FCA) was used; for the 1st to 4th booster immunizations (corresponding to the second, third, fourth, and fifth immunizations, respectively), Freund's incomplete adjuvant (FIA) was used. The adjuvant and purified antigen protein were mixed in equal volumes (each time the antigen protein was diluted to 1000 μg / mL, and the antigen concentration after mixing with the adjuvant was 500 μg / mL). After emulsifying the antigen-adjuvant mixture using an ultrasonic cell disruptor, the New Zealand rabbits were subcutaneously immunized (500 μg / rabbit / time, i.e., 1 mL per rabbit per injection). A booster immunization was given every 21 days. Blood was collected from the ear vein 7 days after immunization, and the serum antibody titer of the immunized rabbits was continuously monitored using indirect ELISA (e.g., 500 μg / rabbit / time). Figure 3 (As shown), 7 days after the fifth immunization, blood was collected from the marginal ear vein and the antibody titer was found to be higher than 10. 5 A shock immunization can then be performed. During the shock immunization, 2000 μg of protein without adjuvant is injected subcutaneously into the back. Seven days later, blood is collected from the marginal ear vein for three consecutive days, 5–10 mL each time. After serum separation, IgG is crudely purified from the serum using the caprylic acid-saturated ammonium sulfate precipitation method. After dialyzing with double-distilled water at 4°C for 24 h, it is purified using a protein G affinity column, followed by dialyzing with double-distilled water at 4°C for 48 h. BCA detection shows that the final purified mouse anti-bovine IgG polyclonal antibody concentration is 4.6 mg / mL. After sterilization using a bacterial filter, it is stored at -20°C for later use. The specificity of the rabbit anti-bovine IgG polyclonal antibody against bovine IgG and sheep IgG is detected using an indirect ELISA, and the results are analyzed using a t-test. Figure 4 As shown (** indicates a highly significant difference compared to the negative control group, i.e., P<0.01, *** indicates a highly significant difference compared to the negative control group, i.e., P<0.001), rabbit anti-bovine IgG polyclonal antibody has a certain specificity for bovine IgG, but still reacts to sheep IgG. It is not sufficient to be used as a gold standard detection antibody, and can only be used as an antibody for detection lines or quality control lines.

[0048] Table 1. Immunization schedule for rabbits

[0049]

[0050] Note: FCA was mixed with FIA and bovine IgG and immunized by subcutaneous injection into the neck and back of the neck using ultrasonic emulsification.

[0051] Example 2: Preparation of Monoclonal Antibodies

[0052] 1. Mouse immunization

[0053] Using the purified bovine IgG protein obtained in the "Antigen Preparation" step of Example 1 as the immunogen, five healthy 6-week-old BALB / c mice were selected. The mice were immunized according to the immunization schedule in Table 2. For the first immunization, the antigen and an equal volume of Freund's complete adjuvant were mixed (the immunogen was diluted to 200 μg / mL before mixing the adjuvant, and to 100 μg / mL after mixing the adjuvant). After thorough emulsification using an ultrasonic cell disruptor, the mice were subcutaneously injected at multiple points on the back (50 μg / mouse / time, 0.5 mL per mouse per injection). After the first immunization, the mice were boosted every 14 days. For booster immunizations, Freund's incomplete adjuvant was used. The antigen and adjuvant were mixed in equal volumes and thoroughly emulsified using an ultrasonic cell disruptor. The antigen injection dose per mouse was 50 μg per injection. Seven days after each immunization, blood was collected from the tail vein and serum was separated. The antibody titer in the serum of the immunized mice was determined using an indirect ELISA (e.g., 50 μg of antigen per mouse per injection). Figure 5 As shown in the figure, 7 days after the third booster immunization, the antibody titer in mouse serum exceeded 1:10. 5 Pre-fusion shock immunization can be performed without adjuvants. A single subcutaneous injection of 200 μg of antigen solution (purified bovine IgG diluted to 400 μg / mL, 0.5 mL per mouse) is administered directly to the back of the mouse. Three days after shock immunization, blood is collected from the tail vein of the mice and serum is separated. The anti-bovine IgG titer of the mouse serum is determined by ELISA. The mice with the highest serum antibody titer are selected as donors for obtaining immune lymphocytes (Note: serum from unimmunized mice is considered negative serum. If the OD value of the immunized mouse serum is >2.1 times the OD value of the negative serum, it is considered positive).

[0054] Table 2. Immunization schedule for mice

[0055]

[0056] Note: Mice were immunized with a mixture of FCA, FIA, and bovine IgG and emulsified using ultrasound at multiple subcutaneous injections at the nape of the neck.

[0057] 2. Hybridoma cell fusion

[0058] 1) Culture of myeloma cells

[0059] Frozen myeloma cells (SP2 / 0) were revived in 60 mm cell culture dishes using 2 mL of high-glucose DMEM (Gibco, USA) containing 20% ​​fetal bovine serum (Gibco) and 1% penicillin-streptomycin-amphoteric acid B (Beyotime Biotechnology, Shanghai). After continuous culture for a period of time, the cell growth status was observed. Cells were passaged when the confluence reached about 90%. During passage, the cells were gently washed twice with PBS preheated to 37°C. The cleaned adherent cells were then repeatedly washed with 1500 μL of 20% serum high-glucose complete medium to suspend the cells before passage. Cells that reached 90% confluence in one 60 mm cell culture dish could be seeded into three 60 mm cell culture dishes. When the cell confluence reached about 90%, the cells could be passaged again. After 2-3 passages, once the cells show good growth and uniform morphology, the medium is replaced with high-glucose DMEM (Gibco, USA) containing a mixture of 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin-amphoteric acid B (Beyotime Biotechnology, Shanghai) for continuous passage. Cell growth is then observed. Once the cells show good morphology, uniform size, and optimal growth in the 10% serum high-glucose complete medium, they can be used for cell fusion.

[0060] 2) Obtaining feeder cells

[0061] (1) Preparation of mouse ascites model: Three healthy adult ICR mice aged 10 weeks and above were selected. The preparation of feeder cells was started 7 days before cell fusion. Each mouse was injected with 0.5 mL of liquid paraffin to induce the ascites model (the ascites model was considered to be successfully induced when the ascites cavity of the mouse was significantly enlarged 6 days after the liquid paraffin injection). The mice were anesthetized 1 day before cell fusion and their ascites was collected to separate peritoneal macrophages as feeder cells.

[0062] (2) Collection of peritoneal macrophages: The ascites model mice prepared in the previous step were anesthetized with ether and euthanized by dislocation. They were then quickly immersed in 75% alcohol pre-cooled at 4°C for about 5 minutes to thoroughly disinfect their body surface. The disinfected mouse carcasses were transferred to a clean bench and fixed to a disinfected mouse board with sterilized thumbtacks. The abdominal skin was cut along the midline of the abdominal cavity to expose the peritoneum. The peritoneum on the ventral side was gently lifted with ophthalmic forceps, and a small incision was made at the highest point of the lift. The small incision was lifted continuously. 5 mL of preheated (37°C) high-glucose DMEM (Gibco, USA) was injected into the peritoneal cavity using a 5 mL syringe. The abdomen was gently massaged to fully suspend the macrophages in the ascites fluid. The ascites fluid was collected using a sterile syringe, filtered through a 200-mesh cell sieve, and transferred to a 50 mL centrifuge tube. After aspirating the ascites fluid, another 5 mL of preheated high-glucose DMEM was injected to flush the peritoneal cavity. This flushing process was repeated three times to collect peritoneal macrophages. After filtration through a 200-mesh cell sieve, the cells were centrifuged at 800 rpm for 10 min to collect the cell pellet. The cells were then resuspended in high-glucose DMEM and washed twice. Cells were suspended in a selective high-glucose complete medium (HAT selective high-glucose complete medium) containing 1×HAT medium additive (HAT medium additive, purchased from Suzhou Bio-Long Company), 10% fetal bovine serum (Gibco, USA), and 1% penicillin-streptomycin-amphoteric acid B mixture (Beyotime Biotechnology, Shanghai) to obtain a cell suspension.

[0063] (3) Plating: 15 μL of cell suspension was stained with 15 μL of 2× trypan blue solution, followed by viable cell counting. The cell suspension was then diluted to 2×10⁻⁶ using HAT selective high-glucose complete medium. 5 Cells were cultured at a density of 100 μL / well in 96-well cell culture plates and then incubated at 37°C in a constant temperature cell culture incubator containing 10% CO2 for 12 h. Cell growth was observed to ensure that there was no bacterial or fungal contamination and that the cells were growing well.

[0064] 3) Preparation of mouse spleen cells

[0065] BALB / c mice with the highest titer after immunization were selected as donors to isolate and prepare mouse spleen lymphocytes. The specific operation steps are as follows:

[0066] (1) After anesthetizing mice with ether, blood was collected by enucleating the eyeballs to collect hyperimmune serum. When collecting blood, gently press the mouse's abdomen with your fingers to allow as much blood as possible to flow out of the body to reduce the number of blood cells remaining in the spleen. After the blood stops flowing out, the mouse was euthanized by dislocation of the neck. The mouse was then immediately immersed in 75% alcohol pre-cooled at 4°C for about 3 to 5 minutes to thoroughly sterilize its body surface.

[0067] (2) After the mice were sterilized, they were transferred into a laminar flow hood and fixed with sterile mouse plates and thumbtacks. Ophthalmic surgical scissors and forceps were used to lift the abdominal skin and cut it longitudinally along the midline of the abdomen. Then the skin was peeled off to expose the abdominal wall. After spraying the abdominal wall with alcohol for 15 seconds, new clean scissors were used to cut open the abdominal wall and peritoneum. New clean forceps were used to carefully remove the swollen spleen (the spleen tissue of successfully immunized mice will show obvious swelling 3 days after the shock immunization) and placed in a sterile petri dish containing sterile physiological saline.

[0068] (3) After rinsing the spleen surface 3-4 times with sterile saline, immediately transfer it to a new sterile culture dish. Then, use clean forceps to hold the spleen directly above the sterile culture dish. Use a 5ml syringe to draw 3mL of high-glucose DMEM preheated at 37℃, gently insert the needle into the spleen and slowly inject it to flush out most of the lymphocytes in the spleen. Repeat the injection and rinsing 3 times. Use a 1mL pipette to draw out the liquid from the culture dish and filter it through a 200-mesh cell sieve. Transfer the filtrate to a clean 50mL centrifuge tube. Then, place the remaining spleen tissue on a 200-mesh cell sieve and gently grind it with a clean syringe stopper to fully rupture the spleen tissue and allow the lymphocytes in the spleen to fully infiltrate. Next, use high-glucose DMEM to rinse the cells ground out from the remaining spleen tissue on the cell sieve. Transfer the filtrate and the filtrate collected when rinsing the spleen to the same 50mL centrifuge tube and mix them evenly.

[0069] (4) The spleen tissue was rinsed and ground into a cell suspension and centrifuged at 800 r / min for 10 min. The supernatant was discarded and the precipitated spleen cells were resuspended in 10 mL of high glucose DMEM and centrifuged at 800 r / min for 10 min. This process was repeated twice. Then, the cells were resuspended in 2 mL of 10% serum high glucose complete medium. 15 μL of the cell suspension was stained with trypan blue and viable cell count was performed.

[0070] 4) Cell fusion

[0071] (1) Cell counting: SP2 / 0 cells with good growth and normal cell morphology were selected from 60 mm culture dishes, washed and suspended with high glucose DMEM, and then transferred to 15 mL centrifuge tubes. After centrifugation at 800 r / min for 5 min, the supernatant was discarded, and the cells were resuspended with 2 mL of high glucose DMEM. A small amount of cell suspension was stained with trypan blue and viable cell counting was performed.

[0072] (2) Cell mixing: Based on the live cell count results, the spleen cells were adjusted to 6 × 10⁶. 7 SP2 / 0 cells were adjusted to 6 × 10⁶ cells / mL. 6 Take 3 mL of each of the 15 mL centrifuge tubes and mix them. Use a 1 mL pipette to gently mix the mixture. Centrifuge at 800 rpm for 10 min, discard the supernatant, and gently tap the bottom of the centrifuge tube with tweezers to loosen the precipitated cell clumps for later use.

[0073] (3) Cell fusion: Take 1 mL of 50% PEG4000 (PBS solution containing 50% PEG4000) preheated at 38℃, and slowly add PEG4000 to the cell pellet at a rate of 1 mL / min. Be careful to stir evenly while adding the PEG4000, and then transfer it to a 37℃ incubator and let it stand for 10 min.

[0074] (4) Termination of fusion: Use a 10mL electric pipette to slowly add 10mL of high-glucose DMEM to the fusion cell mixture to terminate the fusion reaction. Note that the addition must be slow. Initially, the addition speed should be controlled at about 1mL / min. When adding the first mL, mix gently while adding. Then increase the addition speed to 2mL / min and add 2mL while mixing. Finally, increase the addition speed to 7mL / min and add while mixing to terminate the cell fusion reaction.

[0075] (5) Centrifugation: After the fusion reaction is terminated, centrifuge the cell suspension at 800 r / min for 3 min and carefully discard the supernatant;

[0076] (6) Cell resuspension: Resuspend the cells in 2 mL of HAT selective high glucose complete medium, and then add 100 μL per well to two 96-well cell culture plates with feeder cells, and transfer to a 37°C cell culture incubator.

[0077] 3. Screening of monoclonal hybridoma cells and identification of monoclonal antibodies

[0078] 1) Hybridoma cell screening

[0079] After cell fusion, half of the culture medium in the 96-well cell culture plate was replaced every 72 hours (100 μL of the old medium was removed from each well and 100 μL of fresh HAT-selective high-glucose complete medium was added). Before each medium change, cell growth was observed using an inverted microscope. Successfully fused hybridoma cells may exhibit proliferation of monoclonal cell clusters (e.g., Figure 6As shown, timely labeling is possible. Hybridoma cells have the characteristic of being able to proliferate in large quantities in HAT selective high-glucose complete medium. Hybridoma cell lines with good growth status are screened and labeled. After changing the medium twice, when changing the medium for the third time (9 days after fusion), 100 μL of supernatant is taken from the wells of cells with good proliferation for indirect ELISA detection to screen hybridoma cell lines that can secrete mouse anti-bovine IgG antibodies. The detection is repeated after 72 hours, and the detection is repeated 3 times to screen out cell wells with strong antibody detection.

[0080] 2) Subcloning

[0081] When cells in a 96-well cell culture plate reach 50% confluence, subcloning of strongly positive antibody-detected cell lines can be performed. First, feeder cells need to be prepared (as described in step 2). Then, the strongly positive hybridoma cell lines are resuspended and viable cell counts are performed. Based on cell density, the cells are diluted to approximately one cell per 200 μL. 200 μL of the diluted cells are seeded into each well of a 96-well cell culture plate containing feeder cells and cultured in HAT selective high-glucose complete medium. After 24 hours, cell growth is observed using an inverted microscope. Wells containing single clones are selected and marked. The medium in each well containing a single clone is replaced with 100 μL every 72 hours. The supernatant from the single clone wells is collected and analyzed by indirect ELISA. Single-cell clones and strongly positive antibody-detected cell lines in the cell supernatant are selected. The seeding and screening of subcloned cell lines is repeated 2–3 times to ensure the selection of strongly positive single clones. Note that a portion of the strongly positive cells should be frozen for later use during the screening process. Strongly positive cell lines that have undergone three rounds of subcloning selection were transferred to 48-well plates for expansion culture, taking care to freeze any excess strongly positive monoclonal cells. Once the cells in the 48-well cell culture plates reached a confluence of 80%, they were transferred to 24-well cell culture plates for further expansion culture. After the 24-well cell culture plates reached confluence, they were transferred to 6-well cell culture plates for further expansion culture, and finally transferred to 60mm cell culture dishes for further expansion culture.

[0082] 3) Monoclonal antibody specificity identification

[0083] The supernatant of the monoclonal cell line was collected, centrifuged, and filtered for sterilization. Then, purified bovine IgG and goat IgG (purified from the serum of healthy dairy cows and goats using a protein G antibody purification kit, at concentrations of 0.6 mg / mL and 0.5 mg / mL, respectively) were coated onto an ELISA 96-well plate. The specificity of the obtained mouse anti-bovine IgG monoclonal antibody was then detected using an indirect ELISA method. Figure 7As shown, the obtained monoclonal antibody showed a strong positive reaction to bovine IgG and a negative reaction to goat IgG (OD450 < 0.5). Therefore, the antibodies produced by the three selected monoclonal cell lines (1-D6, 2-D7, and 2-E3) can all specifically recognize bovine IgG. The specificity of the mouse anti-bovine monoclonal antibody in differentiating between bovine IgG and dairy goat IgG was detected using indirect ELISA; the results were analyzed using a t-test, with *** indicating a highly significant difference compared to the negative control group (P < 0.001), and ns indicating no significant difference compared to the negative control group (P > 0.05).

[0084] The accession number of hybridoma cell line 2-D7 is CCTCC NO: C2024137. It has been deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, on November 29, 2024. It is classified and named as Hybridoma cell line 2-D7.

[0085] 4) Selection of methods for monoclonal antibody subtype identification and purification

[0086] (1) Identification of antibody subtypes produced by hybridoma cells

[0087] Collect the supernatant of monoclonal cell lines and use a mouse monoclonal antibody subtype identification kit (Wuhan Sanying CatNo.PK20002) to detect the antibody subtypes of the obtained monoclonal cell lines. Figure 8 As shown, the monoclonal antibodies produced by the three selected cell lines are all of type IgG1 heavy chain and type Kappa light chain, so they can be purified using a protein G purification column.

[0088] (2) Karyotype identification of hybridoma cell lines

[0089] Cells were seeded into 6-well plates and cultured in complete medium containing 10% FBS for about 24 hours. When the cell confluence reached about 70% to 80%, the medium was replaced with complete medium containing 0.2 μg / mL colchicine and cultured for another 2 to 4 hours. The cells were then digested, centrifuged and the supernatant was discarded. The cells were resuspended in 1 mL of PBS and gently mixed. Then, 5 to 7 mL of preheated (37°C for 30 min) hypotonic treatment solution (0.075 M KCl) was added dropwise. The plates were gently tilted to the horizontal 2 to 3 times to mix. The plates were then transferred to a 37°C water bath for 15 min. Then, add 200 μL of 0.4% paraformaldehyde for pre-fixation for 2-3 min, centrifuge at 800 rpm for 10 min, discard the supernatant, gently tap the bottom of the tube to resuspend the cells, and then add 6 mL of 0.4% paraformaldehyde fixative dropwise along the tube wall. Fix at room temperature for 40 min, centrifuge at 800 rpm for 8 min, discard the supernatant, and repeat the fixation 2-3 times. Finally, gently resuspend the cells with 0.5 mL of fixative. Pre-cool the slide at 4℃ for 5 min, then tilt it at about 15° on the table. Aspirate 20 μL of cell suspension and drop it about 20-30 cm above the slide. Add about 200 μL of Giemsa dye to the area on the slide with adhered cells, stain for 20-30 min, rinse with distilled water to stop staining, and dry in a 35℃ oven. Use neutral resin as a mounting medium and cover with a coverslip. Then, observe and count the chromosome number and acquire images (e.g., ...). Figure 9 (As shown). The chromosome counts of the three selected cell lines were all around 2n = 92, which is consistent with the chromosome number after the fusion of SP20 (2n = 52) and BALB / c mouse spleen B lymphocytes (2n = 40).

[0090] 5) Preparation of monoclonal antibody ascites fluid

[0091] (1) As described in (1) Ascites modeling in the acquisition of feeder cells in (2), a mouse ascites model was prepared by injecting 6 adult BALB / c mice with liquid paraffin.

[0092] (2) Seven days after liquid paraffin injection, strongly positive monoclonal cells with good growth, high antibody specificity, and IgG antibody subtype were selected and diluted to a concentration of 2.5 × 10⁻⁶. 6 1 mL of cell suspension was injected intraperitoneally into each mouse with ascites model.

[0093] (3) Observe the growth and health status of mice after injection. The abdomen of mice will gradually swell after 3-4 days. When the abdominal swelling is large, the ascites should be collected immediately. Note that the hybridoma cell inoculation should not exceed 6 days to prevent mouse death. Collect mouse ascites into centrifuge tubes according to a method similar to that used in 2) for obtaining feeder cells. After centrifugation at 1000 r / min for 10 min, collect the supernatant and filter it for sterilization. Store it at -20℃ for later use.

[0094] 6) Monoclonal antibody purification

[0095] The ascites fluid after centrifugation and filtration was purified using a protein G affinity column, followed by dialyzing with double-distilled water at 4°C for 24–48 h. After determining the protein concentration (4.3 ng / μL), the fluid was frozen at -20°C for later use.

[0096] 7) Monoclonal antibody sandwich ELISA for specific detection

[0097] A 96-well ELISA plate was coated with mouse anti-bovine IgG monoclonal antibody, and rabbit anti-bovine IgG polyclonal antibody was used as the secondary antibody to detect different ratios of mixed raw cow's milk and goat's milk. Figure 10 As shown, the purified mouse anti-bovine IgG monoclonal antibody can specifically identify pure milk, 80% milk adulterated with goat milk, 50% milk adulterated with goat milk, 40% milk adulterated with goat milk, 25% milk adulterated with goat milk, and 10% milk adulterated with goat milk.

[0098] Example 3: Preparation of Colloidal Gold Immunochromatographic Test Strips

[0099] like Figure 13 As shown, the prepared mouse anti-bovine IgG monoclonal antibody was made into a gold-labeled antibody and immersed in a glass fiber membrane (gold-labeled pad). Rabbit anti-bovine IgG polyclonal antibody was streaked onto a nitrocellulose membrane (NC membrane) as the detection line (T line). Purified bovine IgG served as the control line (C line). The sample pad was immersed in the sample to be tested. The proteins in the sample diffused sequentially in the direction of "gold-labeled pad → detection line → control line → absorbent pad" under the action of capillary action and the absorbent pad. In the milk sample, bovine IgG first reacted with the gold-labeled mouse anti-bovine IgG monoclonal antibody, forming an "antigen-gold-labeled monoclonal antibody" complex, which continued to diffuse. Subsequently, it gradually and specifically bound to the rabbit anti-bovine polyclonal antibody on the NC membrane detection line, forming a "gold-labeled monoclonal antibody-antigen-polyclonal antibody" complex. Color development occurred when the antigen concentration in the sample reached the requirement of the detection line. The color deepened with increasing antigen concentration; while unbound gold-labeled mouse anti-bovine IgG monoclonal antibody continued to bind to the bovine IgG on the control line, demonstrating the good performance of the test strip.

[0100] 1) Preparation and optimization of gold-labeled conjugates

[0101] (1) Determination of pH value during labeling

[0102] To determine the optimal antibody labeling concentration and obtain stable gold-labeled monoclonal antibodies, the colloidal gold-labeled monoclonal antibody system needs to be optimized. The specific steps are as follows: Take five 1.5 mL centrifuge tubes and add 1 mL of colloidal gold solution (Shaanxi Kalinro Biotechnology Co., Ltd., catalog number GN0101-01) to each tube; adjust the pH to 6.5, 7.5, 8, 9, and 10 respectively using 0.1 mol / L K₂CO₃ solution; take a 96-well ELISA plate and add 100 μL of the above colloidal gold solution to each well according to pH from low to high, with three replicates for each pH; add 100 μL of 1 mg / mL mouse anti-bovine IgG monoclonal antibody to each well, mix well, and incubate at room temperature for 10–15 min; then add 20 μL of 10% NaCl solution to each well, gently shake to mix, and incubate at room temperature for 10 min; observe the color change of the colloidal gold and measure the OD250 using a UV spectrophotometer, recording the lowest pH value at which the red color is maintained. Figure 11 As shown, the optimal labeling pH is 7.5.

[0103] (2) Determination of the minimum concentration of gold-labeled antibody

[0104] Take one 96-well ELISA plate and add 100 μL of monoclonal antibody at concentrations of 40 μg / mL, 30 μg / mL, 20 μg / mL, 10 μg / mL, and 5 μg / mL, respectively, with three replicates for each concentration. Then, add 100 μL of colloidal gold solution adjusted to pH 7.5 with 0.1 mol / L K₂CO₃ to each well, gently vortex to mix, and incubate at room temperature for 15 min. Add 20 μL of 10% NaCl solution to each well and incubate at room temperature for 10 min. Observe the color change and measure the OD₂50 using a UV spectrophotometer. The minimum primary antibody concentration is defined as a bright red color with an OD₄50 > 0.2. Figure 12 As shown, 10 ng / mL was selected as the optimal colloidal gold labeling concentration for monoclonal antibodies.

[0105] (3) Colloidal gold-labeled monoclonal antibodies

[0106] After determining the optimal labeling pH and optimal labeling concentration, gold-labeled antibodies can be prepared in large quantities. Take 5 mL of colloidal gold solution into a 15 mL centrifuge tube and adjust the pH to 7.5 with 0.1 M K2CO3 solution. Take 5 mL of 10 μg / mL mouse anti-bovine IgG monoclonal antibody, mix by inversion, and let stand at room temperature for 30 min. Add polyethylene glycol-20000 (PEG-20000) (final concentration 0.05%), mix, and let stand at room temperature for 30 min. Centrifuge at 10000 r / min for 30 min at 4℃, and discard the supernatant. Reconstitute with 15 mM Tris buffer containing 0.05% PEG-20000, mix, and let stand at room temperature for 30 min. Centrifuge at 10000 r / min for 30 min at 4℃, discard the supernatant, and store the precipitate at 20℃ or 4℃ for later use.

[0107] 2) Preparation of gold-labeled polyester film

[0108] The gold-labeled antibody was resuspended in 10 mL of reconstitution solution (PBS solution containing 0.05% PEG20000). The pretreated polyester membrane was cut into strips 6 mm wide and 10 cm long and immersed in the liquid containing the gold-labeled antibody for about 10 minutes. Then it was transferred to a 37°C oven to dry. After drying, it was made into a test strip.

[0109] 3) Determination of the optimal antibody concentration for coating the detection line

[0110] NC membranes exhibit high affinity for most proteins and demonstrate good homogeneity and stability; therefore, they were selected as the chromatographic membrane. Rabbit anti-bovine IgG polyclonal antibody solutions at concentrations of 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, and 2.0 mg / mL were streaked with the solution, and the results were detected using a 50 μg / mL bovine IgG antigen standard solution. The color development of the NC membrane was observed, and the experiment was performed in triplicate. The lowest concentration with the best color development was selected as the optimal coating concentration for the detection line. The results showed that the optimal antibody concentration for coating the detection line was 1.5 mg / mL.

[0111] 4) Determination of the optimal coating antibody concentration for the quality control line

[0112] Bovine IgG at concentrations of 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, and 2.0 mg / mL were used to streak the control lines on the NC membrane. The color development of the control lines was observed using a gold-labeled antibody standard solution. The lowest concentration with the best color development was selected as the optimal coating concentration for the control lines. The results showed that the optimal coating antibody concentration for the control lines was 1.0 mg / mL.

[0113] 5) Test strip assembly

[0114] like Figure 13As shown, when assembling the colloidal gold test strip, the absorbent paper (model: H5072), the NC membrane (model: pall vivid 120) after the optimal concentration is traced, the gold-labeled binding pad (model: GL0194) with the optimal concentration, and the sample pad (model: XQ-Y8 sample pad) should be pasted onto the base plate (model: DB-6) in sequence. Each membrane should overlap by about 1-2 mm, and the overlapping area should be fixed with tape. Then, the assembled test card should be cut into test strips with a width of about 4 mm and embedded into the plastic card slot to prepare the colloidal gold detection kit.

[0115] 6) Clinical sample testing

[0116] Fresh goat milk and Holstein milk were collected from farms. Raw goat milk was mixed with 50%, 25%, 10%, and 5% raw cow milk to prepare adulterants. Assembled colloidal gold test strips were used to detect the adulteration of cow milk with goat milk. Approximately 500 μL of sample was added each time, and the sample was allowed to stand for 3–5 minutes before observing the results. As shown in Table 3, this test strip can accurately identify adulteration with a milk adulteration rate of 10% or higher.

[0117] Table 3 Results of the detection of adulterated cow's milk and goat's milk using the colloidal gold reagent kit.

[0118]

[0119] Note: When using clinical fresh cow's milk and goat's milk mixed in a certain proportion, adulteration can be detected if cow's milk is mixed with 10% or more of goat's milk.

[0120] The above description of the disclosed embodiments enables those skilled in the art to make or use 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 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 disclosed herein.

Claims

1. A hybridoma cell line, characterized in that, The hybridoma cell line was obtained by fusing splenic lymphocytes of BALB / c mice immunized with bovine IgG with mouse myeloma cells SP2 / 0 and then screening them. It can stably proliferate and secrete mouse monoclonal antibodies that specifically recognize bovine IgG. Its accession number is CCTCC NO: C2024137.

2. A mouse anti-bovine IgG monoclonal antibody, characterized in that, The monoclonal antibody is produced by the hybridoma cell line of claim 1, and is capable of specifically recognizing bovine IgG, without producing detectable cross-reactivity with sheep IgG, and retains its specific immune recognition ability under unwashed conditions.

3. The application of the mouse anti-bovine IgG monoclonal antibody as described in claim 2 in the specific detection of bovine IgG.

4. The application of the mouse anti-bovine IgG monoclonal antibody as described in claim 2 in the detection of adulterated cow's milk in goat's milk.

5. A colloidal gold reagent kit for rapid detection of adulterated cow's milk in goat milk based on mouse anti-bovine IgG monoclonal antibody, characterized in that, It contains the mouse anti-bovine IgG monoclonal antibody as described in claim 2.

6. The application of the kit according to claim 5 in the detection of adulterated goat milk and cow milk.

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