Hybridoma cell strain, anti-fox skeletal muscle troponin monoclonal antibody generated by hybridoma cell strain and application of antibody

Abstract

The invention relates to the technical field of immunology and meat source identification of adulterated meat products. The invention provides a hybridoma cell strain, an anti-fox skeletal muscle troponin monoclonal antibody generated by the hybridoma cell strain and application of the antibody, the hybridoma cell strain is preserved in China Center for Type Culture Collection on October 10, 2023, and the preservation number of the hybridoma cell strain is skTn-3A4. The preservation number of the strain is CCTCC (China Center For Type Culture Collection) The anti-fox skeletal muscle troponin monoclonal antibody skTn-3A4 provided by the invention can be used for preparing an enzyme linked immunosorbent assay kit, a colloidal gold chromatography test strip and an evanescent wave fluorescence immunosensor for detecting fox skeletal muscle troponin, so that the aim of quickly and sensitively detecting fox skeletal muscle troponin in food is fulfilled.

Description

Technical Field

[0001] The invention relates to the technical field of immunology and meat source identification of adulterated meat products, and in particular to a hybridoma cell line and anti-fox skeletal muscle troponin monoclonal antibody produced by the hybridoma cell line and the application of the antibody. Background Art

[0002] In recent years, "adulterated meat" has continuously impacted the meat market, and meat safety issues have received increasing attention. Under the temptation of high profits, some unscrupulous merchants use low-value meats such as fox meat to impersonate mutton, horse meat to impersonate beef, and raccoon meat to impersonate mutton. Among them, the most notable is the adulteration of rat meat, fox meat, raccoon meat and mink meat. The meat sources may carry a large number of bacteria, viruses, parasites and other substances that are harmful to the human body, because there is no strict quarantine procedure in the process of raising fur animals. Therefore, meat certification methods and animal meat traceability analysis are increasingly hot topics in the food industry. Meat source components are qualitative components used to distinguish and identify different animal species, and are widely used in meat certification and quality control of meat products. Therefore, developing a method to characterize and quantify the source of meat products is necessary for authenticating authenticity and controlling the quality of meat.

[0003] Enzyme-linked immunosorbent assay (ELISA) is one of the most specific and direct assays for detecting biomolecules in research and clinical settings. With the advancement of scientific research, ELISA assays have been continuously optimized to improve their sensitivity, and different types of ELISAs are now available to detect a variety of biomarkers. Skeletal troponin is heat-stable and can be used as a marker protein to identify the composition of raw and heat-treated meat ingredients. Skeletal troponin is a structural protein that regulates the contractile function of skeletal muscle and is composed of three subunits: troponin T, with a molecular weight of 37kD, and troponin I, with a molecular weight of 24kD and troponin C, with a molecular weight of 18kD.

[0004] In the present invention, the fox skeletal muscle tissue is completely crushed, cooked and homogenized by protein extraction to obtain immunogens, and monoclonal antibodies are prepared by immunizing mice, and the immunological properties such as the titer, subtype, affinity, etc. of the monoclonal antibodies are detected, analyzed and evaluated. This study is of great significance for establishing a series of immunological methods for identifying fox meat ingredients. Summary of the invention

[0005] The purpose of the present invention is to provide a hybridoma cell line and an anti-fox skeletal muscle troponin monoclonal antibody produced by the hybridoma cell line and the application of the antibody. The quality of the anti-fox skeletal muscle troponin monoclonal antibody provided has strong controllability and repeatability. Through preliminary analysis and identification of the obtained antibody characteristics, experimental basis and basic raw materials are provided for the further establishment and application of a specific and sensitive fox skeletal muscle troponin immunoassay method. It can be used to prepare an enzyme-linked immunosorbent assay kit, colloidal gold chromatography test strips and evanescent wave fluorescence immunosensor for detecting fox skeletal muscle troponin, so as to achieve the purpose of quickly and sensitively detecting fox meat adulteration in meat products.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a hybridoma cell strain, the deposited name of the hybridoma cell strain is: hybridoma cell strain skTn-3A4 of anti-fox skeletal muscle troponin monoclonal antibody, the deposited location is: China Center for Type Culture Collection, the deposited date is: October 10, 2023, and the deposited number is CCTCC NO: C2023302.

[0008] The present invention also provides the use of the hybridoma cell line in preparing anti-fox skeletal muscle troponin monoclonal antibody.

[0009] The present invention also provides an anti-fox skeletal muscle troponin monoclonal antibody produced by the hybridoma cell line skTn-3A4.

[0010] The present invention also provides the use of the anti-fox skeletal muscle troponin monoclonal antibody in preparing a product for detecting fox skeletal muscle troponin.

[0011] Preferably, the products include enzyme-linked immunosorbent assay kits, colloidal gold chromatography test strips, and evanescent wave fluorescence immunosensors.

[0012] The present invention also provides an enzyme-linked immunosorbent assay kit for detecting fox skeletal muscle troponin, wherein the enzyme-linked immunosorbent assay kit comprises the anti-fox skeletal muscle troponin monoclonal antibody.

[0013] The present invention also provides a colloidal gold chromatography test strip for detecting fox skeletal muscle troponin, wherein the colloidal gold chromatography test strip comprises the anti-fox skeletal muscle troponin monoclonal antibody.

[0014] The present invention also provides an evanescent wave fluorescence immunosensor for detecting fox skeletal muscle troponin. The evanescent wave fluorescence immunosensor comprises the anti-fox skeletal muscle troponin monoclonal antibody.

[0015] The present invention provides a hybridoma cell line and an anti-fox skeletal muscle troponin monoclonal antibody produced by the hybridoma cell line and an application of the antibody. The deposited name of the hybridoma cell line is: hybridoma cell line skTn-3A4 of anti-fox skeletal muscle troponin monoclonal antibody, deposited at China Center for Type Culture Collection, deposited on October 10, 2023, and deposited with CCTCC NO: C2023302. The anti-fox skeletal muscle troponin monoclonal antibody skTn-3A4 provided by the present invention can be used to prepare an enzyme-linked immunosorbent assay kit, colloidal gold chromatography test strips, and evanescent wave fluorescence immunosensor for detecting fox skeletal muscle troponin, so as to achieve the purpose of quickly and sensitively detecting fox skeletal muscle troponin in food. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a gel electrophoresis identification diagram of fox skeletal muscle troponin immune antigen and detection antigen prepared in Example 1 of the present invention;

[0017] Figure 2 This is an immunoblot identification diagram of fox skeletal muscle troponin detection antigen prepared in Example 1 of the present invention;

[0018] Figure 3 This is a diagram for determining subtypes of the anti-fox skeletal muscle troponin monoclonal antibody prepared in Example 2 of the present invention;

[0019] Figure 4 This is a graph showing the affinity determination of the anti-fox skeletal muscle troponin monoclonal antibody prepared in Example 2 of the present invention;

[0020] Figure 5 This is a standard curve diagram of the reaction between the anti-fox skeletal muscle troponin monoclonal antibody prepared in Example 2 of the present invention and the antigen.

[0021] Collection Instructions

[0022] The hybridoma cell line skTn-3A4 of anti-fox skeletal muscle troponin monoclonal antibody was deposited in China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, with a deposit date of October 10, 2023, and a deposit number of CCTCCNO: C2023302. DETAILED DESCRIPTION

[0023] The present invention provides a hybridoma cell strain, the deposited name of the hybridoma cell strain is: hybridoma cell strain skTn-3A4 of anti-fox skeletal muscle troponin monoclonal antibody, the deposited location is: China Center for Type Culture Collection, the deposited date is: October 10, 2023, and the deposited number is CCTCC NO: C2023302.

[0024] The present invention also provides the use of the hybridoma cell line in preparing anti-fox skeletal muscle troponin monoclonal antibody.

[0025] The present invention also provides an anti-fox skeletal muscle troponin monoclonal antibody produced by the hybridoma cell line skTn-3A4.

[0026] The present invention also provides the use of the anti-fox skeletal muscle troponin monoclonal antibody in preparing a product for detecting fox skeletal muscle troponin.

[0027] In the present invention, the products include an enzyme-linked immunosorbent assay kit, a colloidal gold chromatography test strip, and an evanescent wave fluorescent immunosensor.

[0028] The present invention also provides an enzyme-linked immunosorbent assay kit for detecting fox skeletal muscle troponin, wherein the enzyme-linked immunosorbent assay kit comprises the anti-fox skeletal muscle troponin monoclonal antibody.

[0029] The present invention also provides a colloidal gold chromatography test strip for detecting fox skeletal muscle troponin, wherein the colloidal gold chromatography test strip comprises the anti-fox skeletal muscle troponin monoclonal antibody.

[0030] The present invention also provides an evanescent wave fluorescence immunosensor for detecting fox skeletal muscle troponin. The evanescent wave fluorescence immunosensor comprises the anti-fox skeletal muscle troponin monoclonal antibody.

[0031] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0032] Example 1 Preparation of fox skeletal muscle troponin immune antigen

[0033] (1) Preparation of immune antigens

[0034] Take 10g of fresh fox meat, remove fat and connective tissue, chop it, add 20ml of 0.15mol / LNaCl solution, and homogenize it with a homogenizer for 1h. Ultrasonic treatment, 100W, 5min, after boiling water bath for 20min, centrifuge at 6000r / min for 30min. Take the supernatant and treat it with high pressure at 121℃ for 30min. Filter with Whatman No.1 filter paper, add 90% ethanol (1:3.74v / v) to the filtrate, centrifuge at 8000r / min for 20min, discard the supernatant, dissolve the precipitate in 0.01mol / L PBS buffer, and the obtained pig skeletal muscle extract is the pig skeletal muscle troponin immunogen. Measure the protein concentration and store it at -80℃ for later use.

[0035] (2) Preparation of detection antigen

[0036] Take some fresh fox meat, cut it into pieces and store it at -80℃. Thaw it completely at room temperature for 3 hours before use. Weigh 10g of each meat, chop it, and put it in centrifuge tubes. After boiling the tubes in a water bath for 15 minutes, immediately cool it with ice water, add 20ml of 0.01mol / LPBS buffer containing 0.5mol / LNaCl to the centrifuge tubes, and homogenize for 1 hour. Centrifuge at 6000r / min for 30 minutes, discard the precipitate, take the supernatant, and obtain the skeletal muscle extracts of the above species, which are the skeletal muscle troponin detection precursors for each species. Measure the protein concentration and store it at -80℃ for later use.

[0037] (3) Identification of the prepared fox skeletal muscle troponin immune antigen and detection antigen:

[0038] Tricine-SDS-PAGE identification

[0039] Tricine-SDS-PAGE was used to identify fox skeletal muscle troponin immune antigen and detection antigen. According to the conventional Tricine-SDS-PAGE method, 4% concentrated gel, 10% gap gel, and 15% separation gel were prepared at a voltage of 80V until the protein band entered the separation gel, and then the voltage was adjusted to 120V and continued electrophoresis for 2h. After staining and decolorization.

[0040] The results are as follows Figure 1 As shown in the Tricine-SDS-PAGE results (see Figure 1 The results of Tricine-SDS-PAGE identification of fox skeletal muscle troponin immune antigen and detection antigen; Lane 1 is fox skeletal muscle troponin detection antigen, and Lane 2 is fox skeletal muscle troponin immune antigen). After electrophoresis of fox skeletal muscle troponin immune antigen, there are two clear and dark bands, located at 37kD and 24kD, respectively, which are consistent with the size of sTnT and sTnI subunits recorded in the literature. After electrophoresis of fox skeletal muscle troponin detection antigen, there is one main band, located at 37kD, which is the sTnT subunit. There is also a band at 24kD, but it is not as dark as the band at 37kD, which is the sTnI subunit.

[0041] Example 2

[0042] 1. Preparation of anti-fox skeletal muscle troponin monoclonal antibody

[0043] (1) Animal immunization

[0044] The fox skeletal muscle troponin immune antigen prepared in Example 1 was used to immunize female Balb / c mice aged 6 to 8 weeks, once every 2 weeks. The immunization process is shown in Table 1. After three immunizations, the tails were cut off and blood was collected to determine the antiserum titer. The mice with the best results were selected for booster immunization and used as fusion mice.

[0045] Table 1 Immunization process chart

[0046] Immunization time Immunization times Immune site Immunization dose Remark 0 First immunization Neck injection 40μg / piece 2 weeks Second immunization Neck injection 40μg / piece 4 weeks The third immunization Neck injection 40μg / piece Determination of antiserum titer 6 weeks Strengthen immunity Abdominal injection 20μg / each

[0047] (2) Cell fusion

[0048] The fusion mice selected in step (1) were eyeballed and bled, and the spleens were removed under sterile conditions after being killed by cervical dislocation. Splenocytes were prepared and fused with SP2 / 0 cells at a ratio of 5:1 by PEG. The fused cell suspension was added to a 96-well plate already plated with feeder cells and cultured in an incubator at 37°C and 5% CO2.

[0049] (3) Screening of positive hybridoma cell lines

[0050] After fusion, the cells were checked for contamination the next day, and HT medium was used on the 10th day after fusion. Three days after the medium change, the cell supernatant was detected by indirect ELISA method, and the wells with positive test results were screened. The cells were subcloned by limiting dilution method to obtain a hybridoma cell line with stable antibody secretion. It was deposited in the China Center for Type Culture Collection and named as the hybridoma cell line skTn-3A4 of anti-fox skeletal muscle troponin monoclonal antibody, with the deposit number CCTCC NO: C2023302.

[0051] (4) Preparation and purification of anti-fox skeletal muscle troponin monoclonal antibody

[0052] Using the method of inducing ascites in mice, healthy Balb / c female mice were selected and 0.5 mL of paraffin oil was injected into each mouse. After 7 days, 10 cloned positive hybridoma cells were adjusted. 6 / mL, 800 μL was injected intraperitoneally into each mouse. After 12 days, the ascites was collected and purified by the caprylic acid-ammonium sulfate method to obtain the monoclonal antibody. The concentration was detected and stored at -20°C for later use. The purified anti-fox skeletal muscle troponin monoclonal antibody was obtained.

[0053] 2. Identification of the characteristics of the prepared anti-fox skeletal muscle troponin monoclonal antibody

[0054] (1) Potency determination

[0055] The indirect ELISA method was used to determine the titer of monoclonal antibodies: the concentration of the original test was diluted to 2 μg / ml, added to a 96-well plate, 100 μL per well, and coated at 37°C for 1 hour; Each well was washed 3 times with PBS and patted dry; the monoclonal antibody was diluted at 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600, 1:51200, 1:102400, 1:204800, 1:409600, and 1:819200, 100 μL was added to each well, incubated at 37°C for 45 min, washed, and patted dry; 100 μL of goat anti-mouse IgG secondary antibody (diluted 10,000 times) was added to each well, incubated at 37°C for 30 min, washed, and patted dry; 100 μL of color development solution was added to each well, and reacted at 37°C in the dark for 12 min; 50 μL of stop solution was added to each well, and the wavelength was set to 450 nm to measure the absorbance value. The absorbance value of the negative control well is A0, and the absorbance value of each assay well is A t , with A t The dilution ratio corresponding to / A0≥2.1 is taken as the result of the titer test. The titer of the anti-fox skeletal muscle troponin monoclonal antibody sTn-3A4 is 1:1×10 5 , see Table 2.

[0056] Table 2 Titer and concentration of monoclonal antibody sTn-3A4

[0057] Monoclonal antibodies Concentration / (mg / ml) Potency sTn-3A4 1.76 <![CDATA[1:1×10 5 ]]>

[0058] (2) Subtype determination

[0059] The subtype determination was performed using a mouse monoclonal antibody subtype identification kit purchased from Sigma. The anti-fox skeletal muscle troponin monoclonal antibody secreted by the hybridoma cells had obvious differences in color development with the secondary antibodies of different subtypes, among which the A450nm value with the IgM secondary antibody was the highest, the color development with the IgG1 secondary antibody was weak, and the color development with the IgG2a, IgG2b, IgG3 and IgA secondary antibodies was almost nonexistent. Therefore, the antibody type secreted by the hybridoma cells was mainly IgM. The results are shown in Figure 2 Therefore, the subtype of the anti-fox skeletal muscle troponin monoclonal antibody of the present invention is IgM.

[0060] (3) Affinity determination

[0061] The original was coated with four concentration gradients (1μg / ml, 0.5μg / ml, 0.25μg / ml, 0.125μg / ml), and the monoclonal antibody was diluted by 10 times, 30 times, 90 times, 270 times, 810 times, 2430 times, 7290 times, 21870 times, 65610 times, 196830 times, 590490 times, 1771470 times, 5314410 times, 15943230 times, 47829690 times, and determined by indirect ELISA method. The logarithm of the antibody mass concentration was used as the abscissa and the corresponding absorbance value was used as the ordinate to draw the curve.

[0062] The affinity constant was calculated according to the following formula: Ka = (n-1) / [2(nAb'-Ab)]

[0063] In the formula, Ab' and Ab represent the antibody concentrations at OD = 1 / 2ODmax at two different concentrations of antigen dilution; n represents the dilution multiple between the corresponding antigen concentrations. At four concentrations, the average value is calculated after two-by-two calculations to make the results more reliable.

[0064] The affinity constant of the anti-fox skeletal muscle troponin monoclonal antibody was determined by indirect enzyme-linked immunosorbent assay. Figure 3 The affinity curve of monoclonal antibody sTn-3A4 is shown in Figure 1. The calculated affinity constant Ka is 3.16×10 6 L / mol.

[0065] (4) Identification by immunoblotting

[0066] The specific reaction of the prepared anti-fox skeletal muscle troponin monoclonal antibody sTn-3A4 with the target protein was identified by immunoblotting.

[0067] After the detection antigens of various species (skeletal muscle troponin detection antigens of pigs, cattle, sheep, chickens, ducks, and foxes) were subjected to Trcine-SDS-PAGE electrophoresis, the membrane was transferred: the gel was stripped, the concentrated gel and the gap gel were cut off, the marker and the target protein were retained, a piece of PVDF membrane of the same size as the gel was cut, and it was soaked in methanol for 5s, and 6 pieces of filter paper of the same size as the gel were cut, and the PVDF membrane, gel, and filter paper were placed in the membrane transfer buffer and soaked for 30min. On the cathode plate of the electrotransfer instrument, 3 layers of filter paper, gel, PVDF membrane, and 3 layers of filter paper were placed from bottom to top, and the clamps were placed in the electrotransfer mold, and the membrane transfer buffer was added, and the constant current was set to 92mA. After the transfer process was carried out for 80min, the PVDF membrane was placed in the blocking solution and kept overnight at 4℃. The membrane was washed with the washing buffer 3 times, 10min each time. The monoclonal antibody sTn-3A4 was diluted 1:2000 with the blocking solution, and the PVDF membrane was placed in it, and incubated with shaking at 37℃ for 1h. After washing the membrane three times, dilute the goat anti-mouse IgG secondary antibody with blocking solution at 1:3000, place the PVDF membrane in it, and incubate with shaking at 37°C for 1 hour. After washing the membrane three times, develop the color with an ultrasensitive color development kit and take pictures.

[0068] The results of immunoblotting showed that at 37kD, the monoclonal antibody skTn-3A4 reacted significantly with fox skeletal muscle troponin detector, and the band was clear.

[0069] (Refer to Figure 4 The immunoblot profile of the specific reaction of the monoclonal antibody sTn-3A4 with each test source, wherein lane 1 is the porcine skeletal muscle troponin test source; lane 2 is the bovine skeletal muscle troponin test source; lane 3 is the sheep skeletal muscle troponin test source; lane 4 is the chicken skeletal muscle troponin test source; lane 5 is the duck skeletal muscle troponin test source; lane 6 is the fox skeletal muscle troponin test source)

[0070] The reaction with pig and raccoon skeletal muscle troponin detectors showed distinguishable slight bands, while the reaction with sheep, chicken and duck skeletal muscle troponin detectors showed no obvious bands, and the reaction with cattle skeletal muscle troponin detectors showed no bands. This shows that skTn-3A4 can specifically bind to fox skeletal muscle troponin T subunit, and to a certain extent distinguish fox skeletal muscle troponin from skeletal muscle troponin of other species.

[0071] (5) Specificity determination

[0072] The cross-reactivity of monoclonal antibody skTn-3A4 with porcine, bovine, mutton, chicken and duck meat detectors was determined by indirect ELISA: the concentration of each species detector was diluted to 2 μg / ml, added to 96-well plates, 100 μL per well, and coated at 37°C for 1 hour; the washing solution (0.01 M containing 0.1% Tween-20) was used to wash the plate. Each well was washed 3 times with PBS and patted dry; the monoclonal antibody was diluted at 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600, 1:51200, 1:102400, 1:204800, 1:409600, and 1:819200, 100 μL was added to each well, incubated at 37°C for 45 min, washed, and patted dry; 100 μL of goat anti-mouse IgG secondary antibody (diluted 10,000 times) was added to each well, incubated at 37°C for 30 min, washed, and patted dry; 100 μL of color development solution was added to each well, and reacted at 37°C in the dark for 12 min; 50 μL of stop solution was added to each well, and the wavelength was set to 450 nm to measure the absorbance value. The cross-reactivity results of anti-fox skeletal muscle troponin monoclonal antibody skTn-3A4 with the detectors of various species are shown in Table 3.

[0073] Table 3 Cross-reaction results of monoclonal antibody skTn-3A4 with various species detectors

[0074]

[0075]

[0076] Example 3

[0077] The anti-fox skeletal muscle troponin monoclonal antibody skTn-3A4 of the present invention is used in an enzyme-linked immunosorbent assay kit. The detection principle of the kit of the present invention is a sandwich ELISA method.

[0078] The skeletal muscle troponin in the meat product to be tested was extracted according to the preparation method of the test antigen in Example 1 to prepare a test sample.

[0079] Label the anti-fox skeletal muscle troponin monoclonal antibody skTn-3A4 with horseradish peroxidase. Weigh 4 mg of horseradish peroxidase (antibody: enzyme = 2:1) according to the amount of monoclonal antibody used, dissolve in 0.5 ml of distilled water, add 0.5 ml of the newly prepared 0.06M sodium periodate aqueous solution, mix well, and place at 4°C for 30 minutes. Then add 0.5 ml of 0.16M ethylene glycol aqueous solution, place at room temperature for 30 minutes, and the enzyme solution will be activated. Take an appropriate amount of activated enzyme solution, add monoclonal antibody solution, mix well and adjust the pH to about 9 with 0.05M pH10 carbonate buffer, place at 4°C for 24 hours to allow it to fully act. Then add sodium borohydride, the amount is calculated according to 0.2 mg per milligram of enzyme, slowly invert and shake well, and place at 4°C for 2 hours. Then add an equal volume of saturated ammonium sulfate solution to the resulting enzyme conjugate mixture, and place at 4°C for 30 minutes. Then centrifuge at 3000rpm for 40 minutes. The obtained precipitate was dissolved in a small amount of 0.02M pH 7.4 phosphate buffer and dialyzed against the same buffer overnight to obtain the horseradish peroxidase-labeled anti-fox skeletal muscle troponin monoclonal antibody skTn-3A4 working solution.

[0080] Anti-porcine skeletal muscle troponin T monoclonal antibody skTnT-2E8 was coated on a microwell strip at a concentration of 2μg / ml, 100μl per well, and the strip was removed as needed and placed on an ELISA plate; washed 3 times, patted dry, and the sample to be tested was added at a concentration of 4μg / ml, 100μl per well, and incubated at 37℃ for 45min; washed 3 times, patted dry, and the anti-fox skeletal muscle troponin monoclonal antibody skTn-3A4 working solution labeled with horseradish peroxidase was added at a concentration of 0.2μg / ml, and incubated at 37℃ for 30min; washed 3 times, patted dry, and the color development solution A and the color development solution B were mixed at a ratio of 1:1, 100μL per well, and incubated at 25℃ in the dark for 15min; stop solution was added, 50μL per well, and after the reaction was stopped, the absorbance value was measured using an ELISA reader, and the wavelength was set to 450nm. The sample to be tested was quantitatively or qualitatively determined according to the standard curve.

[0081] Result determination: (a) Quantitative analysis: Calculate the average absorbance of fox skeletal muscle troponin detection antigen (standard substance) with different concentrations, and draw a standard curve with the absorbance as the ordinate and the concentration of fox skeletal muscle troponin detection antigen standard substance as the abscissa. Figure 5(b) Qualitative analysis: Compare the average absorbance value of the sample to be tested with the absorbance value of the standard sample to obtain the concentration range of fox skeletal muscle troponin in the sample to be tested.

[0082] The calculation principle of the minimum detection limit of this embodiment is: the lowest concentration corresponding to the value that is significantly different from the blank control value. Calculate the mean (m) and variance (σ) of 30 blank control values, and obtain the value of m+3σ, and the corresponding concentration is the minimum detection limit. The linear test range of this embodiment is 0.4μg / ml~12μg / mL.

[0083] The fox meat adulteration in beef was determined by the established sandwich ELISA method. The preparation method of the beef test sample adulterated with fox meat is as follows: According to the method for extracting the detection antigen in Example 1, the beef detection antigen and the fox detection antigen were extracted respectively, and the fox detection antigen was added to the beef detection antigen at three concentrations of 2μg / ml, 6μg / ml, and 10μg / ml. The test results of the test samples at these three adulteration concentrations are shown in Table 4.

[0084] Table 4 Results of determination of fox skeletal muscle troponin concentration in adulterated test samples

[0085]

[0086]

[0087] Example 4

[0088] The anti-fox skeletal muscle troponin monoclonal antibody of the invention is applied to detection by a colloidal gold chromatography test strip method.

[0089] Reaction principle: The sandwich ELISA method is used to perform semi-quantitative detection of fox skeletal muscle troponin. The fox skeletal muscle troponin present in the sample first binds to the antibody skTnT-2E8 labeled with colloidal gold particles during the process of moving up along the test strip, and reaches the T position (i.e., the detection line: coated with anti-fox skeletal muscle troponin monoclonal antibody sTn-3A4). The intensity of the color development of the detection line is positively correlated with the content of fox skeletal muscle troponin added to the sample. If there is no adulteration of fox meat in the sample, the protein in the sample and the antibody skTnT-2E8 labeled with colloidal gold cannot react with the antibody sTn-3A4 coated at the T position during their respective upward movement, and the T position of the test strip does not develop color.

[0090] Detection: Take out the anti-fox skeletal muscle troponin colloidal gold chromatography test strip, insert the sample end into the sample liquid to be tested, the insertion depth should not exceed the marked line, take out the test strip after about 15 seconds, place it horizontally, observe and determine the test result after 3 minutes, and the result will be invalid after 10 minutes.

[0091] Result determination: If a brown-red line is displayed at the corresponding quality control area C position (quality control line) on the coating membrane, and a brown-red line is displayed at the detection area T position (detection line), it means that the test result is positive, indicating that the sample to be tested contains fox skeletal muscle troponin; if a brown-red line is displayed at position C on the coating membrane, and no band is displayed at position T (detection line) in the detection area, it means that the result is negative, indicating that the sample to be tested does not contain fox skeletal muscle troponin; when the quality control area C does not show a brown-red band, the test will be deemed invalid regardless of whether the detection area T shows a brown-red band.

[0092] Example 5

[0093] The anti-fox skeletal muscle troponin monoclonal antibody skTn-3A4 of the present invention is applied to detection by an evanescent wave fluorescence immunosensor method.

[0094] Reaction principle: The evanescent wave fluorescence analysis principle is combined with the bioaffinity reaction principle, that is, the fluorescently labeled anti-fox skeletal muscle troponin monoclonal antibody skTn-3A4 reacts with the skeletal muscle troponin in the sample, and then combines with the anti-porcine skeletal muscle troponin monoclonal antibody skTnT-2E8 on the surface of the optical fiber biosensor. After the excitation light is introduced into the optical fiber probe, an evanescent wave is generated, which makes the fluorescent labeled antibody bound to the surface emit light. The fluorescence collected by the photodetector is processed and the results are automatically displayed.

[0095] The instrument includes: a flow injection system, a fiber optic biosensor, an optical path system, and a signal processing system. The flow injection system is used to transport buffer solutions, samples to be tested, and regeneration solutions. The surface of the fiber optic biosensor is modified with the anti-porcine skeletal muscle troponin monoclonal antibody skTnT-2E8 through an optical fiber probe to achieve specific detection of the drug. The fiber optic probe modification uses N, N′-disuccinimidyl carbonate (DSC) to replace glutaraldehyde, and the anti-porcine skeletal muscle troponin monoclonal antibody skTnT-2E8 is covalently fixed to the surface of the fiber optic biosensor. The optical path system uses an optical switch to control the excitation light to alternately enter the mode fiber at intervals of 1 second. After the mode fiber introduces the excitation light into the fiber optic probe, an evanescent wave is generated on the surface of the fiber, causing the fluorescent-labeled anti-fox skeletal muscle troponin monoclonal antibody skTn-3A4 bound to the test sample on the surface to emit light. The photodetector detects the collected fluorescence at intervals of 1 second, and the signal processing system processes the data and automatically displays the results.

[0096] Detection: Take 50 μL of fox skeletal muscle troponin solution of different concentrations or the sample to be tested and 50 μL of Cy5.5 fluorescently labeled anti-fox skeletal muscle troponin monoclonal antibody skTn-3A4 respectively and mix them in 0.5 mL brown centrifuge tubes. After shaking and mixing with an oscillator, stand in the dark and avoid light for 6 minutes. After the reaction, perform instrument detection. According to the pre-set program, first pass 10mM PBS buffer solution into the optical fiber reaction tank in the optical fiber biosensor to clean the surface optical fiber (30s) to restore the baseline to a stable state. After that, the system program automatically draws the mixed solutions of Cy5.5 fluorescently labeled anti-fox skeletal muscle troponin monoclonal antibody skTn-3A4 and different concentrations of fox skeletal muscle troponin solution or the mixed solutions of the sample to be tested into the optical fiber reaction tank. Then stabilize the reaction for 4 minutes, at which time the detection signal is observed to gradually rise and is recorded by the computer. Then, a regeneration solution (0.5% (pH 1.9) SDS solution) was pumped into the optical fiber reaction tank for 3 minutes to remove the fluorescent labeled antibodies that have been bound to the optical fiber surface. Finally, a 10mM PBS buffer solution was passed through for 1 minute to restore the pH value of the optical fiber surface, and the detection was completed.

[0097] Result determination: Taking the fluorescence signal (e0) of the blank sample (PBS) as the benchmark, the signal value (e) obtained after the reaction of different concentrations of fox skeletal muscle troponin solutions with Cy5.5 fluorescently labeled anti-fox skeletal muscle troponin monoclonal antibody skTn-3A4 was compared with it to obtain the standardized value (e / e0). The Logistic function was used for fitting to establish the functional relationship between the standardized value (e / e0) and the concentration of fox skeletal muscle troponin, which is the standard detection curve. By comparing the average signal value of the sample to be tested with the signal value of the standard, the concentration of fox skeletal muscle troponin in the sample to be tested can be obtained.

[0098] The beef test sample mixed with fox meat was extracted according to the method of extracting the test antigen in Example 1, and the fox test antigen was added to the beef test antigen at three concentrations of 2μg / ml, 6μg / ml, and 10μg / ml. The test results of the evanescent wave immunofluorescence technique for the test samples at these three adulteration concentrations are shown in Table 5.

[0099] Table 5 Detection results of fox meat adulteration test samples by evanescent wave immunofluorescence technique

[0100]

[0101] It can be seen from the above embodiments that the present invention provides a hybridoma cell line and an anti-fox skeletal muscle troponin monoclonal antibody and an application of the antibody produced by the hybridoma cell line. The deposited name of the hybridoma cell line is: hybridoma cell line skTn-3A4 of anti-fox skeletal muscle troponin monoclonal antibody, deposited at China Center for Type Culture Collection, deposited on October 10, 2023, and deposited with the number CCTCC NO: C2023302. The anti-fox skeletal muscle troponin monoclonal antibody skTn-3A4 provided by the present invention can be used to prepare an enzyme-linked immunosorbent assay kit, colloidal gold chromatography test strips, and evanescent wave fluorescence immunosensor for detecting fox skeletal muscle troponin, so as to achieve the purpose of quickly and sensitively detecting fox skeletal muscle troponin in food.

[0102] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A hybridoma cell line, characterized in that The deposited name of the hybridoma cell line is: hybridoma cell line skTn-3A4 of anti-fox skeletal muscle troponin monoclonal antibody, the deposited location is: China Center for Type Culture Collection, the deposited date is: October 10, 2023, and the deposited number is CCTCCNO: C2023302.

2. Use of the hybridoma cell line according to claim 1 in the preparation of anti-fox skeletal muscle troponin monoclonal antibodies.

3. An anti-fox skeletal muscle troponin monoclonal antibody, characterized in that: Produced by the hybridoma cell line skTn-3A4 according to claim 1.

4. Use of the anti-fox skeletal muscle troponin monoclonal antibody according to claim 3 in the preparation of a product for detecting fox skeletal muscle troponin.

5. The use according to claim 4, characterized in that: The products include enzyme-linked immunosorbent assay kits, colloidal gold chromatography test strips, and evanescent wave fluorescent immunosensors.

6. An enzyme-linked immunosorbent assay kit for detecting fox skeletal muscle troponin, characterized in that: The enzyme-linked immunosorbent assay kit comprises the anti-fox skeletal muscle troponin monoclonal antibody according to claim 3.

7. A colloidal gold chromatography test strip for detecting fox skeletal muscle troponin, characterized in that: The colloidal gold chromatography test strip comprises the anti-fox skeletal muscle troponin monoclonal antibody according to claim 3.

8. An evanescent wave fluorescence immunosensor for detecting fox skeletal muscle troponin, characterized in that: The evanescent wave fluorescent immunosensor comprises the anti-fox skeletal muscle troponin monoclonal antibody according to claim 3.

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