WO2018095254A1

WO2018095254A1 - Monoclonal cell line c4 capable of secreting monoclonal antibody recognizing methylene blue and application thereof

Info

Publication number: WO2018095254A1
Application number: PCT/CN2017/111098
Authority: WO
Inventors: 胥传来; 匡华; 陈燕妮; 徐丽广; 刘丽强; 宋珊珊; 吴晓玲
Original assignee: 江南大学
Priority date: 2016-11-28
Filing date: 2017-11-15
Publication date: 2018-05-31
Also published as: CN106434569A; CN106434569B

Abstract

The invention discloses a monoclonal cell line C4 capable of secreting a monoclonal antibody recognizing methylene blue and an application thereof in the technical field of immunochemistry. The monoclonal cell line C4 provided by the invention has been deposited with the general microbial center of the China General Microbial Culture Collection Committee under Accession Number CGMCC No.12026. The monoclonal antibody secreted by the monoclonal cell line C4 can specifically recognize methylene blue with good specificity and high sensitivity, and IC₅₀ is 21.13ng/ml. The monoclonal antibody can be used to establish immune detection technology for methylene blue.

Description

Monoclonal cell line C4 capable of secreting monoclonal antibody recognizing methylene blue and application thereof

Technical field

The invention relates to a monoclonal cell strain C4 capable of secreting a monoclonal antibody recognizing methylene blue and an application thereof, and belongs to the technical field of immunochemistry.

Background technique

Methylene blue belongs to the thiazide dye. Since the malachite green has been banned for aquaculture, the residue of the substitute methylene blue has attracted extensive attention and attention. Methylene blue was originally used for the prevention and treatment of urinary tract infections and malaria. With the deepening of research, it has been found that methylene blue can kill tumor cells and have a higher affinity for tumor cells. Therefore, methylene blue is used for diagnosis after radioisotope labeling. , locate and treat a variety of tumors. Methylene blue is also oxidizing and reducing, and can be used as an antidote in clinical use for drug poisoning such as cyanide and nitrite. Since methylene blue is in an ionic state in an aqueous solution, hydrogen ions are competed with the enzyme system of the surrounding microorganisms, resulting in inactivation of the microbial enzymes. Therefore, methylene blue is also used as a disinfectant in the veterinary field, especially in aquaculture. Studies have shown that methylene blue also has a good effect on some fish diseases such as tuberculosis, small clawworm, and water mold. Therefore, when some triphenylmethane dyes, such as malachite green and crystal violet, are explicitly banned from aquaculture, methylene blue is an object of choice for farmers.

In addition to disinfectants and fish disease prevention, methylene blue can also be used as an antifungal agent during the transport of aquatic products to reduce mortality. The toxicity of methylene blue is lower than that of malachite green. However, any unreasonable use of methylene blue in aquaculture may cause the accumulation of methylene blue. In addition, with the wide application of dyes, methylene blue as a cationic dye enters the surrounding waters with wastewater. It causes the concentration of methylene blue in the environment to exceed the standard. When human or animal is exposed to high concentration of methylene blue for a long time, it is easy to cause poisoning, toxic side effects are mutagenic, red blood cell morphology changes or anemia, necrotic abscess, etc., can cause death in severe cases. Therefore, countries or organizations including the United States, Japan, and the European Union do not allow methylene blue to be used in aquaculture. In China, methylene blue is widely used because of its low price and clear curative effect, and the farmers have less understanding of its toxic and side effects. Methylene blue is widely used as a disinfectant and a substitute for malachite green in aquaculture. Therefore, the residual problem cannot be underestimated. The research on its residual detection technology is of great significance to China's food safety and export trade. Although China has not yet introduced relevant laws and regulations, the research of detection technology is a pioneer in supervision. Facing the technical barriers of international trade, research on methylene blue and its metabolic residue detection technology, especially the rapid and simple screening technology, cannot be delayed.

At present, the detection technology of methylene blue is still stuck in the instrument method, such as liquid chromatography, capillary electrophoresis and ion chromatography. The detector is generally a mass spectrometer or an optical detector. The pretreatment technology has liquid-liquid extraction and solid phase extraction. . At present, the main detection method for methylene blue is mainly high performance liquid chromatography tandem mass spectrometry, except that the steps of the detector and sample pretreatment such as extraction and purification are slightly different. Research base for immunoassay of methylene blue in aquatic products This is in a blank stage.

Summary of the invention

In view of the above problems, the present invention provides a monoclonal cell line C4 capable of producing a monoclonal antibody that specifically recognizes methylene blue, and can be used for an immunodetection technique for establishing methylene blue.

The monoclonal cell line C4 capable of secreting a monoclonal antibody recognizing methylene blue has been deposited at the General Microbiology Center of the China Microbial Culture Collection Management Committee on January 20, 2016, and the deposit number is CGMCC No. 12026, and the deposit address is Beijing. No. 3, Beichen West Road, Chaoyang District, No. 3, Institute of Microbiology, Chinese Academy of Sciences.

The present invention also provides a specific monoclonal antibody recognizing methylene blue which is secreted by a monoclonal cell line C4 having the accession number CGMCC No. 12026.

The specific monoclonal antibody recognizing methylene blue can be used for detecting methylene blue or for preparing an immunological tool for detecting methylene blue. For example, it is used to detect the content of methylene blue in fish.

The specific monoclonal antibody produced by the monoclonal cell line C4 provided by the invention can specifically recognize methylene blue, and has good sensitivity, and the half-inhibitory concentration (IC ₅₀ ) is 21.13 ng/mL; and provides an immunological tool for detecting methylene blue detection in food. Raw materials, such as enzyme-linked immunosorbent assays and immunochromatographic strips.

Biomaterial preservation

Hybridoma cell line C4, classified as a monoclonal cell line, was deposited on January 20, 2016 at the General Microbiology Center of China Microbial Culture Collection Management Committee, referred to as CGMCC, at No. 1 Beichen West Road, Chaoyang District, Beijing. No. 3, Institute of Microbiology, Chinese Academy of Sciences, with the preservation number CGMCC No.12026.

DRAWINGS

Figure 1. Standard curve of methylene blue mAb.

Figure 2. UV identification of complete antigen.

detailed description

The present invention provides a monoclonal antibody cell C4 and a monoclonal antibody specific for its secretion against methylene blue. The present invention will be further described in conjunction with the specific embodiments, and the specific examples are all conventional methods unless otherwise specified.

Example 1 Preparation of monoclonal cell line C4

1. Mouse immunization and antiserum screening

The hapten (MB-HS) was coupled to hemocyanin (KLH) and ovalbumin (OVA) by an activated ester method to form an immunogen (MB-HS-KLH) and a coating (MB-HS-OVA). ). As shown in Fig. 2, before and after cross-linking, the absorption peaks of haptens and antigens were found to be significantly shifted, indicating that the hapten was successfully coupled with hemocyanin (KLH) or ovalbumin (OVA).

The first use of a complete adjuvant and immunogen 1..1 volume mixture, subcutaneous immunization of BALB / c mice, immunization 4 weeks after the interval, 7-10 days after the 6th exemption, the original package was coated with the enzyme plate, the titer and inhibition of the serum were determined by indirect competitive enzyme-linked immunosorbent assay, and the mice with high titer and inhibition were selected for fusion.

Among them, the hapten is a methylene blue derivative, and the structure of the hapten is:

2, cell fusion

On the 21st day after evaluation of mouse antiserum, selected mice were selected for intraperitoneal spur immunization. The specific procedure is as follows: the immunogen is diluted with physiological saline to a volume of 200 μL, and the dose is half of the dose of the last booster. The diluted immunogen is injected into a 1 mL syringe and slowly injected into the mouse from the left abdominal cavity of the mouse.

Three days after the mice were vaccinated, the mice were sacrificed by cervical dislocation and quickly placed in 100 mL of 75% alcohol. The mice were immersed in alcohol for 5-10 min and transferred to a clean bench to remove the spleen and placed in 200 mesh stainless steel. The mesh was gently ground with a 5 mL syringe core, and rinsing with RPMI1640 medium was continued. The spleen cell suspension was collected into a 50 mL centrifuge tube, centrifuged at 1200 r/min for 8 min, the supernatant was discarded, and the bottom of the tube was gently blown. The cells were dispersed, spleen cells were resuspended in RPMI 1640 medium, and transferred to a new 50 mL centrifuge tube. After repeating centrifugation three times, 10 mL of RPMI1640 medium was added, and the mixture was mixed, and 100 μL of the mixture was taken out and diluted 1000 times to be counted, and the rest was used. After the spleen cells are collected, the tumor cells in the logarithmic growth phase are collected. Tumor cells and spleen cells were mixed evenly at a ratio of 1..2-10, centrifuged at 1200r/min for 8min, the supernatant was discarded, and the bottom of the centrifuge tube was gently bounced with the index finger to make the cells evenly dispersed, ready for fusion: within 1 min, pre- Heat 1 mL PEG slowly and gently drip into the mixed cells, gently shake the tube while adding; in the 2 min, hold the tube in the hand and let it stand; 3-4 min, slowly at 1 mL/min 2 mL of RPMI 1640 was added dropwise, while shaking the tube; while in the 5-6 min, 4 mL of RPMI 1640 was slowly added dropwise at a rate of 1 mL/30 s, while shaking; and within 7 min, at a rate of 1 mL/10 s. RPMI 1640. Finally, add RPMI 1640 to 20mL, seal the centrifuge tube with the sealing membrane and put it in the incubator for 5min, centrifuge at 800r/min for 8min, discard the supernatant, gently bounce the bottom of the centrifuge tube with the index finger to spread the cells, slowly join The medium was selected by HAT, gently mixed, and transferred to a 96-well cell plate at 200 μL/well (the number of plating plates was determined according to the number of cells), and cultured in a CO ₂ incubator.

On the third day after cell fusion, the HAT selection medium was semi-exchanged, and on the sixth day, the HAT medium was completely exchanged, and on the seventh day, the cell supernatant was taken for cell selection.

On the 7th day after the fusion, according to the titer of the mouse antiserum and the inhibition assay result, the coating concentration required for the cell screening was determined, and the plate was closed. First, the cell positive assay is performed, and the cell supernatant of the positive well is taken out, and the inhibition assay is performed. The inhibition is determined by appropriate dilution so that the OD ₄₅₀ values of all the wells are as high as about 1.5, and a certain concentration is added to the 96-well plate. The methylene blue standard and the standard dilution, 50 μL/well, were added to the diluted cell supernatant at 50 μL/well, incubated at 37 ° C for 30 min in an oven, and then the secondary antibody was added to the plate by a conventional ELISA method until the absorbance was measured. According to the test results, 4-6 wells with higher inhibition positives were selected for the first subcloning. After three subcloning, the selected single cells were expanded and cultured in RPMI 1640 medium containing 10% fetal bovine serum. When the cells are spread over the bottom of the culture flask and the cells are in the logarithmic growth phase, the cells are collected for cryopreservation and preparation of ascites.

Example 2 Preparation and purification of specific monoclonal antibodies

1. Preparation of monoclonal antibodies

Ascites was prepared by in vivo induced ascites tumor method. Paraffin oil is sterilized for use. BALB/c mice in good condition were selected, and each mouse was intraperitoneally injected with 0.5 mL of sterile paraffin oil for pre-stimulation. Paraffin oil is injected because paraffin oil can reduce the immunity of mice, weaken the rejection of injected hybridoma cells, and stimulate the production of interleukins in mice, providing a good growth environment for injected hybridoma cells. Conducive to the production of ascites. After 7-10 days, the expanded hybridoma cells were collected, washed three times with RPMI 1640 medium, and then injected into the abdominal cavity of the mouse at 0.5 mL/tube. After 7-10 days, the mice began to produce ascites tumors. The state of the mice was observed at any time. After the abdominal cavity was swollen, ascites was taken from the abdominal cavity of the mice with a 5 mL syringe, dispensed into a 1.5 mL centrifuge tube, and centrifuged at 8000 r/min for 10 min. The supernatant was aspirated, dispensed into a 1.5 mL sterile tube, and stored at -20 ° C until use.

2. Monoclonal antibody purification

Ascites was purified by caprylic acid-ammonium sulfate precipitation. Take 1 mL of ascites in a centrifuge tube, add an equal volume of sodium acetate solution (0.01M, pH 4.0), mix and shake, slowly add 33 μL of n-octanoic acid, seal the sealing film, and place on a shaker at room temperature for 30 min. After centrifugation at 8000 r/min for 10 min, the supernatant was taken to a new 1.5 mL centrifuge tube and an equal volume of saturated ammonium sulfate solution was added. After standing overnight in a refrigerator at 4 ° C, the precipitate was sufficiently precipitated, centrifuged at 8000 r/min for 15 min, and the supernatant was discarded. The pellet was reconstituted with 1 mL of potassium salt-containing PBS (0.01 M, pH 7.4), placed in a dialysis bag boiled in water, dialyzed against 0.01 M PBS dialysate for 3 days, and dialyzed every 12 hours. After the end, it was dispensed in a 1.5 mL sterile tube and stored at -20 ° C until use.

Example 3 Antibody Performance Evaluation

1, subtype determination

Subtypes of prepared monoclonal antibodies were tested using a purchased subtype identification kit. The specific process is as follows: Add 0.2μg/mL of diluted antibody to the microplate coated with 0.1μg/mL, 50μL/well, incubate in an oven at 37°C for 30min, wash the plate and dry it, respectively Add IgM, IgG1, IgG2a, IgG2b, IgG3, IgA and other enzyme-labeled secondary antibody types, 100μL/well, incubate in an oven at 37°C for 30min, and stop the coloration after washing the plate, and the enzyme standard corresponding to the color-developing hole The type of resistance is the A subtype of a given antibody.

2. Cross reaction (CR)

Azure A, Azure B, Azure C, Malachite Green and Crystal Violet were separately added to the C4 secreting antibody at a concentration of 10 μg/mL for cross-reactivity test. The results are shown in Table 1 below, and C4 secreted antibodies against other structural analogs. No cross-reaction, highly specific.

Table 1 Cross-reaction of anti-methylene blue monoclonal antibody

3. Sensitivity determination

The sensitivity of the antibody is expressed by the half-inhibitory concentration (IC ₅₀ ). IC ₂₀ - IC ₈₀ represents the linear range of the method.

Both the conjugate (MB-HS-KLH) and the conjugate (MB-HS-OVA) can be obtained by an activated ester method.

The methylene blue-succinic anhydride hapten was synthesized by sodium dithionite reduction, and then used as an immunogen by an activated ester method and hemocyanin coupling to obtain a monoclonal antibody specifically recognizing methylene blue.

The original concentration of the coating was 0.1 μg/mL, the protein concentration of the antibody was 0.3 μg/mL, and the standard methylene blue was diluted to a series of concentrations: 0, 2.333, 3, 21, 63, 189, 567 ppb, and then subjected to indirect competition ELISA. The OD _{450 was} determined by fitting the data using the origin software to obtain a standard curve of the methylene blue monoclonal antibody, as shown in Fig. 1, the IC ₅₀ was 21.13 ng/mL, and the linear range (IC ₂₀ -IC ₈₀ ) was 4.26-104.83 ng/mL. The correlation coefficient is 0.997.

Although the present invention has been disclosed in the above preferred embodiments, the present invention is not limited thereto, and various modifications and changes can be made thereto without departing from the spirit and scope of the invention. The scope of the invention should be determined by the scope of the claims.

Claims

A monoclonal cell line C4 capable of secreting a monoclonal antibody recognizing methylene blue was deposited on January 20, 2016 at the General Microbiology Center of the China Microbial Culture Collection Management Committee, referred to as CGMCC, at Beichen West Road, Chaoyang District, Beijing. No. 3, No. 3, Institute of Microbiology, Chinese Academy of Sciences, with the preservation number CGMCC No.12026.
A specific monoclonal antibody recognizing methylene blue, which is secreted by the monoclonal cell line C4 of the accession number CGMCC No. 12026 of claim 1.
Use of a specific monoclonal antibody recognizing methylene blue according to claim 2, characterized in that it is used for preparing an immunological tool for detecting methylene blue or directly for detecting methyl blue.
Use of the monoclonal cell line C4 according to claim 1, characterized in that it is used for the preparation of an immunological tool for detecting methylene blue or directly for detecting methyl blue.
The use according to claim 4, characterized in that it is used for detecting methyl blue in foods.
A tool for detecting methylene blue, which comprises the monoclonal cell line C4 of claim 1 or the specific monoclonal antibody of claim 2.
A tool for detecting methylene blue according to claim 6, wherein the tool is based on a kit of enzyme-linked immunosorbent technology or a test strip based on immunochromatography.
Use of the tool of claim 6 or 7 in the detection of methylene blue.