Goat milk adulterated milk rapid detection colloidal gold kit based on mouse anti-bovine IgG monoclonal antibody and application of goat milk adulterated milk rapid detection colloidal gold kit

Abstract

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

Description

Technical Field

[0001] The invention relates to the technical field of animal husbandry and veterinary medicine, and more particularly to a colloidal gold kit for rapid detection of adulterated goat milk based on mouse anti-cow IgG monoclonal antibody and application thereof. Background Art

[0002] The overall nutritional content of goat milk is better than that of cow milk and closer to human milk. It has a broad market prospect and huge development potential. However, the number of dairy goats in stock is limited, and there are limitations such as short lactation period and low milk production, which leads to the market price of goat milk being much higher than that of cow milk. Therefore, driven by interests, the phenomenon of adding cow milk to goat milk is very common, which seriously disrupts the market order and harms the health of the industry. At present, several conventional detection methods for adding cow milk to goat milk (high performance liquid chromatography, mass spectrometry, enzyme-linked immunosorbent assay and PCR, etc.) have the defects of long detection cycle, high cost and cumbersome operation, and cannot quickly and accurately identify whether goat milk is added with cow milk. Therefore, it is particularly necessary to develop an efficient and rapid method for identifying the addition of cow milk to goat milk in the fresh milk storage inspection link.

[0003] Therefore, providing a colloidal gold kit for rapid detection of adulterated goat milk based on mouse anti-bovine IgG monoclonal antibody and its application is a problem that those skilled in the art need to solve urgently. Summary of the invention

[0004] In view of this, the present invention provides a colloidal gold kit for rapid detection of adulterated goat milk based on mouse anti-bovine IgG monoclonal antibody and its application. In view of the problems that the current detection means are cumbersome, the detection time is too slow, and it is difficult to apply to the grassroots, the present invention adopts colloidal gold rapid detection technology, selects IgG protein in milk as antigen, obtains monoclonal antibodies with high specificity through immunological means and hybridoma cell culture technology, and adopts double antibody sandwich method to prepare high-sensitivity colloidal gold rapid detection test paper. Through comparison and optimization, test strips with high sensitivity and strong specificity are screened out.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] A hybridoma cell line is produced by the fusion of lymphocytes from mice immunized with bovine IgG and SP2 / 0 cells. It can proliferate indefinitely and secrete mouse anti-bovine IgG monoclonal antibodies. Its preservation number is CCTCC NO: C2024137. It has been deposited in the China Center for Type Culture Collection, abbreviated as CCTCC, address: Wuhan University, Wuhan, China. The preservation date is November 29, 2024, and it is classified and named Hybridoma cell line 2-D7.

[0007] Furthermore, a mouse anti-bovine IgG monoclonal antibody can produce a specific binding reaction to the species-specific region of bovine IgG and is produced by the hybridoma cell line.

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

[0009] Furthermore, the mouse anti-bovine IgG monoclonal antibody is used in the detection of adulterated goat milk.

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

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

[0012] Through the above technical scheme, it can be known that compared with the prior art, the present invention discloses a colloidal gold kit for rapid detection of adulterated milk in goat milk based on mouse anti-bovine IgG monoclonal antibody and its application. The present invention selects an economical, practical, fast and convenient colloidal gold rapid detection technology, selects IgG in milk as the target antigen, combines hybridoma cell technology, and produces a hybridoma cell strain that can produce highly specific monoclonal antibodies. Mouse anti-bovine IgG monoclonal antibodies are obtained through ascites proliferation and affinity column purification. The gold-labeled mouse anti-bovine IgG monoclonal antibody is further prepared using the immune colloidal gold labeling technology, and with this as the core, rabbit anti-bovine IgG polyclonal antibody is used as the detection line, and purified bovine IgG is used as the quality control line. Through comparison and optimization, a "colloidal gold rapid diagnosis kit for adulterated milk in goat milk" of the double antibody sandwich method is assembled. When the milk is mixed with a ratio of 10% or more, the kit can be accurately and quickly identified as "milk adulteration positive". In short, the present invention achieves the purpose of rapid and convenient detection of adulterated milk in goat milk. Compared with the prior art, it has the advantages of higher sensitivity, simpler operation, no dependence on laboratory conditions, and easy application in production. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the prior art descriptions. Obviously, the drawings described below are only embodiments of the present invention, and for ordinary technicians in this field, other drawings can be obtained based on the provided drawings without creative work.

[0014] Figure 1 The accompanying drawing is a technical roadmap of the present invention;

[0015] Figure 2 The accompanying drawing is the electrophoresis identification of the immunogen-bovine IgG protein purified by the present invention;

[0016] Among them, M is a protein standard marker; both lanes are purified bovine IgG;

[0017] Figure 3 The accompanying drawing shows the change in serum antibody titer of New Zealand rabbits after immunizing New Zealand rabbits with bovine IgG protein in the present invention;

[0018] Figure 4 The accompanying drawing shows the indirect ELISA specific detection of rabbit anti-bovine IgG polyclonal antibodies of the present invention;

[0019] Figure 5 The accompanying drawing shows the change in serum antibody titer of mice after immunizing mice with bovine IgG in the present invention;

[0020] Figure 6 The attached drawings are images of the hybridoma cell line of the monoclonal clone of the present invention (left) and the hybridoma cells in expanded culture (right) observed under an inverted microscope;

[0021] Figure 7 The accompanying drawing shows the specific detection of monoclonal antibodies in the prepared hybridoma cell supernatant by the ELISA method of the present invention;

[0022] Figure 8 The accompanying drawings show the results of the present invention for identifying the subtypes of antibodies in the supernatant of hybridoma cell lines;

[0023] Fig. 9 The accompanying drawings show the results of the hybridoma cell chromosome karyotype analysis of the present invention (left: Giemsa staining 100×, right: chromosome number statistics);

[0024] Fig.10 The accompanying drawing shows the specific detection of clinical samples by the sandwich ELISA method assembled with the prepared monoclonal antibody and polyclonal antibody of the present invention;

[0025] Fig.11 The accompanying drawing shows the optimal pH screening results of the present invention for colloidal gold labeled monoclonal antibodies;

[0026] Fig.12 The accompanying drawing shows the screening of the optimal concentration of monoclonal antibody labeled with colloidal gold of the present invention;

[0027] Fig.13 The accompanying drawing is a schematic diagram of the structure of the sandwich colloidal gold test strip assembled by the present invention. DETAILED DESCRIPTION

[0028] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] A colloidal gold rapid detection method, the technical roadmap is as follows Figure 1 As shown in the figure, IgG protein in milk was selected as antigen, monoclonal antibodies with high specificity were obtained through immunological means and hybridoma cell culture technology, and high-sensitivity colloidal gold rapid detection strips were prepared using double antibody sandwich method or competitive method. Through comparative optimization, test strips with high sensitivity and strong specificity were screened out.

[0030] (1) Selection of antigen

[0031] By comparing the similarities and differences in the milk of dairy cows and dairy goats, it is found that the whey protein of milk contains more bovine IgG (content is about 0.61mg / mL), and the bovine IgG protein has a species-specific amino acid sequence, which has different immune epitopes from the goat IgG and other proteins in goat milk, and can be recognized by its specific antibodies; while the goat IgG and other proteins in goat milk cannot be recognized by antibodies against the species-specific sequence of bovine IgG. Therefore, the present invention selects bovine IgG as a specific antigen, obtains mouse anti-bovine IgG monoclonal antibodies and rabbit anti-bovine IgG polyclonal antibodies with high specificity through immunological means and hybridoma cell culture technology, and prepares highly sensitive colloidal gold test paper.

[0032] (2) Preparation of Antigen

[0033] In order to prepare higher purity bovine IgG as an immunogen, this study collected blood from healthy adult female Holstein cows, separated the serum, and crudely purified it by salting out with caprylic acid-saturated ammonium sulfate salt and dialyzing to obtain crude IgG. The crude IgG was then further purified using a protein G affinity column (Tian Di Ren He, rProtein G Beads gravity column, SA016GC01) to obtain higher 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, and the blood of rabbits with higher antibody titers after multiple immunizations was collected to separate serum to obtain high-immune serum, which was crudely purified by caprylic acid-saturated ammonium sulfate salt precipitation, dialyzed, and protein G affinity chromatography to obtain purified rabbit anti-bovine IgG polyclonal antibodies.

[0036] (4) Preparation of monoclonal antibodies

[0037] Mice were immunized with a mixture of Freund's complete adjuvant and purified bovine IgG protein, and B lymphocytes that could produce highly mature, high-affinity antibodies were obtained through shock immunization. Hybridoma cells were selected to obtain a monoclonal cell line, which was then subjected to ascites proliferation, caprylic acid-saturated ammonium sulfate salt precipitation, dialysis, and protein G affinity chromatography to prepare mouse anti-bovine IgG protein monoclonal antibodies.

[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 antibodies. At the same time, the prepared rabbit anti-bovine IgG polyclonal antibodies were used as the detection line and the purified bovine IgG was used as the quality control line to assemble immunochromatographic test strips to test their sensitivity and specificity. Through comparative optimization, the most suitable detection system was screened out.

[0040] Key point 1: High specificity in target selection;

[0041] Key point 2: The production of highly specific monoclonal antibodies against target antigens has strong sensitivity and specificity;

[0042] Point three: The assembly is a colloidal gold method, which is easier to operate, has a shorter detection time and lower cost.

[0043] Example 1 Preparation of polyclonal antibodies

[0044] 1. Antigen Preparation

[0045] An adult healthy female Holstein cow was selected, and 40 mL of blood (10 mL per tube, 4 tubes in total) was collected by jugular vein puncture using a disposable vacuum blood collection tube. After standing at room temperature for 30 min, the blood was centrifuged at 3500 r / min for 10 min to obtain bovine serum. After crude purification by salting out of caprylic acid-saturated ammonium sulfate salt and dialysis to obtain crude IgG purified material, the bovine IgG in the bovine 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 identified by SDS. Figure 2 As shown, the Coomassie brilliant blue staining results showed that the purified protein solution had only two bands of about 25 kDa and about 55 kDa, so a relatively pure bovine IgG purification band was obtained. The protein concentration was determined by the Bicinchoninic Acid Assay (BCA) protein concentration determination kit (Jiangsu Keygene Biotechnology) to be 6.2 mg / mL.

[0046] 2. Preparation of Polyclonal Antibodies

[0047] The bovine IgG protein purified in the above steps was used as an immunogen, and healthy adult New Zealand rabbits were immunized according to the procedure in Table 1: Freund's complete adjuvant (FCA) was used for the primary immunization (first immunization), and Freund's incomplete adjuvant (FIA) was used for the 1st to 4th booster immunizations (corresponding to the second, third, fourth, and fifth immunizations, respectively). When used, the adjuvant and the purified antigen protein were mixed in equal volumes (the antigen protein needed to be diluted to 1000 μg / mL each time, and the antigen concentration after mixing with the adjuvant was 500 μg / mL). After emulsifying the antigen-adjuvant mixture with an ultrasonic cell disruptor, the New Zealand rabbits were subcutaneously immunized (500 μg / rabbit / time, that is, each experimental rabbit was vaccinated with 1 mL each time), and booster immunization was performed 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 tracked and detected using indirect ELISA (such as Figure 3 7 days after the fifth immunization, blood was collected from the ear vein and the antibody titer was higher than 10 5 , then shock immunization can be performed; during shock immunization, 2000 μg protein is used without adding adjuvants and injected subcutaneously on the back. After 7 days, blood is collected from the ear vein for 3 consecutive days, and 5-10 mL is collected each time; after separating the serum, IgG is crudely purified from the serum using the caprylic acid-saturated ammonium sulfate precipitation method, dialyzed with double distilled water at 4°C for 24 hours, purified using a protein G affinity column, and then dialyzed with double distilled water at 4°C for 48 hours. After BCA detection, the final purified mouse anti-bovine IgG polyclonal antibody concentration is 4.6 mg / mL. After sterilization by filtering with a bacteria filter, it is frozen at -20°C for future use. Indirect ELISA is used to detect the specificity of rabbit anti-bovine IgG polyclonal antibodies to bovine IgG and sheep IgG, and the results are tested using T. Figure 4 As shown in the figure (** indicates a very significant difference compared with the negative control group, i.e. P < 0.01, *** indicates a very significant difference compared with the negative control group, i.e. P < 0.001), the rabbit anti-bovine IgG polyclonal antibody has a certain specificity for bovine IgG, but still reacts to sheep IgG, which is not enough to be used as a gold standard detection antibody and can only be used as an antibody for the detection line or quality control line.

[0048] Table 1 Rabbit immunization program

[0049]

[0050] Note: FCA is mixed with FIA and bovine IgG and injected subcutaneously at the back of the neck using phacoemulsification to immunize New Zealand rabbits.

[0051] Example 2 Preparation of monoclonal antibodies

[0052] 1. Mouse Immunization

[0053] The bovine IgG protein purified in the "antigen preparation" step of Example 1 was used as an immunogen, and 5 healthy BALB / c mice aged 6 weeks were selected; the mice were immunized according to the immunization program in Table 2, and the antigen and an equal volume of Freund's complete adjuvant were mixed for the first immunization (the immunogen was diluted to 200 μg / mL before mixing with the adjuvant, and 100 μg / mL after mixing with the adjuvant), and after being fully emulsified using an ultrasonic cell disruptor, the immunized 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 once every 14 days, and Freund's incomplete adjuvant was used for the booster immunization. The antigen and adjuvant were mixed in equal volumes and fully emulsified using an ultrasonic cell disruptor. The antigen injection amount per mouse was 50 μg per time. After each immunization for 7 days, the mouse blood was collected from the tail vein and the serum was separated. The serum antibody titer of the immunized mice was determined using indirect ELISA (such as Figure 5 ), 7 days after the third booster immunization, the antibody titer of mouse serum was detected to be more than 1:10 5 The shock immunization before cell fusion can be carried out. No adjuvant is used during the shock immunization. A single injection of 200 μg of antigen solution is given subcutaneously on the back of the mouse (the purified bovine IgG is diluted to 400 μg / mL, and 0.5 mL is injected into each mouse); 3 days after the shock immunization, the mouse blood is collected from the tail vein and the serum is separated. The anti-bovine IgG titer of the mouse serum is determined by the ELISA method, and the mouse with the highest serum antibody titer is selected as the donor for obtaining immune lymphocytes (Note: the serum of the unimmunized mouse is negative serum. If the OD value of the serum of the immunized mouse is >2.1 times the OD value of the negative serum, it is positive).

[0054] Table 2 Mouse immunization program

[0055]

[0056] Note: FCA is mixed with FIA and bovine IgG and injected subcutaneously at multiple points on the back of the neck using phacoemulsification to immunize mice.

[0057] 2. Hybridoma Cell Fusion

[0058] 1) Myeloma cell culture

[0059] The frozen myeloma cells (SP2 / 0) were revived in a 60 mm cell culture dish with 2 mL of complete medium containing 20% ​​fetal bovine serum (Gibco, USA) and 1% penicillin-streptomycin-amphotericin B (Shanghai Beyotime Biotechnology Co., Ltd.) of high-glucose DMEM (Gibco, USA) (hereinafter referred to as 20% serum high-glucose complete medium). After continuous culture for a period of time, the cell growth state was observed. When the cell confluence reached about 90%, the cells could be passaged. During passage, the cells were gently rinsed twice with PBS preheated at 37°C, and the washed adherent cells were repeatedly rinsed with 1500 μL of 20% serum high-glucose complete medium. The cells could be suspended and passaged. During passage, the cells grown to 90% confluence in one 60 mm culture dish could be inoculated into three 60 mm cell culture dishes. When the cell confluence reached about 90%, they could be passaged again. After 2 to 3 generations of culture, the cells were observed to be in good growth state and uniform in morphology, and then replaced with a complete medium (referred to as 10% serum high-glucose complete medium) containing a mixture of 10% fetal bovine serum (Gibco, USA) and 1% penicillin-streptomycin-amphotericin B (Shanghai Biyuntian Biotechnology Co., Ltd.) in high-glucose DMEM (Gibco, USA) for continuous subculture, and the cell growth state was observed. When the cells grew in good morphology, uniform size, and reached the optimal growth state in the 10% serum high-glucose complete medium, they could be used for cell fusion.

[0060] 2) Acquisition of feeder cells

[0061] (1) Preparation of mouse ascites model: Three healthy adult ICR mice aged 10 weeks or older were selected. The feeder cell preparation experiment was started 7 days before cell fusion. Each mouse was intraperitoneally injected with 0.5 mL of liquid paraffin to induce the ascites model (the induction was successful when the mouse's abdominal cavity was obviously swollen with fluid 6 days after liquid paraffin injection). One day before cell fusion, the mice were anesthetized and their ascites were collected to isolate 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 killed by dislocating the neck. They were then quickly immersed in 75% alcohol precooled at 4°C for about 5 min. The body surface was thoroughly disinfected. The disinfected mouse carcasses were transferred to an ultra-clean workbench and fixed to a disinfected mouse board with sterilized thumbtacks. Ophthalmic scissors were used to cut the abdominal skin along the midline of the abdominal cavity to expose the peritoneum. The ventral peritoneum was gently lifted with ophthalmic forceps, and a small incision was made at the highest point of the lift. Care was taken to continue lifting the incision. Use a 5mL syringe to inject 5mL of 37℃ preheated high-glucose DMEM (Gibco, USA) into the abdominal cavity, gently massage the abdomen to fully suspend the macrophages in the ascites, use a sterile syringe to collect the ascites, filter the collected ascites with a 200-mesh cell sieve, transfer it to a 50mL centrifuge tube, and after absorbing the ascites, inject 5mL of preheated high-glucose DMEM again to flush the abdominal cavity. Repeat the flushing to collect peritoneal macrophages three times; after filtering with a 200-mesh cell sieve, centrifuge at 800r / min for 10min to collect the precipitated cells, resuspend and wash twice with high-glucose DMEM, and then The cells were suspended in a high-glucose DMEM selective complete medium (referred to as HAT selective high-glucose complete medium) containing 1×HAT medium additive (a cell culture medium additive containing hypoxanthine, aminopterin, and thymidine, referred to as HAT medium additive, purchased from Suzhou Biolong Company), 10% fetal bovine serum (Gibco Company, USA), and 1% penicillin-streptomycin-amphotericin B (Shanghai Biotech Company) to obtain a cell suspension;

[0063] (3) Plating: Take 15 μL of cell suspension and use 15 μL of 2× Trypan blue staining solution to stain the cells and count the live cells. Dilute the cell suspension to 2×10 using HAT selective high-glucose complete medium. 5 The cell suspension was then inoculated into a 96-well cell culture plate at 100 μL / well and cultured in a constant temperature cell culture incubator at 37°C containing 10% CO2 for 12 h. The cell growth status was observed to ensure that there was no bacterial or fungal contamination and that the cell growth status was good.

[0064] 3) Preparation of mouse spleen cells

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

[0066] (1) After the mice are anesthetized with ether, their eyeballs are removed and blood is collected for the collection of high-immune serum. When collecting blood, press the mouse's abdomen gently with your fingers to make the blood in the body flow out as much as possible to reduce the residual blood cells in the spleen. When the blood stops flowing out, the mouse is killed by dislocating the neck. Then, the mouse is immediately placed in 75% alcohol precooled at 4°C and soaked for about 3 to 5 minutes to thoroughly sterilize its body surface.

[0067] (2) After the surface of the sterilized mice was sterilized, they were moved into the clean bench and fixed with a sterilized mouse board and thumbtacks. The abdominal skin was lifted with ophthalmic surgical scissors and forceps, and cut longitudinally along the midline of the abdomen. The skin was then peeled off to expose the abdominal wall. The abdominal wall was sprayed with alcohol for disinfection for 15 seconds, and then new clean scissors were used to cut the abdominal wall and peritoneum. The enlarged spleen was carefully removed with new clean forceps (the spleen tissue of the successfully immunized mice will show obvious enlargement 3 days after the shock immunization) and placed in a sterile dish containing sterile saline.

[0068] (3) Rinse the surface of the spleen with sterile saline for 3 to 4 times and immediately transfer it to a new sterile culture dish. Then, pick up the spleen with clean tweezers and place it directly above the sterile culture dish. Use a 5 ml syringe to draw 3 ml of high-glucose DMEM preheated at 37°C. Gently insert the needle into the spleen and slowly push it in to flush out most of the lymphocytes in the spleen. After repeated injection and flushing for 3 times, use a 1 ml pipette to draw the liquid flushed out of the culture dish and filter it through a 200-mesh cell sieve. Transfer the filtrate to a clean 50 ml centrifuge tube. Then, place the remaining spleen tissue in a 200-mesh cell sieve and gently grind it with a clean syringe stopper to fully rupture the spleen tissue and fully exude the lymphocytes in the spleen. Then, use high-glucose DMEM to rinse the ground cells of the spleen tissue remaining on the cell sieve. Transfer the filtrate and the filtrate collected when flushing the spleen to the same 50 ml centrifuge tube and mix them evenly.

[0069] (4) The cell suspension obtained by flushing and grinding the spleen tissue was centrifuged at 800 r / min for 10 min, and the supernatant was discarded. The precipitated spleen cells were resuspended in 10 mL of high-glucose DMEM and centrifuged at 800 r / min for 10 min. This was repeated twice, and then resuspended in 2 mL of 10% serum high-glucose complete medium. 15 μL of the cell suspension was taken and stained with trypan blue for viable cell counting.

[0070] 4) Cell fusion

[0071] (1) Cell counting: Select SP2 / 0 cells with good growth status and normal cell morphology cultured in a 60 mm culture dish, rinse and suspend them with high-glucose DMEM, transfer all of them to a 15 mL centrifuge tube, centrifuge at 800 r / min for 5 min, discard the supernatant, resuspend the cells with 2 mL of high-glucose DMEM, take a small amount of the cell suspension, stain it with trypan blue, and count the live cells;

[0072] (2) Cell mixing: According to the live cell count results, spleen cells were adjusted to 6×10 7 SP2 / 0 cells were adjusted to 6×10 6 / mL, take 3mL of each, mix them in a 15mL centrifuge tube, use a 1mL pipette to gently blow and mix, centrifuge at 800r / min for 10min, discard the supernatant, tap the bottom of the centrifuge tube with tweezers to flick the precipitated cell clumps to a loose state for later use.

[0073] (3) Cell fusion: Pipette 1 mL of 50% PEG4000 (PBS solution containing 50% PEG4000) preheated at 38°C, slowly add PEG4000 to the cell pellet at a rate of 1 mL / min, stirring evenly while adding, and then transfer to a 37°C incubator and let stand for 10 min;

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

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

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

[0077] 3. Monoclonal hybridoma cell screening and monoclonal antibody identification

[0078] 1) Hybridoma cell screening

[0079] After cell fusion, replace half of the culture medium in the 96-well cell culture plate every 72 hours (absorb and discard 100 μL of the old culture medium in each well, and add 100 μL of new HAT selective high-glucose complete culture medium). Before each medium change, observe the cell growth under an inverted microscope. Successfully fused hybridoma cells may show proliferation of monoclonal cell clusters (such as Figure 6Hybridoma cells have the property 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 marked. After replacing the medium twice, when the medium is replaced for the third time (9 days after fusion), 100 μL of supernatant is taken from the well-proliferated cell wells 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 in a row to screen out cell wells with strong positive antibody detection.

[0080] 2) Subcloning

[0081] When the cells in the 96-well cell culture plate grow to 50% confluence, the cell lines with strong positive antibody detection can be subcloned. First, it is necessary to prepare feeder cells (prepare feeder cells as described in step 2), then resuspend the strong positive hybridoma cell lines, and count the viable cells. According to the cell density, dilute the cells to about 1 cell per 200 μL, and plate 200 μL per well on a 96-well cell culture plate containing feeder cells, and culture with HAT selective high-sugar complete medium. After 24 hours, use an inverted microscope to observe the cell growth status, select the monoclonal cell wells, and mark them. Replace 100 μL of the culture medium in the monoclonal cell wells every 72 hours, collect the supernatant of the monoclonal cell wells and perform indirect ELISA detection, screen single cell clones and cell supernatant antibody detection strong positive cell lines. Repeat the plating and screening of subclone cell lines 2 to 3 times to ensure that strongly positive monoclonal cell lines are screened. Note that a portion of the strong positive cells are frozen for standby during the screening period. After three rounds of subclone screening, the strong positive cell lines were transferred to 48-well plates for expansion culture. The excess strong positive monoclonal cells were frozen. When the cells in the 48-well cell culture plate grew to more than 80%, they could be transferred to the 24-well cell culture plate for expansion culture. After the 24-well cell culture plate was full, they were transferred to the 6-well cell culture plate for expansion culture, and finally transferred to the 60mm cell culture dish for expansion culture.

[0082] 3) Monoclonal antibody specificity identification

[0083] The supernatant of the monoclonal cell line was collected, centrifuged and filtered for sterilization, and then the purified bovine IgG and goat IgG (the same as the "antigen preparation" step in Example 1, purified from healthy cow and goat serum using a protein G antibody purification kit, with concentrations of 0.6 mg / mL and 0.5 mg / mL, respectively) were coated on an enzyme-linked immunosorbent 96-well enzyme-labeled colorimetric plate, and then the specificity of the obtained mouse anti-bovine IgG monoclonal antibody was detected using an indirect ELISA method, as shown in FIG. Figure 7As shown, the monoclonal antibody obtained has 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 specifically recognize bovine IgG. Indirect ELISA was used to detect the specificity of mouse anti-bovine monoclonal antibodies for identifying bovine IgG and dairy goat IgG; the results were tested using T test, *** indicates that the difference is extremely significant compared with the negative control group (P<0.001), and ns indicates that the difference is not significant compared with the negative control group (P>0.05).

[0084] The deposit number of hybridoma cell line 2-D7 is CCTCCNO: C2024137. It has been deposited in China Center for Type Culture Collection, referred to as CCTCC, address: Wuhan University, Wuhan, China. The deposit date is November 29, 2024. It is classified and named Hybridoma cell line 2-D7 Hybridoma cell line2-D7.

[0085] 4) Monoclonal antibody subtype identification and purification method selection

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

[0087] The supernatant of the monoclonal cell line was collected and tested using a mouse monoclonal antibody subtype identification kit (Cat No. PK20002, Wuhan Mitaka) to distinguish the antibody subtypes of the monoclonal cell line obtained. Figure 8 As shown, the heavy chain type of the monoclonal antibodies produced by the three selected cell lines are all IgG1, and the light chain type is all Kappa light chain, so they can be purified using a protein G purification column.

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

[0089] The cells were inoculated into 6-well plates and cultured with complete medium containing 10% FBS for about 24 h. When the cell confluence reached about 70% to 80%, the complete medium containing 0.2 μg / mL colchicine was replaced and cultured for another 2 to 4 h. The cells were then digested, the supernatant was discarded by centrifugation, resuspended in 1 mL of PBS and gently mixed, and then 5 to 7 mL of preheated (37°C for 30 min) hypotonic treatment solution (0.075 M KCl) was added dropwise. The plate was gently tilted to a horizontal position for 2 to 3 times to mix, and then transferred to a 37°C water bath for treatment for 15 min. Subsequently, 200 μL of 0.4% paraformaldehyde was added for pre-fixation for 2 to 3 minutes, and then centrifuged at 800 r / min for 10 minutes, the supernatant was discarded, the bottom of the shaking tube was tapped to resuspend the cells, and then 6 mL of 0.4% paraformaldehyde fixative was added drop by drop along the wall of the tube, fixed at room temperature for 40 minutes, and then centrifuged at 800 r / min for 8 minutes, and the supernatant was discarded. The fixation was repeated 2 to 3 times, and finally the cells were gently resuspended with 0.5 mL of fixative; the slide was pre-cooled at 4°C for 5 minutes, and then tilted at about 15° on the desktop, 20 μL of cell suspension was drawn, and the suspension was about 20 to 30 cm above the slide; about 200 μL of Giemsa dye was added to the area with cells adhered to the slide, and the staining was stopped for 20 to 30 minutes, and the staining was terminated by rinsing with distilled water, and the slide was dried in a 35°C oven; a neutral resin was used as a sealing agent, and the slide was sealed with a cover glass; then the number of chromosomes was counted and images were collected using a microscope (such as Fig. 9 The chromosome counts of the three selected cell lines were all around 2n=92, which was 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

[0091] (1) As described in (1) Ascites modeling in 2) Obtaining feeder cells, 6 adult BALB / c mice were injected with liquid paraffin to prepare a mouse ascites model;

[0092] (2) Seven days after the injection of liquid paraffin, strong positive monoclonal cells with good growth status, strong secretion of antibodies with strong specificity and IgG antibody subtype were selected and diluted to 2.5×10 6 Each mouse with ascites model was intraperitoneally injected with 1 mL of cell suspension;

[0093] (3) Observe the growth and health of the mice after injection. The abdomen of the mice will gradually swell after 3-4 days. When the abdomen of the mice swells greatly, the ascites should be collected immediately. Note that the hybridoma cell inoculation should not exceed 6 days to prevent the death of the mice. According to the method similar to that of obtaining feeder cells in 2), the ascites of the mice is collected into a centrifuge tube. After centrifugation at 1000r / min for 10 minutes, the supernatant is collected and sterilized by filtration, and then frozen at -20℃ for later use.

[0094] 6) Monoclonal antibody purification

[0095] The ascites after centrifugation and filtration was purified using a protein G affinity column and then dialyzed with double distilled water at 4°C for 24 to 48 h. The protein concentration was detected (4.3 ng / μL) and then frozen at -20°C for later use.

[0096] 7) Monoclonal antibody sandwich ELISA specific detection

[0097] Mouse anti-bovine IgG monoclonal antibody was used to coat a 96-well enzyme-labeled colorimetric plate, and rabbit anti-bovine IgG polyclonal antibody was used as the secondary antibody to detect different proportions of raw bovine milk and goat milk, such as Fig.10 As shown, the purified mouse anti-bovine IgG monoclonal antibody can specifically identify pure cow's milk, 80% cow's milk adulterated goat's milk, 50% cow's milk adulterated goat's milk, 40% cow's milk adulterated goat's milk, 25% cow's milk adulterated goat's milk and 10% cow's milk adulterated goat's milk.

[0098] Example 3 Preparation of colloidal gold immunochromatography test strips

[0099] like Fig.13 As shown, the prepared mouse anti-bovine IgG monoclonal antibody is made into a gold-labeled antibody and soaked on a glass cellulose membrane (gold-labeled pad), and the rabbit anti-bovine IgG polyclonal antibody is streaked on a nitrocellulose membrane (NC membrane) as the test line (T line). The purified bovine IgG is used as the quality control line (C line). The sample pad is immersed in the sample to be tested. The protein in the sample to be tested is affected by the capillary action and diffuses in the direction of "gold-labeled pad→test line→quality control line→water-absorbent pad" in sequence under the action of the absorbent pad. The bovine IgG in the milk sample first reacts with the gold-labeled mouse anti-bovine IgG monoclonal antibody to form an "antigen-gold-labeled monoclonal antibody" complex and continues to diffuse, and then gradually specifically binds to the rabbit anti-bovine polyclonal antibody on the NC membrane test line to form a "gold-labeled monoclonal antibody-antigen-polyclonal antibody" complex. When the antigen concentration in the sample reaches the requirement of the test line, it will develop color. As the antigen concentration increases, its color deepens; while the unbound gold-labeled mouse anti-bovine IgG monoclonal antibody continues to bind to the bovine IgG on the quality control line to develop color, proving that the test strip has good performance.

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

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

[0102] In order to determine the optimal antibody labeling concentration and obtain a stable gold-labeled monoclonal antibody, it is necessary to optimize the system of colloidal gold-labeled monoclonal antibodies. The specific steps are as follows: take 5 1.5mL centrifuge tubes and add 1mL of colloidal gold solution (Shaanxi Karinluo Biotechnology Co., Ltd., catalog number GN0101-01) to each well; adjust the pH to 6.5, 7.5, 8, 9, and 10 with 0.1mol / L K2CO3 solution; take a 96-well enzyme-labeled colorimetric plate, and add 100μL of the above colloidal gold solution to the wells according to the pH from low to high, and set 3 replicates for each pH; add 100μL of 1mg / mL mouse anti-bovine IgG monoclonal antibody to each well, mix well, and stand at room temperature for 10 to 15min; then add 20μL of 10% NaCl solution to each well, shake gently to mix, and stand at room temperature for 10min; observe the color change of colloidal gold, and use a UV spectrophotometer to detect OD250, and record the lowest pH value that remains red, such as Fig.11 As shown, the optimal labeling pH is 7.5.

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

[0104] Take a 96-well enzyme-labeled colorimetric plate, add 100 μL of monoclonal antibodies at 40 μg / mL, 30 μg / mL, 20 μg / mL, 10 μg / mL and 5 μg / mL in sequence, and repeat 3 times 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 shake to mix, and let stand at room temperature for 15 minutes; add 20 μL of 10% NaCl solution to each well, let stand at room temperature for 10 minutes; then observe the color change and use a UV spectrophotometer to detect its OD250. The minimum primary antibody dosage with bright red color and OD450>0.2 is the minimum labeling concentration. Fig.12 As shown, 10 ng / mL was selected as the optimal colloidal gold labeling concentration of the monoclonal antibody.

[0105] (3) Colloidal gold labeled monoclonal antibody

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

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

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

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

[0110] NC membrane shows high affinity for most proteins, and has good uniformity and stability, so NC membrane is selected as the chromatographic membrane. The lines were drawn with rabbit anti-bovine IgG polyclonal antibody solutions with concentrations of 0.5mg / mL, 1.0mg / mL, 1.5mg / mL, and 2.0mg / mL, and detected with 50μg / mL bovine IgG antigen standard solution. The color development of the NC membrane was observed, and the parallel test was performed three times. The lower concentration and better color development were selected as the optimal coating concentration of the detection line. The results showed that the optimal detection line coating antibody concentration was 1.5mg / mL.

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

[0112] The lines were drawn with bovine IgG at concentrations of 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, and 2.0 mg / mL, respectively, and detected with gold-labeled antibody standard solution. The color development of the quality control line on the NC membrane was observed, and the one with lower concentration and better color development was selected as the optimal coating concentration of the quality control line. The results showed that the optimal coating antibody concentration of the quality control line was 1.0 mg / mL.

[0113] 5) Test strip assembly

[0114] like Fig.13As shown in the figure, when assembling the colloidal gold test strip, the absorbent paper (model: H5072), the NC membrane with the optimal concentration line (model: Pall Vivid 120), the gold label binding pad marked with the optimal concentration (model: GL0194), and the sample pad (model: XQ-Y8 sample pad) need to be pasted to the bottom plate (model: DB-6) in sequence, with each membrane overlapping by about 1 to 2 mm, and the overlapping area is fixed with tape, and then the assembled test card is cut into test strips with a width of about 4 mm and embedded in the plastic card slot to prepare a colloidal gold detection kit.

[0115] 6) Clinical sample testing

[0116] Fresh goat milk and Holstein milk were collected from the farm, and raw goat milk was mixed with 50%, 25%, 10%, and 5% raw cow milk. The assembled colloidal gold test strips were used to detect the adulteration of cow milk with goat milk. Each time, about 500 μL of the sample was added, and it was placed flat and still for 3 to 5 minutes to observe the test results. As shown in Table 3, the test strips can be used to accurately identify the adulteration ratio of milk 10% and above.

[0117] Table 3 Results of Colloidal Gold Kit for Detecting Adulterated Milk from Goat Milk

[0118]

[0119] Note: Use clinical fresh cow's milk and goat's milk and mix them in proportion. Adulteration can be detected if the cow's milk is adulterated with goat's milk by 10% or more.

[0120] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one 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 present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A hybridoma cell line, characterized in that Its deposit number is CCTCCNO: C2024137.

2. A mouse anti-bovine IgG monoclonal antibody, characterized in that: Produced by the hybridoma cell line of claim 1.

3. Use of the mouse anti-bovine IgG monoclonal antibody according to claim 2 in the specific detection of bovine IgG.

4. Use of the mouse anti-bovine IgG monoclonal antibody according to claim 2 in the detection of adulterated goat milk.

5. A colloidal gold kit for rapid detection of adulterated goat milk based on mouse anti-bovine IgG monoclonal antibody, characterized in that: Contains the mouse anti-bovine IgG monoclonal antibody according to claim 2.

6. Use of the kit according to claim 5 in detecting adulterated goat milk.

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