A hybridoma cell strain and the anti-fox skeletal muscle troponin monoclonal antibody and application of the antibody produced by the hybridoma cell strain

Abstract

The present application relates to the technical field of immunology and meat product adulteration meat source identification, and provides a hybridoma cell strain, an anti-fox skeletal muscle troponin monoclonal antibody produced by the hybridoma cell strain and application of the antibody, the preservation name of the hybridoma cell strain is: the hybridoma cell strain skTn-3A4 of the anti-fox skeletal muscle troponin monoclonal antibody, the preservation place is China Center for Type Culture Collection, the preservation date is October 10, 2023, and the preservation number is CCTCC NO: C2023302.The anti-fox skeletal muscle troponin monoclonal antibody skTn-3A4 provided by the present application can be used for preparing an enzyme-linked immunoassay kit, a colloidal gold chromatography test strip and an evanescent wave fluorescence immune sensor for detecting fox skeletal muscle troponin, so that the fox skeletal muscle troponin in food can be rapidly and sensitively detected.

Description

Technical Field

[0001] This invention relates to the fields of immunology and meat source identification technology for adulterated meat products, and particularly to a hybridoma cell line and the monoclonal antibody against fox skeletal muscle troponin produced therefrom, and the application of the antibody. Background Technology

[0002] In recent years, adulterated meat has been a persistent problem in the meat market, raising concerns about meat safety. Driven by high profits, some unscrupulous merchants use low-value meats like fox meat to impersonate lamb, horse meat to impersonate beef, and raccoon meat to impersonate lamb. Most alarmingly, these adulterates include rat, fox, raccoon, and mink meat, which may carry large amounts of bacteria, viruses, parasites, and other harmful substances because there are no strict quarantine procedures during the raising of fur-bearing animals. Therefore, meat certification methods and animal traceability analysis are increasingly important topics in the food industry. Meat composition, a qualitative measure used to distinguish and identify different animal species, is widely used in meat certification and quality control. Therefore, developing a method to characterize and quantify the origin of meat products is essential for authenticating their authenticity and controlling meat quality.

[0003] Enzyme-linked immunosorbent assay (ELISA) is one of the most specific and direct methods for detecting biomolecules in research and clinical practice. With advancements in scientific research, ELISA assays have been continuously optimized to improve their sensitivity, and now different types of ELISA can be used to detect a wide variety of biomarkers. Skeletal troponin is thermally stable and can serve as a marker protein for identifying components in raw and heat-treated meat. Skeletal troponin is a structural protein that regulates the contractile function of skeletal muscle and consists of three subunits: troponin T (37 kD), troponin I (24 kD), and troponin C (18 kD).

[0004] In this invention, fox skeletal muscle tissue was completely pulverized, cooked, and homogenized using a protein extraction method to obtain an immunogen. Monoclonal antibodies were then prepared by immunizing mice, and their immunological properties, such as titer, subtype, and affinity, were detected, analyzed, and evaluated. This research is of great significance for establishing a series of immunological methods for identifying components derived from fox meat. Summary of the Invention

[0005] The purpose of this invention is to provide a hybridoma cell line and the resulting anti-fox skeletal muscle troponin monoclonal antibody, as well as the application of the antibody. The provided anti-fox skeletal muscle troponin monoclonal antibody exhibits highly controllable and reproducible quality. Preliminary analysis and identification of the antibody's characteristics provide experimental basis and fundamental raw materials for the further establishment and application of a specific and sensitive immunoassay method for fox skeletal muscle troponin. It can be used to prepare enzyme-linked immunosorbent assay kits, colloidal gold chromatography test strips, and evanescent fluorescence immunosensors for detecting fox skeletal muscle troponin, achieving rapid and sensitive detection of fox meat adulteration in meat products.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

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

[0008] The present invention also provides the application of the hybridoma cell line described above in the preparation of monoclonal antibodies against fox skeletal muscle troponin.

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

[0010] The present invention also provides the application of the aforementioned anti-fox skeletal muscle troponin monoclonal antibody in the preparation of products for detecting fox skeletal muscle troponin.

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

[0012] The present invention also provides an enzyme-linked immunosorbent assay (ELISA) kit for detecting troponin in fox skeletal muscle, the ELISA kit comprising the aforementioned 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, the colloidal gold chromatography test strip comprising the aforementioned anti-fox skeletal muscle troponin monoclonal antibody.

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

[0015] This invention provides a hybridoma cell line, its generated anti-fox skeletal muscle troponin monoclonal antibody, and the application of the antibody. The hybridoma cell line is deposited at the China Center for Type Culture Collection (CCTCC) on October 10, 2023, with accession number CCTCC NO: C2023302. The anti-fox skeletal muscle troponin monoclonal antibody skTn-3A4 provided by this invention can be used to prepare enzyme-linked immunosorbent assay (ELISA) kits, colloidal gold chromatography test strips, and evanescent fluorescence immunosensors for the detection of fox skeletal muscle troponin, achieving rapid and sensitive detection of fox skeletal muscle troponin in food. Attached Figure Description

[0016] Figure 1 This is a gel electrophoresis image of the fox skeletal muscle troponin immunogen and detection antigen prepared in Example 1 of the present invention.

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

[0018] Figure 3 This is a diagram showing the subtype determination 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 assay of the anti-fox skeletal muscle troponin monoclonal antibody prepared in Example 2 of this invention;

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

[0021] Preservation Instructions

[0022] The hybridoma cell line skTn-3A4, containing a monoclonal antibody against fox skeletal muscle troponin, is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on October 10, 2023, with accession number CCTCCNO: C2023302. Detailed Implementation

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

[0024] The present invention also provides the application of the hybridoma cell line described above in the preparation of monoclonal antibodies against fox skeletal muscle troponin.

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

[0026] The present invention also provides the application of the aforementioned anti-fox skeletal muscle troponin monoclonal antibody in the preparation of products for detecting fox skeletal muscle troponin.

[0027] In this invention, the products include an enzyme-linked immunosorbent assay (ELISA) kit, a colloidal gold chromatography test strip, and an evanescent fluorescence immunosensor.

[0028] The present invention also provides an enzyme-linked immunosorbent assay (ELISA) kit for detecting troponin in fox skeletal muscle, the ELISA kit comprising the aforementioned 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, the colloidal gold chromatography test strip comprising the aforementioned anti-fox skeletal muscle troponin monoclonal antibody.

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

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

[0032] Example 1: Preparation of fox skeletal muscle troponin immunogen

[0033] (1) Preparation of immunoantigens

[0034] Take 10 g of fresh fox meat, remove fat and connective tissue, chop finely, add 20 ml of 0.15 mol / L NaCl solution, and homogenize for 1 h. Sonicate at 100 W for 5 min, then boil in a water bath for 20 min, followed by centrifugation at 6000 r / min for 30 min. Collect the supernatant and autoclave at 121 ℃ for 30 min. Filter with Whatman No. 1 filter paper, add 90% ethanol (1:3.74 v / v) to the filtrate, centrifuge at 8000 r / min for 20 min, discard the supernatant, and dissolve the precipitate in 0.01 mol / L PBS buffer. The resulting fox skeletal muscle extract is the fox skeletal muscle troponin immunogen. Measure the protein concentration and store at -80 ℃ for later use.

[0035] (2) Preparation of detection antigen

[0036] Take a quantity of fresh fox meat, cut it into pieces, and store it at -80 ℃. Thaw it completely at room temperature for 3 hours before use. Weigh 10 g of each type of meat, chop it, and place it in a centrifuge tube. After boiling in a water bath for 15 minutes, immediately cool it with ice water. Add 20 ml of 0.01 mol / L PBS buffer containing 0.5 mol / L NaCl to the centrifuge tube and homogenize for 1 hour. Centrifuge at 6000 r / min for 30 minutes, discard the precipitate, and collect the supernatant to obtain the skeletal muscle extracts of each species, which are their respective skeletal muscle troponin assays. Measure the protein concentration and store at -80 ℃ for later use.

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

[0038] Tricine-SDS-PAGE Identification

[0039] Tricine-SDS-PAGE was used to identify the immunogenic and detection antigens of fox skeletal muscle troponin. Following standard Tricine-SDS-PAGE procedures, a 4% stacking gel, a 10% gap gel, and a 15% separating gel were prepared. Electrophoresis was performed at 80V until the protein bands entered the separating gel, then the voltage was adjusted to 120V and electrophoresis continued for 2 hours. The gels were then stained and destained.

[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 immunoantigen and detection antigen are shown. Lane 1 is the fox skeletal muscle troponin detection antigen, and lane 2 is the fox skeletal muscle troponin immunoantigen. Two clear and dark bands were observed after electrophoresis of the fox skeletal muscle troponin immunoantigen, located at 37 kD and 24 kD respectively, consistent with the sizes of the sTnT and sTnI subunits described in the literature. One dominant band was observed after electrophoresis of the fox skeletal muscle troponin detection antigen, located at 37 kD, which is the sTnT subunit. A band was also observed at 24 kD, but less dark than the band at 37 kD, which is the sTnI subunit.

[0041] Example 2

[0042] 1. Preparation of monoclonal antibodies against fox skeletal muscle troponin

[0043] (1) Animal immunization

[0044] Female Balb / c mice aged 6-8 weeks were immunized with the fox skeletal muscle troponin immunogen prepared in Example 1. The mice were immunized once every 2 weeks. The immunization procedure is shown in Table 1. After three immunizations, blood was collected from the tail to determine the antiserum titer. The mice with the best results were selected for booster immunization as fusion mice.

[0045] Table 1 Immune Flowchart

[0046]

[0047] (2) Cell fusion

[0048] The selected fusion mice in step (1) were euthanized by removing the eyeballs and bleeding. After cervical dislocation, the spleen was removed under sterile conditions to prepare spleen cells. These cells were then fused with SP2 / 0 cells at a ratio of 5:1 using PEG. The fused cell suspension was added to a 96-well plate that had been coated 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 following day, and the medium was changed to HT medium on day 10 post-fusion. Three days after the medium change, the cell supernatant was examined using an indirect ELISA method. Wells with positive results were screened, and cell subcloning was performed using limiting dilution to obtain a hybridoma cell line with stable antibody secretion. This line was deposited at the China Center for Type Culture Collection (CCTCC) and named skTn-3A4, an anti-fox skeletal muscle troponin hybridoma cell line, with accession number CCTCC NO: C2023302.

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

[0052] Ascites was induced in mice using an in vivo method. Healthy female Balb / c mice were injected with 0.5 mL of paraffin oil each. After 7 days, 10 cloned positive hybridoma cells were added. 6 / mL, 800μL was injected intraperitoneally into each mouse, and ascites was collected after 12 days. The ascites was purified by the caprylic acid-ammonium sulfate method to obtain the monoclonal antibody. The concentration was detected and stored at -20 ℃ for later use, thus obtaining the purified anti-fox skeletal muscle troponin monoclonal antibody.

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

[0054] (1) Valence determination

[0055] The titer of monoclonal antibodies was determined using an indirect ELISA method: the detector was diluted to 2 μg / ml and added to 100 μL per well of a 96-well plate, and incubated at 37 ℃ for 1 h; each well was washed three times with washing buffer (0.1% Tween-20 in 0.01 M PBS) and patted dry; the monoclonal antibody was serially 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, and 100 μL was added to each well, incubated at 37 ℃ 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, and incubated at 37 ℃ for 30 min. After washing and patting dry, add 100 μL of chromogenic solution to each well and incubate at 37 ℃ in the dark for 12 min. Add 50 μL of stop solution to each well, set the wavelength to 450 nm, and measure the absorbance. The absorbance value of the negative control well is recorded as A0, and the absorbance value of each measurement well is recorded as A... t , with A t The dilution ratio corresponding to A0 ≥ 2.1 is used as the result of the titer test. The titer of the anti-fox skeletal muscle troponin monoclonal antibody skTn-3A4 is 1:1 × 10⁻⁶. 5 See Table 2.

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

[0057]

[0058] (2) Subtype determination

[0059] Isotype determination was performed using a murine monoclonal antibody subtype identification kit purchased from Sigma. The anti-fox skeletal muscle troponin monoclonal antibody secreted by hybridoma cells showed significant differences in color development with different subtypes of secondary antibodies. The highest A450nm value was observed with IgM secondary antibody, weak color development with IgG1 secondary antibody, and almost no color development with IgG2a, IgG2b, IgG3, and IgA secondary antibodies. Therefore, the antibody type secreted by these hybridoma cells is predominantly IgM. The results are shown in [Figure number missing]. Figure 2 The subtype detection results of the monoclonal antibody skTn-3A4 show that the subtype of the anti-fox skeletal muscle troponin monoclonal antibody of this invention is IgM.

[0060] (3) Affinity test

[0061] The detection base was coated with four concentration gradients (1 μg / ml, 0.5 μg / ml, 0.25 μg / ml, and 0.125 μg / ml). Monoclonal antibodies were serially diluted 10-fold, 30-fold, 90-fold, 270-fold, 810-fold, 2430-fold, 7290-fold, 21870-fold, 65610-fold, 196830-fold, 590490-fold, 1771470-fold, 5314410-fold, 15943230-fold, and 47829690-fold, and the results were measured using an indirect ELISA method. A curve was plotted with the logarithm of antibody concentration on the x-axis and the corresponding absorbance value on the y-axis.

[0062] The affinity constant can be calculated using the following formula: Ka =( n -1) / [2( nAb' - Ab )]

[0063] In the formula, Ab' and Ab represent two different concentrations of antigen at different concentrations in the dilution. OD =1 / 2 ODmax The corresponding antibody concentration is given by n; n represents the dilution factor between the corresponding antigen concentrations. The values ​​are calculated pairwise at the four concentrations, and the average is taken to make the results more reliable.

[0064] The affinity constant of the anti-fox skeletal muscle troponin monoclonal antibody was determined using an indirect enzyme-linked immunosorbent assay (ELISA). The results are as follows: Figure 3 The affinity curve of the monoclonal antibody skTn-3A4 is shown in the figure. The calculated affinity constant Ka is 3.16 × 10⁻⁶. 6 L / mol.

[0065] (4) Identification by immunoblotting

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

[0067] After Trcine-SDS-PAGE electrophoresis, the detection antigens for each species (swine, cattle, sheep, chicken, duck, and fox skeletal muscle troponin detection antigens) were transferred to membranes: the gel was peeled off, the stacking gel and gap gel were removed, and the marker and target protein were retained. A PVDF membrane of the same size as the gel was cut and soaked in methanol for 5 s. Six pieces of filter paper of the same size as the gel were cut. The PVDF membrane, gel, and filter paper were placed in membrane transfer buffer and soaked for 30 min. On the cathode plate of the electroporator, three layers of filter paper, gel, PVDF membrane, and three layers of filter paper were placed from bottom to top, clamped, and placed in the electroporation mold. Membrane transfer buffer was added, and a constant current of 92 mA was set. After the transfer process lasted for 80 min, the PVDF membrane was placed in blocking buffer and incubated overnight at 4 °C. The membrane was washed three times with washing buffer for 10 min each time. The monoclonal antibody skTn-3A4 was diluted 1:2000 with blocking buffer, and the PVDF membrane was placed in it and incubated at 37 °C with shaking for 1 h. After washing the membrane three times, goat anti-mouse IgG secondary antibody was diluted 1:3000 with blocking buffer, and the PVDF membrane was placed in it and incubated at 37°C with shaking for 1 h. After washing the membrane three times, the membrane was developed using an ultrasensitive colorimetric kit and photographed.

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

[0069] (refer to Figure 4 Immunoblot patterns of the specific reactions of the monoclonal antibody skTn-3A4 with each detector were shown, where lane 1 is the porcine skeletal muscle troponin detector; lane 2 is the bovine skeletal muscle troponin detector; lane 3 is the sheep skeletal muscle troponin detector; lane 4 is the chicken skeletal muscle troponin detector; lane 5 is the duck skeletal muscle troponin detector; and lane 6 is the fox skeletal muscle troponin detector.

[0070] The skTn-3A4 assay showed a distinguishable, slight band with the troponin receptors from pigs and raccoons, but no obvious band with the troponin receptors from sheep, chickens, and ducks, and no band with the troponin receptor from bovines. This indicates that skTn-3A4 can specifically bind to the T subunit of fox troponin, and to some extent distinguish fox troponin from troponin in other species.

[0071] (5) Specificity assay

[0072] The cross-reactivity of the monoclonal antibody skTn-3A4 with the detection sources of pig, cattle, sheep, chicken, and duck meat was determined using an indirect ELISA method: The concentration of the detection source for each species was diluted to 2 μg / ml, and 100 μL was added to each well of a 96-well plate. The plates were incubated at 37 ℃ for 1 h. Each well was washed three times with washing buffer (0.01 M PBS containing 0.1% Tween-20) and patted dry. The monoclonal antibody was serially 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, and 100 μL was added to each well. The plates were incubated at 37 ℃ for 45 min, washed, and patted dry. 100 μL of the antibody was added to each well. Incubate μL of goat anti-mouse IgG secondary antibody (diluted 10,000 times) at 37 ℃ for 30 min, wash, and blot dry. Add 100 μL of chromogenic solution to each well and incubate at 37 ℃ in the dark for 12 min. Add 50 μL of stop solution to each well, set the wavelength to 450 nm, and measure the absorbance. The cross-reactivity results of the anti-fox skeletal muscle troponin monoclonal antibody skTn-3A4 with the detectants of each species are shown in Table 3.

[0073] Table 3. Cross-reactivity results of monoclonal antibody skTn-3A4 with the detection sources of various species.

[0074]

[0075] Example 3

[0076] The anti-fox skeletal muscle troponin monoclonal antibody skTn-3A4 of the present invention is used in an enzyme-linked immunosorbent assay (ELISA) kit. The detection principle of the kit in the present invention is the sandwich ELISA method.

[0077] Skeletal muscle troponin was extracted from the meat product to be tested according to the preparation method of the antigen in Example 1 to prepare the test sample.

[0078] Horseradish peroxidase was used to label the anti-fox skeletal muscle troponin monoclonal antibody skTn-3A4. 4 mg of horseradish peroxidase (antibody:enzyme = 2:1) was weighed and dissolved in 0.5 ml of distilled water. 0.5 ml of freshly prepared 0.06 M sodium periodate aqueous solution was added, mixed well, and incubated at 4 °C for 30 min. Then, 0.5 ml of 0.16 M ethylene glycol aqueous solution was added, and the mixture was incubated at room temperature for 30 min to activate the enzyme. An appropriate amount of the activated enzyme solution was taken, and the monoclonal antibody solution was added. The mixture was mixed well and the pH was adjusted to approximately 9 with 0.05 M pH 10 carbonate buffer. The mixture was incubated at 4 °C for 24 h to allow for complete activation. Then, sodium borohydride was added at a dosage of 0.2 mg per mg of enzyme, and the mixture was gently inverted and incubated at 4 °C for 2 h. Finally, an equal volume of saturated ammonium sulfate solution was added to the resulting enzyme conjugate mixture, and the mixture was incubated at 4 °C for 30 min. The mixture was then centrifuged at 3000 rpm for 40 min. The resulting precipitate was dissolved in a small amount of 0.02M pH 7.4 phosphate buffer and dialyzed overnight with the same buffer to obtain the working solution of horseradish peroxidase-labeled anti-fox skeletal muscle troponin monoclonal antibody skTn-3A4.

[0079] Anti-porcine skeletal muscle troponin T monoclonal antibody skTnT-2E8 was coated onto microplate strips at a concentration of 2 μg / ml, 100 μl per well. Strips were removed as needed and placed on an ELISA plate. After washing three times and patting dry, 100 μl of the sample to be tested was added at a concentration of 4 μg / ml per well, and incubated at 37 ℃ for 45 min. After washing three times and patting dry, working solution of horseradish peroxidase-labeled anti-fox skeletal muscle troponin T monoclonal antibody skTn-3A4 was added at a concentration of 0.2 μg / ml, and incubated at 37 ℃ for 30 min. After washing three times and patting dry, chromogenic solution A and chromogenic solution B were mixed at a 1:1 ratio, 100 μl per well, and incubated at 25 ℃ in the dark for 15 min. Stop solution was added at 50 μl per well to terminate the reaction. The absorbance was measured using an ELISA reader at a wavelength of 450 nm. Quantitative or qualitative determination of the sample was performed according to the standard curve.

[0080] Results determination: (a) Quantitative analysis: Calculate the average absorbance value of different concentrations of fox skeletal muscle troponin detection antigen (standard), and plot a standard curve with absorbance value on the ordinate and concentration of fox skeletal muscle troponin detection antigen standard on the abscissa. Figure 5(a) Standard curve for sandwich ELISA detection of fox skeletal muscle troponin. The sample is diluted at different factors, and the absorbance value of the sample is substituted into the standard curve to calculate the concentration of fox skeletal muscle troponin in the sample. Multiplying this by the dilution factor gives the actual content of fox skeletal muscle troponin in the sample. (b) Qualitative analysis: By comparing the average absorbance value of the sample with the absorbance value of the standard, the concentration range of fox skeletal muscle troponin in the sample can be determined.

[0081] The principle for calculating the limit of detection (LOD) in this embodiment is: the lowest concentration corresponding to a value that is significantly different from the blank control value. The mean (m) and variance (σ) of 30 blank control values ​​are calculated, and the concentration corresponding to the value of m+3σ is the LOD. The linear testing range of this embodiment is 0.4 μg / ml to 12 μg / mL.

[0082] The established sandwich ELISA method was used to determine the adulteration of beef with fox meat. The preparation method for beef test samples adulterated with fox meat was as follows: Following the antigen extraction method in Example 1, beef and fox detection antigens were extracted separately. The fox detection antigen was then adulterated with the beef detection antigen at three concentrations: 2 μg / ml, 6 μg / ml, and 8 μg / ml. The test results for these three adulteration concentrations are shown in Table 4.

[0083] Table 4. Results of troponin concentration determination in fox skeletal muscle of adulterated test samples

[0084]

[0085] Example 4

[0086] The anti-fox skeletal muscle troponin monoclonal antibody of the present invention was applied to the detection method of colloidal gold chromatography test strip.

[0087] Reaction principle: A sandwich ELISA method is used for semi-quantitative detection of fox skeletal muscle troponin. As the fox skeletal muscle troponin in the sample moves upwards along the test strip, it first binds to the colloidal gold-labeled antibody skTnT-2E8, reaching the T position (i.e., the test line: coated with the anti-fox skeletal muscle troponin monoclonal antibody skTn-3A4). The intensity of the test line's color development is positively correlated with the amount of fox skeletal muscle troponin adulterated in the sample. If the sample is not adulterated with fox meat, the protein in the sample and the colloidal gold-labeled antibody skTnT-2E8 cannot react with the antibody skTn-3A4 coated at the T position during their upward movement, and the T position of the test strip will not show color.

[0088] Test: Take out the anti-fox skeletal muscle troponin colloidal gold chromatography test strip, insert the sample end into the sample solution to be tested, and insert it to a depth not exceeding the marked line. After about 15 seconds, take out the test strip, place it horizontally, and observe and judge the test results after 3 minutes. The results are invalid after 10 minutes.

[0089] Result Interpretation: A positive result indicates the presence of fox skeletal muscle troponin in the sample solution if a brown-red line appears at position C (control line) on the coated membrane and a brown-red line appears at position T (detection line). A negative result indicates the absence of fox skeletal muscle troponin in the sample solution if a brown-red line appears at position C and no band appears at position T (detection line). If no brown-red band appears at control line C, the test is considered invalid regardless of whether a brown-red band appears at position T.

[0090] Example 5

[0091] The anti-fox skeletal muscle troponin monoclonal antibody skTn-3A4 of the present invention was applied to the evanescent wave fluorescence immunosensor method for detection.

[0092] Reaction Principle: The evanescent wave fluorescence analysis principle is combined with the bioaffinity reaction principle. Specifically, a fluorescently labeled anti-fox skeletal muscle troponin monoclonal antibody (skTn-3A4) reacts with skeletal muscle troponin in the sample. Then, an anti-porcine skeletal muscle troponin monoclonal antibody (skTnT-2E8) on the surface of the fiber optic biosensor binds to this antibody, generating an evanescent wave that causes the bound fluorescently labeled antibody to glow. The fluorescence collected by the photodetector is processed, and the results are automatically displayed.

[0093] The instrument comprises a flow sample delivery system, a fiber optic biosensor, an optical path system, and a signal processing system. The flow sample delivery system delivers buffer solution, the test sample, and a regeneration solution. The fiber optic biosensor achieves specific detection of the anti-porcine skeletal muscle troponin monoclonal antibody skTnT-2E8 by modifying the surface of the fiber optic probe. The fiber optic probe modification utilizes N,N′-disuccinimidyl carbonate (DSC) to replace glutaraldehyde, covalently immobilizing the anti-porcine skeletal muscle troponin monoclonal antibody skTnT-2E8 onto 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 1-second intervals. After the mode fiber introduces the excitation light into the fiber optic probe, it generates an evanescent wave on the fiber surface, causing the fluorescently labeled anti-fox skeletal muscle troponin monoclonal antibody skTn-3A4 bound to the test sample to emit light. A photodetector detects the collected fluorescence at 1-second intervals, and the signal processing system processes the data and automatically displays the results.

[0094] Detection: 50 μL of fox skeletal muscle troponin solutions of different concentrations or the test sample were mixed with 50 μL of Cy5.5 fluorescently labeled anti-fox skeletal muscle troponin monoclonal antibody skTn-3A4 in 0.5 mL brown centrifuge tubes. After mixing with a shaker, the mixtures were incubated in the dark for 6 min. Instrument detection was performed after the reaction. Following the pre-set program, 10 mM PBS buffer solution was first passed through the fiber optic reaction chamber of the fiber optic biosensor to clean the surface of the fiber optic fibers (30 s) to stabilize the baseline. Then, the system automatically aspirated the mixtures of Cy5.5 fluorescently labeled anti-fox skeletal muscle troponin monoclonal antibody skTn-3A4 with different concentrations of fox skeletal muscle troponin solutions or the test sample mixtures into the fiber optic reaction chamber. The reaction was then stabilized for 4 min, at which point the detection signal gradually increased and was recorded by the computer. Next, a regeneration solution (0.5% (pH 1.9) SDS solution) was pumped into the fiber optic reaction vessel for 3 min to regenerate and remove the fluorescently labeled antibody that had bound to the fiber surface. Finally, 10 mM PBS buffer solution was purged for 1 min to restore the pH value of the fiber surface, thus completing the detection process.

[0095] Results Interpretation: Using the fluorescence signal (e0) of the blank sample (PBS) as a benchmark, the signal values ​​(e) obtained after reacting different concentrations of fox skeletal muscle troponin solutions with Cy5.5 fluorescently labeled anti-fox skeletal muscle troponin monoclonal antibody skTn-3A4 were compared with the PBS to obtain the standardized value (e / e0). A logistic function was used to fit the curve, establishing a 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 test sample with the signal value of the standard, the concentration of fox skeletal muscle troponin in the test sample can be determined.

[0096] Beef samples adulterated with fox meat were tested using the antigen extraction method described in Example 1. Beef and fox antigens were extracted separately, and the fox antigen was adulterated with the beef antigen at three concentrations: 2 μg / ml, 6 μg / ml, and 10 μg / ml. The results of evanescent wave immunofluorescence assays for these three adulteration concentrations are shown in Table 5.

[0097] Table 5. Detection results of fox meat adulteration test samples by evanescent wave immunofluorescence technique.

[0098]

[0099] As can be seen from the above embodiments, the present invention provides a hybridoma cell line and the resulting anti-fox skeletal muscle troponin monoclonal antibody and its applications. The hybridoma cell line is deposited under the name skTn-3A4, a hybridoma cell line containing an anti-fox skeletal muscle troponin monoclonal antibody. The deposit location is the China Center for Type Culture Collection (CCTCC), the deposit date is October 10, 2023, and the accession number is CCTCC NO: C2023302. The anti-fox skeletal muscle troponin monoclonal antibody skTn-3A4 provided by the present invention can be used to prepare enzyme-linked immunosorbent assay (ELISA) kits, colloidal gold chromatography test strips, and evanescent fluorescence immunosensors for detecting fox skeletal muscle troponin, to achieve rapid and sensitive detection of fox skeletal muscle troponin in food.

[0100] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A hybridoma cell line, characterized in that, The hybridoma cell line is named skTn-3A4, a hybridoma cell line containing a monoclonal antibody against fox skeletal muscle troponin. It is deposited at the China Center for Type Culture Collection (CCTCC) on October 10, 2023, with accession number CCTCC NO: C2023302.

2. The use of the hybridoma cell line according to claim 1 in the preparation of a monoclonal antibody against fox skeletal muscle troponin.

3. A monoclonal antibody against fox skeletal muscle troponin, characterized in that, Produced from the hybridoma cell line skTn-3A4 as described in claim 1.

4. The use of the anti-fox skeletal muscle troponin monoclonal antibody according to claim 3 in the preparation of products for detecting fox skeletal muscle troponin.

5. The application according to claim 4, characterized in that, The products include enzyme-linked immunosorbent assay (ELISA) kits, colloidal gold chromatography test strips, and evanescent fluorescence immunosensors.

6. An enzyme-linked immunosorbent assay (ELISA) kit for detecting troponin in fox skeletal muscle, characterized in that, The enzyme-linked immunosorbent assay kit includes the anti-fox skeletal muscle troponin monoclonal antibody as described in claim 3.

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

8. An evanescent fluorescence immunosensor for detecting troponin in fox skeletal muscle, characterized in that, The evanescent fluorescence immunosensor includes the anti-fox skeletal muscle troponin monoclonal antibody as described in claim 3.

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