A cell line capable of secreting monoclonal antibodies recognizing methylene blue and its metabolites and its application

Abstract

This invention relates to the field of biotechnology, specifically to a cell line capable of secreting monoclonal antibodies that recognize methylene blue and its metabolites, and their applications. The cell line capable of secreting monoclonal antibodies that recognize methylene blue and its metabolites is deposited at the China Center for Type Culture Collection (CCTCCNO: C2024265). This invention addresses the problem of the lack of highly sensitive, broad-spectrum monoclonal antibodies capable of simultaneously recognizing methylene blue and its metabolites in existing technologies. It provides a cell line capable of secreting broad-spectrum monoclonal antibodies that recognize methylene blue and its metabolites. The monoclonal antibodies secreted by this cell line can be used in the development of immunological detection methods, providing a key technology for the rapid detection of methylene blue and its metabolite residues in aquatic products.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a monoclonal antibody cell line that can secrete antibodies that recognize methylene blue and its metabolites, and its applications. Background Technology

[0002] Methylene blue (MB) is a thiazide dye used to control saprolegniasis in aquaculture and was considered a potential alternative to malachite green (which is now banned). Studies have confirmed that methylene blue has chronic toxicity and carcinogenic, mutagenic, and teratogenic side effects in humans. Many countries have implemented strict control measures for it, and MB has not yet been approved for use in aquaculture in my country. Research shows that after entering the fish's body, methylene blue is metabolized into azure A (AZA), azure B (AZB), and azure C (AZC). Therefore, when testing for methylene blue, it is necessary to simultaneously detect methylene blue and its metabolites AZA, AZB, and AZC.

[0003] In recent years, rapid immunological detection technologies have become a primary means of on-site testing at the grassroots level due to their ease of operation and lack of the need for complex equipment. Chinese invention patent CN 106434569 A discloses a monoclonal cell line C4 that secretes a monoclonal antibody that recognizes methylene blue and its applications. However, this monoclonal antibody can only specifically recognize methylene blue and cannot simultaneously recognize methylene blue and its metabolites. Currently, there is no rapid immunological detection method capable of simultaneously detecting methylene blue and its metabolite residues, mainly because there is a lack of highly sensitive, broad-spectrum monoclonal antibodies that can simultaneously recognize methylene blue and its metabolites. Therefore, preparing a broad-spectrum monoclonal antibody capable of simultaneously recognizing 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 shortcomings in the prior art, the purpose of this invention is to provide a monoclonal antibody cell line that can secrete a recognition of methylene blue and its metabolites, and its application.

[0005] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0006] Firstly, a monoclonal antibody cell line capable of secreting antibodies that recognize methylene blue and its metabolites is provided. This cell line is hybridoma cell line 3E8, which has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:C2024265.

[0007] Secondly, a monoclonal antibody that recognizes methylene blue and its metabolites is provided, which is secreted by a monoclonal cell line with accession number CCTCC NO:C2024265.

[0008] Thirdly, it provides the application 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, its application in detecting methylene blue and its metabolites in aquatic products.

[0010] The beneficial effects of this invention are as follows:

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

[0012] 2. The monoclonal antibody prepared using this invention has an IC10 of 4.07-5.57 ng / mL, which is highly sensitive and can meet the detection requirements.

[0013] Preservation of biological material samples: The hybridoma cell line provided by this invention, classified and named Hybridoma cell line 3E8, was deposited on October 24, 2024, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Luojia Mountain, Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China, with accession number CCTCC NO: C2024265. Attached Figure Description

[0014] Figure 1 This is the competitive inhibition standard curve of methylene blue in the examples;

[0015] Figure 2 The example shows the competition inhibition standard curve of Azure A.

[0016] Figure 3 The example shows the competition inhibition standard curve of Azurite B.

[0017] Figure 4 The example shows the competitive inhibition standard curve of Azure C. Detailed Implementation

[0018] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0019] Example

[0020] 1. Preparation of methylene blue complete 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 into a beaker, add 0.5 mL of dimethylformamide solution and mix well. After complete dissolution, add the mixture dropwise to the dissolved TM solution under magnetic stirring. React at room temperature for 8 hours, then let stand overnight at 4°C. After the reaction, centrifuge at 4°C and 4000 rpm for 10 minutes, and collect the supernatant. Weigh 82 mg of bovine serum albumin (BSA). ) Dissolve the supernatant in 5 mL of phosphate buffer solution and add it dropwise under magnetic stirring. React in an ice-water bath for 6 hours. After the reaction, transfer the solution to a treated dialysis bag and dialyze at 4°C for 3 days using phosphate buffer solution as the dialysate, changing the dialysate 3-4 times daily. After dialysis, centrifuge at 4°C, 4000 rpm for 10 minutes, and collect the supernatant, which is the immunogen (TM-BSA).

[0022]

[0023] The preparation method of coating antigen (TM-VOA) is the same as that of TM-BSA, except that BSA is used to replace oocyte serum albumin (OVA).

[0024] 2. Animal immunization

[0025] Eight-week-old female Balb / c mice were selected. Blood was collected via the orbital venous line before the immunization experiment to obtain negative serum. Balb / c mice were injected intraperitoneally five times, with each mouse receiving 100 μg of immunogen (TM-BSA). The first injection was a mixture of TM-BSA and complete adjuvant, administered intraperitoneally. The second to fourth injections were a mixture of TM-BSA and incomplete adjuvant, administered intraperitoneally at two-week intervals. Three days after the fourth injection, a fifth booster immunization was performed by injecting unadjuvanted TM-BSA into the peritoneal cavity and tail vein of the mice. Serum titers were measured via orbital venous line sampling after the third immunization. After five immunizations, mice with high titers were selected for booster immunization, and their spleens were harvested for cell fusion.

[0026] 3. Establishment of hybridoma cell lines

[0027] 3.1 Myeloma cell resuscitation and culture

[0028] Twenty days before fusion, SP2 / 0 cells frozen in liquid nitrogen were removed and immediately placed in a 40°C water bath for 90 seconds. After centrifugation at 1000 rpm for 3 minutes, the supernatant was aseptically discarded. Cells were transferred to cell culture flasks and cultured in complete medium (20% fetal bovine serum, 2% L-glutamine, 1% penicillin-dextrin) for 6 hours. The medium was then replaced with fresh complete medium. After 1-2 passages, the cells were agitated and the cell count was adjusted to 10⁵ cells / mL. The cells were then inoculated into the peritoneal cavity of female Balb / C mice injected with paraffin one week prior. Six to seven days later, mice with significantly distended peritoneal cavities were sacrificed, and the ascites fluid was aseptically collected and incubated at 37°C with 5% CO₂ for 4 hours. The medium was then changed, and the culture continued.

[0029] 3.2 Preparation of feeder layer cells

[0030] One day before cell fusion, age-appropriate Balb / C mice were selected and euthanized by cervical dislocation. The treated mice were then sterilized by immersing them in 75% alcohol for 5 minutes. On a clean bench, the mice were fixed, wiped again with alcohol swabs for disinfection, and the abdominal skin was removed with scissors and forceps without damaging the peritoneum. 15 mL of incomplete culture medium was injected into the peritoneal cavity using a disposable sterile syringe, and the cells were massaged for 5 minutes. The incomplete culture medium was then aspirated, and the cells were centrifuged at 1000 rpm for 5 minutes. The cells were resuspended in complete culture medium, and 100 μL of complete culture medium was added to each well of a 96-well cell culture plate. The plates were incubated overnight at 37°C in a 5% CO2 incubator.

[0031] 3.3 Cell Fusion

[0032] When fusing, combine three 15cm 2 SP2 / 0 cells from the cell culture flask were combined into a 15 mL centrifuge tube. The spleens of boosted-immune mice were then removed and placed in a small petri dish. Incomplete culture medium was drawn up with a 5 mL syringe, and the spleen cells were dispersed by regurgitation. The dispersed cells were then transferred to a 15 mL centrifuge tube and centrifuged at 1000 rpm for 5-7 minutes. The supernatant was discarded, and serum-free culture medium was added, followed by a second centrifugation. The two cell lines were then resuspended in a 50 mL centrifuge tube, mixed, and centrifuged at 1000 rpm for 10 minutes.

[0033] After centrifugation, discard the supernatant and blot the remaining serum-free medium with filter paper. Then, begin cell fusion. Place the centrifuge tube in a large beaker for a water bath. Slowly add 1 mL of PEG to the bottom of the centrifuge tube over 1 minute, stirring constantly. After complete addition, let stand for 90 seconds. Next, add incomplete medium to the fused cells to stop the reaction. Add 1 mL of serum-free medium slowly at the first minute; 1 mL at the second minute; 3 mL at the third and fourth minutes (average 1 mL / 40 seconds); 5 mL at the fifth minute (average 1 mL / 12 seconds); and a final 30 mL of serum-free medium at the sixth minute. Centrifuge at 1000 rpm for 10 minutes. Remove the supernatant, add 1 mL of complete medium to resuspend the cells, pour into a sample well, and add 40 mL of complete medium containing 20% ​​serum. Use a multichannel pipette to transfer 100 μL / well to a 96-well plate containing feeder cells. Incubate at 37°C in a 5% CO2 incubator. On the second day, 50 μL of 10-fold diluted HAT was added to each well of a 96-well plate. Five days after fusion, cell growth was observed, and wells with obvious cell clusters were numbered. Seven days after fusion, the medium was replaced with complete medium containing HT using a half-medium medium change method. At 10-15 days, wells showing good growth were aspirated for testing and screening. Subcloning was performed on well-grown cells with high positive counts.

[0034] 3.4 Screening of positive hybridoma cells

[0035] Indirect ELISA was used to screen the supernatant of positive hybridoma cells. The specific steps are as follows: 100 μL of TM-OVA (1.0 μg / mL) was added to a 96-well ELISA plate and incubated overnight at 4°C. The next day, the waste liquid was discarded, and 200 μL of PBST (containing 0.5% Tween-20) was added to each well. The plate was washed three times and blotted dry. Next, 200 μL of blocking buffer (containing 5% skim milk PBS) was added to each well and the plate was reacted at 37°C for 1 h. The plate was washed three times and blotted dry. 100 μL of cell supernatant was added to each well as a control, and the plate was reacted at 37°C for 1 h. After washing and blotting dry, enzyme-labeled secondary antibody (1:2000 dilution) was added and the plate was reacted at 37°C for 0.5 h. The plate was washed and blotted dry. Add 100 μL of freshly prepared TMB chromogenic solution to each well, react at 37°C for 10 min, then add 50 μL of 2M H2SO4 to terminate the reaction, and read the value at 450 nm using a microplate reader.

[0036] 3.5 Subclones of positive hybridoma cells

[0037] Hybridoma cell lines were cloned using the limiting dilution method. Feeder cells were prepared 1 day in advance. Cells from wells with good growth and high positive counts were pipetted with complete culture medium, and 100 μL / well was added to 1 mL of complete culture medium. This was repeated 4 times. Subcloning was then performed on the next positive well. After subcloning all wells, the cells were incubated at 37°C in a 5% CO2 incubator for 5 days for observation and selection. Subcloning was repeated after 10 days. After subcloning the selected positive cells twice using the above steps, indirect ELISA was used to detect no negative wells, indicating that a single hybridoma cell line had been selected.

[0038] 3.6 Preparation and purification of ascites fluid

[0039] 100 μL of hybridoma cells (2 × 10⁶ cells / mL) were injected into mice. The mice's peritoneal cavity had been injected with sterile liquid paraffin one week prior. After the mice's abdomens swelled, ascites fluid was drained via puncture using a 50 mL syringe needle. The ascites fluid was centrifuged, and the supernatant was collected. After filtration through a 0.45 μm filter membrane, the supernatant was purified using a Protein G affinity chromatography column. The collected eluent was identified by SDS-PAGE. The purified antibody was aliquoted and stored at -70°C.

[0040] 4. Antibody performance assay

[0041] 4.1 Antibody Sensitivity Assay

[0042] The initial coating concentration was 400 ng / mL, and the optimal working antibody concentration was 64,000. Coating was performed overnight at 4℃, with an antigen-antibody reaction time of 1 h. The enzyme-labeled secondary antibody was diluted 4000-fold, and the reaction times for the secondary antibody and chromogenic solution were 1 h and 15 min, respectively. Under these conditions, competitive standard curves for MB, AZA, AZB, and AZC were established. Standards of different mass concentrations of MB and its metabolites (2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, and 3.91 ng / mL) were prepared. The inhibition rate of the 3E8 antibody against the standards was determined by ELISA, and standard inhibition curves were established. Correlation regression analysis was performed. The limit of detection (LOD) was set at the concentration corresponding to IC10. The ordinate and abscissa of the standard curve are B / B0 and the logarithm of different mass concentrations of MB, 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 for MB was y = 50.048x - 20.515, with a correlation coefficient (R² = 0.9861) corresponding to IC50 and IC10 values ​​of 25.7 ng / mL and 4.07 ng / mL, respectively. The competition saturation regions for AZA, AZB, and AZC with MB were similar; therefore, a standard curve was established within the range of 3.91-125 ng / mL. Figure 1-4 The competition curve for AZA was y = 50.048x - 20.515, with a correlation coefficient (R² = 0.9861) corresponding to IC50 and IC10 values ​​of 33.1 ng / mL and 5.57 ng / mL, respectively. The competition curve for AZB was also y = 50.048x - 20.515, with a correlation coefficient (R² = 0.9861) corresponding to IC50 and IC10 values ​​of 33.0 ng / mL and 5.48 ng / mL, respectively. The competition curve for AZC was y = 50.048x - 20.515, with a correlation coefficient (R² = 0.9861) corresponding to IC50 and IC10 values ​​of 30.2 ng / mL and 4.94 ng / mL, respectively. These results indicate that the prepared methylene blue monoclonal antibody can recognize not only methylene blue but also its metabolites AZA, AZB, and AZC, demonstrating strong recognition ability.

[0044] 4.2 Antibody Specificity

[0045] The cross-reactivity of one thiazide dye and six common drug dyes in aquatic products, including Laureth violet, colored malachite green, colorless malachite green, colored crystal violet, colorless crystal violet, brilliant green, and latent brilliant green, was determined using an optimized (competitive enzyme-linked immunosorbent assay) ic-ELISA method. The results (Table 1) showed that the monoclonal antibody of this method cross-reacted with Laureth violet (a thiazide dye) at a cross-reactivity rate of 100.4%, and there was no cross-reactivity with other detected drugs, indicating that this method has broad-spectrum recognition capability for thiazide dyes and good specificity for other drugs.

[0046] Table 1. Cross-reactivity rates of antibody 3E8 with other drugs

[0047]

[0048] In summary, this invention provides a key technology for the rapid detection of methylene blue and its metabolites 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 invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0050] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

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

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

3. The application of the monoclonal antibody for recognizing methylene blue and its metabolites as described in claim 2, characterized in that, Application in the preparation of immunoassay tools for detecting methylene blue and its metabolites azure A, azure B and azure C.

4. The application 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 azure A, azure B and azure C in the detection of aquatic products.

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