Cell strain capable of secreting monoclonal antibody capable of recognizing methylene blue and metabolite thereof and application of cell strain

Abstract

The invention relates to the technical field of biology, in particular to a cell strain capable of secreting and recognizing a monoclonal antibody of methylene blue and metabolite of methylene blue and application of the cell strain, the cell strain capable of secreting and recognizing the monoclonal antibody of methylene blue and metabolite of methylene blue is preserved in China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: C2024265. The invention provides a cell strain capable of secreting a broad-spectrum monoclonal antibody capable of recognizing methylene blue and metabolites thereof, aiming at the problem of lack of the broad-spectrum monoclonal antibody which is high in sensitivity and capable of recognizing methylene blue and metabolites thereof at the same time in the prior art. The monoclonal antibody secreted by the cell strain can be applied to development of an immunological detection method, and a key technology is provided for rapid detection of methylene blue and metabolite residues thereof in aquatic products.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a monoclonal antibody cell line capable of secreting and recognizing methylene blue and its metabolites and application thereof. Background Art

[0002] Methylene blue (MB) is a thiazine dye used to prevent and treat Saprolegniasis in aquaculture and is considered a potential substitute for malachite green (which is now banned). Studies have confirmed that methylene blue has chronic toxicity and "three-hazard" side effects on humans. Many countries have implemented strict control measures on it, and MB has not yet been approved for use in aquaculture in my country. Studies have shown that after methylene blue enters the fish body, it will be metabolized into azure A (AZA), azure B (AZB) and azure C (AZC). Therefore, when testing it, it is necessary to simultaneously test methylene blue and its metabolites AZA, AZB and AZC.

[0003] In recent years, rapid immunological detection technology has become the main technical means for on-site detection at the grassroots level due to its advantages of simple operation and no need for complex equipment. Chinese invention patent CN 106434569 A discloses a monoclonal cell line C4 that can secrete a monoclonal antibody that recognizes methylene blue and its application, but the monoclonal antibody can only specifically recognize methylene blue and cannot simultaneously recognize methylene blue and its metabolites. At present, there is no rapid immunological detection method that can simultaneously detect the residues of methylene blue and its metabolites. The main reason is the lack of a high-sensitivity and broad-spectrum monoclonal antibody that can simultaneously recognize methylene blue and its metabolites. Therefore, the preparation of a broad-spectrum monoclonal antibody that can simultaneously recognize methylene blue and its metabolites is a prerequisite for establishing an immunological analysis method. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a cell line capable of secreting monoclonal antibodies that recognize methylene blue and its metabolites and its application.

[0005] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:

[0006] In a first aspect, a cell line capable of secreting a monoclonal antibody that recognizes methylene blue and its metabolites is provided, which is a hybridoma cell line 3E8 (Hybridoma cell line), which has been deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: C2024265.

[0007] In a second aspect, a monoclonal antibody is provided for recognizing methylene blue and its metabolites, which is secreted and produced by a monoclonal cell line with a deposit number of CCTCC NO: C2024265.

[0008] A third aspect provides the use of monoclonal antibodies that recognize methylene blue and its metabolites in the preparation of immunological tools for detecting methylene blue and its metabolites.

[0009] Furthermore, the application of methylene blue and its metabolites in the detection of aquatic products.

[0010] The beneficial effects of the present invention are:

[0011] 1. The monoclonal antibody prepared by the present invention can simultaneously recognize methylene blue and its metabolite residues. Compared with the previous monoclonal antibodies, it has stronger recognition ability and more recognition types.

[0012] 2. The IC10 of the monoclonal antibody prepared by the present invention is 4.07-5.57 ng / mL, with high sensitivity, which can meet the detection requirements.

[0013] Preservation of biological material samples: The hybridoma cell line provided by the present invention is classified and named as hybridoma cell line 3E8 (Hybridoma cell line), which was deposited in the China Center for Type Culture Collection on October 24, 2024. The preservation address is Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University, Postal Code 430072, and the preservation number is CCTCC NO: C2024265. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The competitive inhibition standard curve of methylene blue in the embodiment;

[0015] Figure 2 The competitive inhibition standard curve of Azure A in the examples;

[0016] Figure 3 The competitive inhibition standard curve of Azure B in the examples;

[0017] Figure 4 This is the competitive inhibition standard curve of Azure C in the examples. DETAILED DESCRIPTION

[0018] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0019] Example

[0020] 1. Preparation of complete methylene blue antigen

[0021] Weigh 74.0 mg of hapten (TM) and dissolve it in 0.4 mL of dimethylformamide solution. Then weigh 41.2 mg of dicyclohexylcarbodiimide and 23 mg of N-hydroxysuccinimide in a beaker, add 0.5 mL of dimethylformamide solution and mix well. After it is completely dissolved, add it dropwise to the dissolved TM liquid under magnetic stirring. After reacting at room temperature for 8 hours, let it stand at 4°C overnight. After the reaction, centrifuge at 4°C, 4000 rpm for 10 minutes and take the supernatant. Weigh 82 mg of bovine serum albumin (BSA ) Dissolve in 5 mL of phosphate buffer solution, add the above supernatant dropwise under magnetic stirring, and react in an ice-water bath for 6 hours. After the reaction, transfer the solution to the treated dialysis bag and dialyze at 4°C for 3 days with phosphate buffer solution as the dialysate, and change the dialysate 3-4 times a day. After dialysis, centrifuge at 4°C, 4000r.pm for 10 minutes, and take the supernatant, which is the immunogen (TM-BSA).

[0022]

[0023] The preparation method of the coating agent (TM-VOA) is the same as that of TM-BSA, except that BSA is replaced with hen egg serum albumin (OVA).

[0024] 2. Animal Immunization

[0025] Eight-week-old female Balb / c mice were selected, and blood was collected from the eye sockets before the start of the immunization experiment as negative serum. Balb / C mice were injected intraperitoneally for a total of 5 times, with 100 μg of immunogen (TM-BSA) injected into each mouse each time. The first injection was intraperitoneal injection after TM-BSA was mixed with equal amounts of complete adjuvant, and the second to fourth injections were intraperitoneal injections after TM-BSA was mixed with incomplete adjuvant, with an interval of two weeks. Three days after the fourth injection, TM-BSA without adjuvant was injected into the mouse peritoneal cavity and tail vein for the fifth booster immunization. After the third immunization, blood was collected from the eye sockets to determine the serum titer. After 5 immunizations, mice with high titers were selected, and their spleens were taken for cell fusion after booster immunization.

[0026] 3. Establishment of hybridoma cell lines

[0027] 3.1 Myeloma cell recovery and culture

[0028] 20 days before fusion, SP2 / 0 cells frozen in liquid nitrogen were taken out and immediately placed in a 40°C water bath. After 90 seconds, they were taken out and centrifuged at 1000rpm for 3 minutes. The supernatant was discarded aseptically. Transferred to a cell culture bottle, cultured with complete medium (20% fetal bovine serum, 2% L-glutamine, 1% double antibody) for 6 hours, replaced with new complete medium, and after 1-2 passages, the cells were blown up and the number of cells was adjusted to 105 / mL. They were inoculated into the abdominal cavity of Balb / C female mice injected with paraffin one week in advance. After 6-7 days, mice with obvious abdominal distension were killed, aseptically collected ascites, placed in a 37°C incubator with a CO2 concentration of 5%, and cultured for 4 hours, and then the medium was changed and cultured.

[0029] 3.2 Preparation of feeder cells

[0030] One day before cell fusion, select Balb / C mice of appropriate age and treat them by cervical dislocation. Then, soak the treated mice in 75% alcohol for 5 minutes for sterilization. On the clean bench, fix the mice, wipe them again with alcohol cotton for disinfection, cut the skin of the mouse abdomen with scissors and tweezers without damaging the mouse peritoneum, draw 15mL of incomplete culture medium with a disposable sterile syringe and inject it into the mouse peritoneal cavity and massage for 5 minutes, draw out the incomplete culture medium, centrifuge at 1000rpm for 5 minutes, resuspend the cells with complete culture medium, add 100μL to each well of the 96-well cell culture plate, and culture overnight at 37℃ in a 5% CO2 incubator.

[0031] 3.3 Cell fusion

[0032] When merging, place three 15cm 2 The SP2 / 0 cells in the cell culture flask were combined in a 15 mL centrifuge tube, and then the spleen of the boosted immunized mouse was taken out and placed in a small dish. The incomplete culture medium was sucked with a 5 mL syringe to blow away the spleen cells, and then transferred to a 15 mL centrifuge tube, 1000 rpm, 5-7 minutes, the supernatant was poured out, and serum-free culture medium was added and centrifuged again. Then the two cells were resuspended in a 50 mL centrifuge tube, mixed, and centrifuged at 1000 rpm for 10 minutes.

[0033] After centrifugation, pour out the supernatant, use filter paper to absorb the remaining serum-free medium, and then start cell fusion. Place the centrifuge tube in a large beaker for water bath, slowly add 1mL PEG to the bottom of the centrifuge tube, the time is controlled within 1min, stir while adding, and stand for 90s after complete addition. Then add incomplete medium to the fused cells to terminate the reaction. In the first minute, slowly add 1mL serum-free medium; in the second minute, slowly add 1mL serum-free medium, in the third and fourth minutes, slowly add 3mL serum-free medium (average 1mL / 40s); in the fifth minute, add 5mL medium (average 1mL / 12s), and in the sixth minute, add 30mL serum-free medium at one time. Centrifuge at 1000rpm for 10min. Remove the supernatant, add 1mL complete medium, resuspend the cells, pour into the sample tank, add 40mL complete medium containing 20% ​​serum, add to the 96-well plate with feeder cells with a multichannel pipette, 100μL / well, and place in a 37℃, 5% CO2 incubator for culture. On the second day, add 10-fold diluted HAT, and add 50 μL per well to the 96-well plate. Observe the cell growth status 5 days after fusion, and replace the wells with obvious cell clusters with complete medium containing HT by half-replacement 7 days after fusion. Take the wells with good growth at 10-15 days for detection and screening. Select the wells with good growth and high positive values ​​for subcloning.

[0034] 3.4 Screening of positive hybridoma cells

[0035] Indirect ELISA was used to detect and screen the supernatant of positive hybridoma cells. The specific steps are as follows. Add 100 μL TM-OVA (1.0 μg / mL) to a 96-well ELISA plate and incubate at 4°C overnight. The next day, discard the waste liquid, add 200 μL to each well with PBST (containing 0.5% Tween-20), wash the plate three times, and pat dry. Then, add 200 μL of blocking solution (PBS containing 5% skim milk) to each well at 37°C for 1 hour. Wash the plate three times and pat dry. Add 100 μL of cell supernatant to each well, set up a control, and react at 37°C for 1 hour. Wash the plate, pat dry, then add enzyme-labeled secondary antibody (1:2000 dilution) at 37°C, react for 0.5 hours, wash the plate, and pat dry. Add 100 μL of freshly prepared TMB colorimetric solution to each well, react at 37°C for 10 min, add 50 μL of 2M H2SO4 to terminate the reaction, and read the results at 450 nm using a microplate reader.

[0036] 3.5 Subcloning of positive hybridoma cells

[0037] The limited dilution method was used to clone hybridoma cell lines. Feeder cells were prepared 1 day in advance, and the cells in the wells with good growth and high positive values ​​were blown up with complete culture medium, and the complete culture medium was added to a total volume of 1 mL, 100 μL / well, and added to the first row of the 96-well plate in sequence; the culture medium was added again to 1 mL, 100 μL / well, and added to the second row of the 96-well plate. After repeating 4 times, the next positive well was subcloned. After all wells were subcloned, they were cultured at 37°C in a 5% CO2 incubator for 5 days for observation and screening, and the next subcloning was performed after 10 days. After the positive cells screened were subcloned twice according to the above steps, there were no negative wells in the indirect ELISA test, which meant that a single hybridoma cell was screened.

[0038] 3.6 Preparation and purification of ascites

[0039] 100 μL of hybridoma cells (2×106 cells / mL) were injected into mice, and the mice had been injected with sterilized liquid paraffin into their peritoneal cavity one week in advance. After the abdomen of the mouse was swollen, the ascites was obtained by puncture and drainage with the needle of a 50mL syringe. After the ascites was centrifuged, the supernatant was taken, filtered with a 0.45μm filter membrane, and then purified with a Protein G affinity chromatography column. The collected eluate was identified by SDS-PAGE. The purified antibodies were stored in a -70℃ refrigerator.

[0040] 4. Antibody performance determination

[0041] 4.1 Antibody sensitivity determination

[0042] The original coating concentration was 400 ng / mL, and the optimal working concentration of the antibody was 64000. The coating time was overnight at 4°C, the antigen-antibody reaction time was 1 h, the enzyme-labeled secondary antibody was diluted 4000 times, and the reaction time of the secondary antibody and the colorimetric solution was 1 h and 15 min, respectively. Under these conditions, the competitive standard curves of MB, AZA, AZB and AZC were established. Standards of MB and its metabolites with different mass concentrations (2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91 ng / mL) were prepared, and the inhibition rate of 3E8 antibody on the standard was determined by ELISA, and a standard inhibition curve was established, and a correlation regression analysis was performed. The concentration corresponding to IC10 was used as the method detection limit (LOD). The ordinate and abscissa of the standard curve are B / B0 and the logarithmic value of MB at different mass concentrations, respectively, where B is the OD450 value corresponding to different concentrations of MB, and B0 is the OD450 value corresponding to a concentration of 0 ng / mL.

[0043] The competition curve of MB is y=50.048x-20.515, and the IC50 and IC10 values ​​corresponding to the correlation coefficient (R2=0.9861) are 25.7ng / mL and 4.07ng / mL. The competition saturation region of AZA, AZB and AZC with MB is similar, so the standard curve is established in the range of 3.91-125ng / mL ( Figure 1-4 ), the competition curve of AZA is y=50.048x-20.515, and the IC50 and IC10 values ​​corresponding to the correlation coefficient (R2=0.9861) are 33.1ng / mL and 5.57ng / mL. The competition curve of AZB is y=50.048x-20.515, and the IC50 and IC10 values ​​corresponding to the correlation coefficient (R2=0.9861) are 33.0ng / mL and 5.48ng / mL. The competition curve of AZC is y=50.048x-20.515, and the IC50 and IC10 values ​​corresponding to the correlation coefficient (R2=0.9861) are 30.2ng / mL and 4.94ng / mL. The results show that the prepared methylene blue monoclonal antibody can not only recognize methylene blue, but also its metabolites AZA, AZB and AZC, indicating that the prepared antibody has a strong recognition ability.

[0044] 4.2 Antibody Specificity

[0045] The cross-reaction of 1 thiazine dye and 6 common drug dyes in aquatic products, including Lloyd's violet, colored malachite green, colorless malachite green, colored crystal violet, colorless crystal violet, brilliant green and recessive brilliant green, was determined by optimized (competitive enzyme-linked immunosorbent assay) ic-ELISA method. The results showed (Table 1): The monoclonal antibody of this method cross-reacted with Lloyd's violet (thiazine dye) with a cross-reaction rate of 100.4%, and there was no cross-reaction with other tested drugs, indicating that this method has a broad-spectrum recognition ability for thiazine dyes and good specificity for other drugs.

[0046] Table 1 Cross-reaction rate of antibody 3E8 with other drugs

[0047]

[0048] In summary, the present invention provides a key technology for the rapid detection of methylene blue and its metabolite residues in aquatic products and successfully prepares a broad-spectrum monoclonal antibody.

[0049] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0050] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A cell line capable of secreting monoclonal antibodies that recognize methylene blue and its metabolites, characterized in that: It is a hybridoma cell line 3E8, which has been deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: C2024265.

2. A monoclonal antibody that recognizes methylene blue and its metabolites, characterized in that: It is secreted and produced by a monoclonal cell line with the deposit number of CCTCCNO: C2024265.

3. The use of the monoclonal antibody for recognizing methylene blue and its metabolites according to claim 2, characterized in that: Application in the preparation of immunoassay tools for detecting methylene blue and its metabolites.

4. The use of the monoclonal antibody for recognizing methylene blue and its metabolites according to claim 3, characterized in that: Application of methylene blue and its metabolites in the detection of aquatic products.

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