Silvermectin monoclonal antibody, hybridoma cell strain secreting monoclonal antibody and application of hybridoma cell strain

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a lectin monoclonal antibody, a hybridoma cell strain secreting the monoclonal antibody and application. The monoclonal antibody secreted by the hybridoma cell strain ALS provided by the invention has strong affinity (7.26 * 10 < 8 > L / mol), has good specificity and detection sensitivity (IC50 value is 33.5 [mu] g / L) to the lecigrin, can realize detection of the lecigrin, provides a raw material for immunodetection of the lecigrin in food, and has practical application value.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and in particular relates to a graminic acid monoclonal antibody and a hybridoma cell line secreting the monoclonal antibody and applications thereof. Background Art

[0002] Altenusin (ALS), a toxic secondary metabolite of the fungus Alternaria alternata, is widely found in food and feed. Ingestion of contaminated food can lead to poisoning, illness, and even cancer. Therefore, establishing a rapid and effective method for detecting ALS is crucial for food safety risk prevention and control.

[0003] Currently, ALS detection methods include instrumental analysis and immunoassays. Instrumental analysis methods, such as high-performance liquid chromatography (HPLC) and HPLC-tandem mass spectrometry (HPLC-MS / MS), are not suitable for on-site testing due to their tedious sample pretreatment, time-consuming nature, and high operator skill requirements. Immunoassays offer low cost, high throughput, high sensitivity, and reduced time, making them suitable for rapid, on-site screening of large numbers of samples. Therefore, immunoassays are of great significance for the detection of galactosidase.

[0004] Enzyme-linked immunosorbent assay (ELISA) is a rapid and portable immunological test method with high tolerance to sample matrices and ease of use, making it suitable for rapid on-site testing of large numbers of samples. Screening for highly specific monoclonal antibodies is a crucial prerequisite for establishing efficient immunological testing methods. Summary of the Invention

[0005] The purpose of the present invention is to provide a monoclonal antibody against galactin and a hybridoma cell line secreting the monoclonal antibody and its application, which can specifically detect galactin and have high detection sensitivity for galactin, and establish an immunological detection method for galactin.

[0006] In order to achieve the above object, the present invention provides a hybridoma cell line ALS that secretes a monoclonal antibody against galactosidase, with the deposit number being CGMCC No.46355.

[0007] Preferably, the hybridoma cell line ALS is obtained by fusing mouse spleen cells and mouse myeloma cells; the mouse spleen cells are mouse spleen cells obtained by immunizing mice with a myelomonocytic immunogen;

[0008] The microgliadin immunogen is obtained by coupling 2-(benzo[d][1,3]dioxa-5-yl)benzoic acid with a carrier protein; the carrier protein is bovine serum albumin.

[0009] The present invention also provides a graminic acid monoclonal antibody, which is secreted by the hybridoma cell line ALS described in the above technical solution.

[0010] The present invention also provides a method for preparing the monoclonal antibody of glaucomatin described in the above technical solution, comprising the following steps: culturing the hybridoma cell line ALS and collecting the culture supernatant; the culture supernatant contains the monoclonal antibody of glaucomatin.

[0011] The present invention also provides the use of the hybridoma cell line ALS described in the above technical solution, the graminic acid monoclonal antibody described in the above technical solution, or the graminic acid monoclonal antibody prepared by the preparation method described in the above technical solution in preparing graminic acid inhibitors and / or graminic acid immunoaffinity columns.

[0012] The present invention also provides a kit, which includes the hybridoma cell line ALS described in the above technical solution or the graminic acid monoclonal antibody described in the above technical solution or the graminic acid monoclonal antibody prepared by the preparation method described in the above technical solution.

[0013] Preferably, the kit further comprises a coating source; the coating source is obtained by coupling 3-hydroxybiphenyl-2-carboxylic acid with a carrier protein; and the carrier protein is ovalbumin.

[0014] Preferably, the kit further comprises one or more of carbonate buffer, phosphate buffer, PBST, a tracer-labeled detection antibody, and TMB color development solution.

[0015] The present invention also provides the use of the hybridoma cell line ALS described in the above technical solution, the galactin monoclonal antibody described in the above technical solution, the galactin monoclonal antibody prepared by the preparation method described in the above technical solution, or the kit described in the above technical solution in galactin immunoassay.

[0016] The present invention also provides a method for immunoassay of galactosidase, comprising the following steps: incubating a test sample, a coating source and a galactosidase monoclonal antibody to form a complex; incubating the complex with a detection antibody labeled with a tracer to detect a tracer signal;

[0017] The coating is obtained by coupling 3-hydroxybiphenyl-2-carboxylic acid with a carrier protein; the carrier protein is ovalbumin;

[0018] The galactin monoclonal antibody is the galactin monoclonal antibody secreted by the hybridoma cell line ALS described in the above technical solution, or the galactin monoclonal antibody described in the above technical solution, or the galactin monoclonal antibody prepared by the preparation method described in the above technical solution.

[0019] Beneficial effects:

[0020] The present invention provides a hybridoma cell line ALS that secretes a monoclonal antibody against galactin, with a deposit number of CGMCC No. 46355. The monoclonal antibody secreted by the hybridoma cell line ALS provided by the present invention has a strong affinity (7.26×10 8 L / mol), with good specificity and detection sensitivity for galactosidase (IC 50 The value is 33.5μg / L), which can realize the detection of streptomycin and provide raw materials for the immunoassay of streptomycin in food, and has practical application value.

[0021] Biological deposit information

[0022] The hybridoma cell line ALS, classified and named as hybridoma cell line, was deposited on April 25, 2025 at the General Microbiology Center of China Culture Collection Administration, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, with the deposit number CGMCC No. 46355. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0024] Figure 1 The results are for the subtype identification of the monoclonal antibody against galactin secreted by the hybridoma cell line ALS;

[0025] Figure 2 The results are the affinity determination results of the monoclonal antibody against galactin secreted by the hybridoma cell line ALS;

[0026] Figure 3 This is the standard curve of the inhibition of galactin by the monoclonal antibody against galactin secreted by the hybridoma cell line ALS. DETAILED DESCRIPTION

[0027] The present invention provides a hybridoma cell line ALS secreting a gram-negative monoclonal antibody, with the deposit number being CGMCC No. 46355.

[0028] In one embodiment, the hybridoma cell line ALS described herein is obtained by fusing mouse spleen cells and mouse myeloma cells; the mouse spleen cells are obtained by immunizing mice with a sclerotin immunogen; the sclerotin immunogen is obtained by coupling 2-(benzo[d][1,3]diox-5-yl)benzoic acid to a carrier protein; the carrier protein is bovine serum albumin. The present invention has no strict requirements on the coupling method of 2-(benzo[d][1,3]diox-5-yl)benzoic acid to the carrier protein; the conventional carbodiimide method in the art is employed.

[0029] As an embodiment, the number of times mice are immunized with the sclerotin immunogen according to the present invention is 7 times, consisting of a first immunization, 5 booster immunizations, and a sprint immunization. As an embodiment, during the first immunization of the present invention, the sclerotin immunogen and Freund's complete adjuvant are mixed in a volume ratio of 1:1, emulsified to obtain an injection, and the mouse is subcutaneously injected through the back of the neck at a dose of 100 μg of sclerotin immunogen / mouse. As an embodiment, during the booster immunization of the present invention, the sclerotin immunogen and Freund's complete adjuvant are mixed in a volume ratio of 1:1, emulsified to obtain an injection, and the mouse is subcutaneously injected through the back of the neck at a dose of 50 μg of sclerotin immunogen / mouse. As an embodiment, the first immunization and the first booster immunization of the present invention are separated by one month, and multiple booster immunizations are separated by 21 days. As an embodiment, during the sprint immunization of the present invention, the sclerotin immunogen is diluted with physiological saline to obtain an injection, and the mouse is subcutaneously injected through the back of the neck at a dose of 25 μg of sclerotin immunogen / mouse. As an embodiment, the interval between the last booster immunization and the sprint immunization of the present invention is 21 days.

[0030] The present invention also provides a graminic acid monoclonal antibody, which is secreted by the hybridoma cell line ALS described in the above technical solution.

[0031] The present invention also provides a method for preparing the monoclonal antibody of glaucomatin described in the above technical solution, comprising the following steps: culturing the hybridoma cell line ALS and collecting the culture supernatant; the culture supernatant contains the monoclonal antibody of glaucomatin.

[0032] The present invention has no strict requirements on the culture method of the hybridoma cell line ALS, and conventional steps in the art can be used.

[0033] The hybridoma cell line ALS provided by the present invention can secrete the monoclonal antibody of galactin with strong affinity (7.26×10 8 L / mol), with good specificity and detection sensitivity for galactosidase (IC 50Therefore, the use of the hybridoma cell line ALS described in the above technical solution of the present invention, the graminic acid monoclonal antibody described in the above technical solution, or the graminic acid monoclonal antibody prepared by the preparation method described in the above technical solution in the preparation of graminic acid inhibitors and / or graminic acid immunoaffinity columns also falls within the scope of protection of the present invention.

[0034] The present invention also provides a kit, which includes the hybridoma cell line ALS described in the above technical solution or the graminic acid monoclonal antibody described in the above technical solution or the graminic acid monoclonal antibody prepared by the preparation method described in the above technical solution.

[0035] In one embodiment, the kit of the present invention further comprises a coating agent; the coating agent is obtained by coupling 3-hydroxybiphenyl-2-carboxylic acid to a carrier protein; the carrier protein is ovalbumin. In one embodiment, the kit of the present invention further comprises one or more of carbonate buffer, phosphate buffer, PBST, a tracer-labeled detection antibody, and TMB color development solution. In one embodiment, the tracer-labeled detection antibody of the present invention comprises an HRP-labeled detection antibody; in another embodiment, the tracer-labeled detection antibody of the present invention comprises an HRP-labeled goat anti-mouse IgG.

[0036] The present invention also provides the use of the hybridoma cell line ALS described in the above technical solution, the galactin monoclonal antibody described in the above technical solution, the galactin monoclonal antibody prepared by the preparation method described in the above technical solution, or the kit described in the above technical solution in galactin immunoassay.

[0037] As an embodiment, the immunoassay for glucocortin of the present invention includes the immunoassay for glucocortin in food.

[0038] The present invention also provides a method for immunoassay of galactosidase, comprising the following steps: incubating a test sample, a coating source and a galactosidase monoclonal antibody to form a complex; incubating the complex with a detection antibody labeled with a tracer to detect a tracer signal;

[0039] The coating is obtained by coupling 3-hydroxybiphenyl-2-carboxylic acid with a carrier protein; the carrier protein is ovalbumin;

[0040] The galactin monoclonal antibody is the galactin monoclonal antibody secreted by the hybridoma cell line ALS described in the above technical solution, or the galactin monoclonal antibody described in the above technical solution, or the galactin monoclonal antibody prepared by the preparation method described in the above technical solution.

[0041] In one embodiment, the incubation temperature of the present invention is 30-40° C.; in another embodiment, the incubation temperature of the present invention is 37° C. In one embodiment, the incubation time of the present invention is 20-60 minutes; in another embodiment, the incubation time of the present invention is 25-50 minutes; in another embodiment, the incubation time of the present invention is 30 minutes.

[0042] To further illustrate the present invention, a monoclonal antibody against galactin and a hybridoma cell line secreting the monoclonal antibody and their applications provided by the present invention are described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0043] Example 1

[0044] 1. Preparation of Complete Antigen

[0045] (1) Preparation of immunogen: 2-(Benzo[d][1,3]dioxin-5-yl)benzoic acid (BPA) is a structural analog of ALS. Therefore, using BPA as a hapten, the carboxyl group (-COOH) of BPA was coupled with the amino group (NH2) of bovine serum albumin (BSA) to obtain the complete antigen BP-BSA as an immunogen. The coupling principle and specific preparation method are as follows:

[0046]

[0047] a. Weigh 1.85 mg of the hapten BPA and dissolve it in 200 μL of anhydrous N,N-dimethylformamide. Add 1.6 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and 1.0 mg of N-hydroxysuccinimide (NHS) in this order. Stir and react at room temperature for 0.5 h to obtain Solution A. Weigh 10 mg of BSA and add 2 mL of boric acid buffer solution to obtain Solution B. While stirring, slowly add Solution A dropwise to Solution B and react at room temperature for 2 h to obtain a mixed solution containing BP-BSA.

[0048] b. Dialysis: Take a 10 cm dialysis bag, boil it in boiling water for 5 minutes, then rinse it with 60°C deionized water for 3 minutes, and store it in 4°C deionized water for later use; place the mixed solution containing BP-BSA obtained in step a in the dialysis bag, use 0.01 mol / L PBS as the dialysate, and dialyze it at 4°C for 3 days. The dialysate is replaced three times a day to separate the complete antigen from the uncoupled hapten and other small molecules. The complete antigen BP-BSA is obtained and used for the next step of mouse immunization.

[0049] (2) Preparation of coating agent: 3-Hydroxybiphenyl-2-carboxylic acid (HBC) is a structural analog of ALS. Therefore, HBC was used as a hapten and conjugated with ovalbumin (OVA) to prepare the complete antigen HB-OVA as a coating agent. The coupling principle and specific preparation method are as follows:

[0050]

[0051] a. Weigh 1.63 mg of the hapten HBC (step 1) and dissolve it in 200 μL of anhydrous N,N-dimethylformamide. Add 1.6 mg of EDCI and 1.0 mg of NHS in sequence, and stir at room temperature for 0.5 h to obtain Solution C. Weigh 10 mg of OVA and add 2 mL of boric acid buffer solution to obtain Solution D. While stirring, slowly add Solution C dropwise to Solution D and react at room temperature for 2 h to obtain a mixed solution containing HBC-OVA.

[0052] b. Dialyze the HBC-OVA mixture according to the method in step (1) to obtain HBC-OVA as the coating source.

[0053] 2. Immunization of mice: BALB / c mice were subcutaneously injected with the immunogen BP-BSA obtained in step 1 via the back of the neck. The specific steps are as follows:

[0054] The first immunization (100 μg / animal) was administered with an emulsion of equal volumes of Freund's complete adjuvant and the immunogen BP-BSA. Five booster immunizations (50 μg / animal) were administered with an emulsion of equal volumes of Freund's incomplete adjuvant and the immunogen BP-BSA. One month elapsed between the first and first booster immunizations, and 21 days elapsed between multiple booster immunizations. The final booster immunization was performed with BP-BSA (25 μg / animal, adjuvant-free), diluted in saline to a concentration of 0.5 mg / mL. Serum titers and inhibition rates were determined by indirect competitive enzyme-linked immunosorbent assay (icELISA).

[0055] 3. Cell fusion: Three days after the sprint immunization, cell fusion was performed according to the conventional PEG method. The specific steps are as follows:

[0056] a. Remove the eyeballs to collect blood. After killing the triple-immunized gram-negative mice by cervical dislocation, immediately disinfect them in 75% alcohol and soak for about 5 minutes. Remove the spleen of the mouse aseptically, grind it moderately with the rubber tip of a syringe, and pass it through a 200-mesh cell sieve to obtain a splenocyte suspension. Collect the suspension and centrifuge it at 1200 rpm for 8 minutes. Wash the splenocytes three times with RPMI-1640 medium. After the final centrifugation, dilute the splenocytes to a certain volume, count them, and set aside.

[0057] b. Collecting murine myeloma SP2 / 0 cells: 7-10 days before fusion, incubate SP2 / 0 cells in RPMI-1640 medium containing 10% FBS (fetal bovine serum) in a 5% CO2 incubator. Before fusion, the number of SP2 / 0 cells should reach (1-4) × 10 7 Ensure that SP2 / 0 tumor cells are in the logarithmic growth phase before fusion. At the time of fusion, tumor cells are collected, suspended in RPMI-1640 basal culture medium, and cell counts are performed;

[0058] c. Fusion process: 7 min. In the first minute, add 1 mL of PEG 1500 dropwise to the cells in a gradual, increasing order. In the second minute, allow to stand. In the third and fourth minutes, add 1 mL of RPMI-1640 medium dropwise over 1 min. In the fifth and sixth minutes, add 2 mL of RPMI-1640 medium dropwise over 1 min. In the seventh minute, add 1 mL of RPMI-1640 medium dropwise every 10 s. Then, incubate at 37°C for 5 min. Centrifuge at 800 rpm for 8 min, discard the supernatant, and resuspend the cells in RPMI-1640 selection medium supplemented with 20% fetal bovine serum and 2% 50× HAT. Add 200 μL / well to a 96-well plate and incubate in a 37°C, 5% CO2 incubator.

[0059] 4. Cell Screening and Cell Line Establishment

[0060] On the third day of cell fusion, the fused cells were half-changed with 2% 50×HAT RPMI-1640 screening medium. On the fifth day, the medium was fully changed with 20% fetal bovine serum and 1% 100×HT RPMI-1640 transition medium. On the seventh day, the cell supernatant was collected for screening. The screening was divided into two steps: the first step was to screen the OD 450 nm>0.2 and OD 450 nm / control well OD 450 For cells with a β-catenin level of ≥2.1 nm, the inhibitory effect of galactosidase (GC) on positive cells was determined using an icELISA. The following steps were performed: 50 μL of PBS was added to the positive control wells; 50 μL of a 50 μg / L GC standard solution was added to the test wells. Cell supernatant (100 μL / well) was added and incubated at 37°C for 30 minutes. After thorough washing, 100 μL / well of a 1:3000 dilution of HRP-goat anti-mouse IgG was added and incubated at 37°C for 30 minutes. The colorimetric solution was added and developed at 37°C for 15 minutes. Stop solution was added and the cells were detected using a microplate reader. Cells that showed significant inhibition against the GC standard were selected and subcloned using the limiting dilution method. The assay was then repeated three times to obtain a monoclonal hybridoma cell line, ALS.

[0061] 5. Preparation and identification of monoclonal antibodies against sarcomatin

[0062] (1) Hybridoma cells are placed in a culture flask and cultured. During the culture process, the hybridoma cells produce and secrete monoclonal antibodies. The culture supernatant is collected and centrifuged to remove cells and debris to obtain the desired monoclonal antibody. The monoclonal antibody is purified by the caprylic acid-ammonium sulfate method. The purified monoclonal antibody is finally obtained and stored at -20°C.

[0063] (2) The immunoglobulin subtype of the monoclonal antibody purified from ascites was identified using a mouse monoclonal antibody subtype identification kit. The subtype was IgG2b, and the light chain type was κ, as detected by the mouse monoclonal antibody subtype identification kit. Figure 1 shown.

[0064] 6. Affinity Testing

[0065] The affinity of the monoclonal antibody against galactosidase was determined by indirect ELISA (iELISA) using the following steps:

[0066] (1) Solution preparation

[0067] 0.05 mol / L carbonate buffer (CBS): Weigh 1.59 g of Na2CO3 and 2.93 g of NaHCO3, dissolve them in a small amount of double-distilled water, mix them, add double-distilled water to about 800 mL, adjust the pH to 9.6, add double-distilled water to 1000 mL, and store at 4°C until used;

[0068] Phosphate buffered saline (PBS): Weigh 8.0 g NaCl, 0.2 g KCl, 0.2 g KH2PO4, and 2.9 g Na2HPO4·12H2O, dissolve in 800 mL pure water, adjust the pH to 7.2–7.4 with NaOH or HCl, and make up to 1000 mL.

[0069] Washing solution (PBST): PBS containing 0.05% Tween-20;

[0070] Blocking solution: 0.05 mol L-1 CBS containing 0.2% (w / v) gelatin;

[0071] TMB colorimetric solution: Solution A: Weigh 18.43 g of Na₂HPO₄·12H₂O and 9.33 g of citric acid, and dilute to 1000 mL with pure water. Solution B: Weigh 60 mg of TMB and dissolve it in 100 mL of ethylene glycol. Mix Solution A and Solution B in a 1:1 volume ratio to create the TMB colorimetric solution. Mix immediately before use.

[0072] Stop solution: 2 mol L -1 of H2SO4 solution.

[0073] (2) Coating: The coated HBC-OVA obtained in step 1 was diluted to 0.1 μg / mL and 0.3 μg / mL with 0.05 mol / L carbonate buffer, respectively, and added to a 96-well ELISA plate at 100 μL / well, and reacted at 37°C for 2 h.

[0074] (3) Washing: Pour off the solution in the plate and wash with PBST three times, 3 minutes each time.

[0075] (4) Blocking: Add 200 μL / well of blocking solution and incubate at 37°C for 2 h. Wash with PBST and dry for later use.

[0076] (5) Sample addition: Add 100 μL PBS to the control wells; add 0.01 mol / L PBS to the detection wells, gradient dilution of microglucosidase monoclonal antibody to 0.2, 0.1, 0.05, 0.025, 0.01, 0.005 μg / mL, 100 μL / well, and react at 37°C for 30 min; after thorough washing with PBST, add 1:3000 diluted HRP-goat anti-mouse IgG, 100 μL / well, and react at 37°C for 30 min.

[0077] (6) Color development: Remove the ELISA plate, wash thoroughly with PBST, add 100 μL of TMB color development solution to each well, and incubate at 37°C in the dark for 15 min.

[0078] (7) Termination and measurement: Add 50 μL of stop solution to each well to terminate the reaction, and then measure the OD of each well using a microplate reader. 450 Value, the result is Figure 2 As shown. According to OD 450 The test results show that the affinity of the monoclonal antibody against galactin is 7.26×10 8 L / mol.

[0079] 7. Sensitivity test

[0080] Referring to step 6, an indirect competitive ELISA (icELISA) was used to test the sensitivity of the sclerotin monoclonal antibody to sclerotin. Sample loading was as follows: 100 μL of PBS was added to the positive control well; 100 μL of a gradient of 2.5, 5, 10, 25, 50, 100, and 200 μg / L sclerotin standard solutions were added to the test wells. Then, 100 μL / well of 0.1 μg / mL sclerotin monoclonal antibody was added and incubated at 37°C for 30 min. After thorough washing, 100 μL / well of a 1:3000 dilution of HRP-goat anti-mouse IgG was added and incubated at 37°C for 30 min.

[0081] like Figure 3 As shown, according to the standard equation y = -0.4278x + 1.1522 (R 2=0.9887) to calculate the IC of the monoclonal antibody against galactosidase. 50 It is 33.5μg / L.

[0082] The galactin monoclonal antibody secreted by the monoclonal hybridoma cell line ALS of the present invention has high affinity and sensitivity and can be used for galactin immunoassay detection and preparation of affinity columns.

[0083] Example 2

[0084] The monoclonal antibody against galactosidase produced by in vitro amplification of hybridoma cell line ALS was applied to the recovery test of galactosidase in apples. The specific steps are as follows:

[0085] (1) Sample pretreatment

[0086] Apples purchased from a local market tested negative according to DB34 / T 3855-2021 were homogenized and spiked with 30 μg / kg and 50 μg / kg of alginate (ALS), respectively. Five grams of spiked sample was mixed with 5 mL of ethyl acetate, shaken for 5 minutes, and then centrifuged at 4000 rpm for 5 minutes. After three repetitions, the organic phase was collected, dried, and redissolved in 5 mL of PBS containing 5% methanol to obtain the sample extract for ELISA analysis and testing with the improved device.

[0087] (2) Sample addition: Add 100 μL of PBS to the positive control well; add 100 μL of 30 and 50 μg / kg sample extract to the detection well. Then, dilute the monoclonal antibody against galactosidase secreted by the monoclonal hybridoma cell line ALS of the present invention to 0.1 μg / mL with 0.01 mol / L PBS and add it to the coated wells of each dilution, 100 μL / well, and react at 37°C for 30 minutes. After thorough washing with PBST, add 100 μL / well of 1:3000 diluted HRP-goat anti-mouse IgG and react at 37°C for 30 minutes.

[0088] (7) Color development: Remove the ELISA plate, wash thoroughly with PBST, add 100 μL of TMB color development solution to each well, and incubate at 37°C in the dark for 15 min.

[0089] (8) Termination and measurement: Add 50 μL of stop solution to each well to terminate the reaction, and then measure the OD of each well using a microplate reader. 450 The recovery rate and relative standard deviation (RSD) were calculated and the results are shown in Table 1.

[0090] Table 1 Results of the additive recovery test

[0091] Added value (μg / kg) Detection value (μg / kg)(X±SD) Recovery rate (%) RSD (%) 30.0 27.60±2.28 92.05 8.24 50.0 51.97±3.42 103.9 6.58

[0092] As can be seen from Table 1, the recovery rates of trichomoniasis in apple juice detected by the established ELISA method ranged from 92.05% to 103.9%, and the relative standard deviations were 6.58% to 8.24%, both less than 15%, indicating good stability of the added recovery.

[0093] Based on the above, it can be seen that the monoclonal antibody secreted by the hybridoma cell line ALS provided by the present invention has strong affinity, strong specificity, and high detection sensitivity, and can realize the detection of galactosidase.

[0094] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A hybridoma cell line ALS secreting a monoclonal antibody against galactosidase, with the deposit number being CGMCC No. 46355.

2. The hybridoma cell line ALS according to claim 1, characterized in that The hybridoma cell line ALS is obtained by fusing mouse spleen cells and mouse myeloma cells; the mouse spleen cells are mouse spleen cells obtained by immunizing mice with a myelomonocytic immunogen; The microgliadin immunogen is obtained by coupling 2-(benzo[d][1,3]dioxa-5-yl)benzoic acid with a carrier protein; the carrier protein is bovine serum albumin.

3. A monoclonal antibody against sarcomatin, characterized in that The invention relates to a monoclonal antibody against galactin secreted by the hybridoma cell line ALS according to claim 1 or 2.

4. The method for preparing the monoclonal antibody against galactosidase according to claim 3, characterized in that: The method comprises the following steps: culturing the hybridoma cell line ALS, and collecting the culture supernatant to produce the microgliadin monoclonal antibody.

5. Use of the hybridoma cell line ALS according to claim 1 or 2, the graminic acid monoclonal antibody according to claim 3, or the graminic acid monoclonal antibody prepared by the preparation method according to claim 4 in the preparation of graminic acid inhibitors and / or graminic acid immunoaffinity columns.

6. A kit, characterized in that The kit comprises the hybridoma cell line ALS according to claim 1 or 2, or the galactin monoclonal antibody according to claim 3, or the galactin monoclonal antibody prepared by the preparation method according to claim 4.

7. The kit according to claim 6, characterized in that The kit also includes a coating source; the coating source is obtained by coupling 3-hydroxybiphenyl-2-carboxylic acid with a carrier protein; and the carrier protein is ovalbumin.

8. The kit according to claim 6 or 7, characterized in that The kit further comprises one or more of carbonate buffer, phosphate buffer, PBST, a tracer-labeled detection antibody, and TMB color developing solution.

9. Use of the hybridoma cell line ALS according to claim 1 or 2, the graminic acid monoclonal antibody according to claim 3, the graminic acid monoclonal antibody prepared by the preparation method according to claim 4, or the kit according to any one of claims 6 to 8 in graminic acid immunoassay.

10. A method for immunoassay of microglia, characterized in that: The method comprises the following steps: mixing and incubating a test sample, a coating source and a microglia monoclonal antibody to form a complex; mixing and incubating the complex with a detection antibody labeled with a tracer to detect a tracer signal; The coating is obtained by coupling 3-hydroxybiphenyl-2-carboxylic acid with a carrier protein; the carrier protein is ovalbumin; The galactin monoclonal antibody is the galactin monoclonal antibody secreted by the hybridoma cell line ALS according to claim 1 or 2, or the galactin monoclonal antibody according to claim 3, or the galactin monoclonal antibody prepared by the preparation method according to claim 4.

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