CN106754742B

CN106754742B - Hybridoma cell strain of anti-microcystin antibody and monoclonal antibody secreted by same

Info

Publication number: CN106754742B
Application number: CN201710212862.4A
Authority: CN
Inventors: 齐红莉; 王睿睿; 徐海龙; 王茜; 贾旭颖; 周文礼
Original assignee: Tianjin Agricultural University
Current assignee: Tianjin Agricultural University
Priority date: 2017-04-01
Filing date: 2017-04-01
Publication date: 2020-04-24
Anticipated expiration: 2037-04-01
Also published as: CN106754742A

Abstract

The invention discloses a hybridoma cell strain of an anti-microcystin antibody and a monoclonal antibody secreted by the same, wherein the preservation number of the cell strain is CGMCC NO. 13802. The BALB/c mouse is immunized by using the microcystin-LR-BSA after coupling the BSA and the microcystin-LR, the spleen of the BALB/c mouse is taken to be fused with Sp2/0 myeloma cells, and the BALB/c mouse is selectively cultured by using HAT culture medium. The antigen microcystin-LR-OVA after coupling of OVA and microcystin-LR is used as a coating antigen for enzyme-linked immunosorbent assay, an ELISA method is used for detecting the antibody in the culture supernatant of the hybridoma cells, and the hybridoma cell strain 6G7 which stably secretes the anti-microcystin antibody is obtained by screening. The monoclonal antibody can specifically recognize MC-LR, and the 50% inhibition concentration IC50 of the monoclonal antibody to the MC-LR is 0.26 ng/mL. Can be used for researching the biological activity of the microcystin, establishing an immunological detection method and preparing a sample pretreatment reagent.

Description

Hybridoma cell strain of anti-microcystin antibody and monoclonal antibody secreted by same

Technical Field

The invention belongs to the fields of biotechnology and cell engineering, and particularly relates to a hybridoma cell strain secreting an anti-microcystin antibody and a monoclonal antibody secreted by the hybridoma cell strain.

Background

Microcystin (MC) is a secondary toxic metabolite generated by explosive reproduction of cyanobacteria in water, and MC is hepatotoxin and can be accumulated in digestive glands of mussels and scallops and enter high-grade nutritional organisms such as fish, birds, mammals and people along a food chain to cause poisoning and even death. MC is a cyclic heptapeptide compound with biological activity, and has more than 60 isomers due to different compositions of two variable amino acids in polypeptide. The 3 most common and abundant microcystins MC-LR, MC-RR and MC-YR (L, R, Y represents leucine, arginine and tyrosine, respectively) exist. The toxicity of MC is related to the structure thereof, Adda is an essential group for expressing the toxicity of MC, and researches show that the acute toxicity of MC-LR is the strongest, MC-YR is the second, MC-RR is the weakest, and the above is the most widely distributed hepatotoxin. The microcystin is mainly produced by fresh water algae, namely Microcystis aeruginosa (Microcysticerinunospora), has quite high stability, can strongly inhibit the activity of protein phosphatase, and also has a strong liver cancer promoter.

The accessory of the No. 2001 document 161 of the Ministry of health supervision and administration and the environmental quality standard of surface water GB 3838-.

At present, HPLC-MS or LC-MS and other detection methods are mostly adopted for detecting the phycotoxin in the water body, the detection technology is complex in operation, long in time consumption, high in price and difficult to meet the requirement for precision, the frequency of conventional detection is limited, and the immunodetection technology has a good application prospect in the field of detection of the phycotoxin and is paid attention by researchers at home and abroad.

Disclosure of Invention

The invention aims to solve the technical problem of providing a hybridoma cell strain of an anti-microcystin antibody and a monoclonal antibody secreted by the same.

In order to solve the technical problems, the invention adopts the technical scheme that: a hybridoma cell strain of an anti-microcystin antibody has a preservation number of CGMCC NO. 13802.

The monoclonal antibody for resisting microcystin secreted by the hybridoma cell strain.

The monoclonal antibody is an IgG2b antibody, and the light chain of the antibody is a kappa subtype.

The invention has the beneficial effects that: the antigen preparation method is simple and feasible, and the prepared complete antigen immunized BALB/c mouse generates better immune response; the microcystin monoclonal antibody provided by the invention has strong specificity and simple preparation method, and is used for research of microcystin and establishment of an immunological detection method of microcystin; obtaining hybridoma cell strain 6G7 which stably secretes microcystin monoclonal antibody, and measuring the ELISA titer of 6G7 ascites to be 6.05 multiplied by 10⁵. The monoclonal antibody is identified as an IgG2 b-class antibody, and the light chain of the antibody is a kappa subtype.

Drawings

FIG. 1 shows the electrophoresis of the purified monoclonal antibody (lane 1 is the microcystin antibody).

FIG. 2 is a graph of the semi-inhibitory concentration of microcystin.

Detailed Description

The hybridoma cell strain for resisting the microcystin antibody is preserved in China general microbiological culture Collection center (CGMCC NO. 13802) of China Committee for culture Collection of microorganisms, and the preservation date is as follows: 2017-02-23, and classification and naming: murine hybridoma cell lines.

The preservation number of the hybridoma cell strain of the anti-microcystin antibody is CGMCC NO. 13802.

The monoclonal antibody is a monoclonal antibody 6G7 hybridoma cell strain which takes Bovine Serum Albumin (BSA) as a carrier protein and is coupled with microcystins to prepare a complete antigen, immunizes BALB/c mice and utilizes a hybridoma technology to specifically recognize the microcystins.

The preparation method comprises the following steps:

1) preparation of immune antigen MC-LR-BSA

Preparing dimethyl sulfoxide (DMSO) solution of microcystin-LR, and adding N-hydroxysuccinamide (NHS) and 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) to activate MC-LR. Slowly dripping the solution into Bovine Serum Albumin (BSA) solution to perform coupling of the microcystins and the BSA, and dialyzing a reaction product by using Phosphate Buffer Solution (PBS) to obtain a conjugate of the microcystins and carrier protein.

2) Immunization of mice

The present invention employs a low dose long cycle immunization regimen. The coupling protein MC-LR-BSA is used as an immunizing antigen to immunize female BALB/c mice with the age of 4-6 weeks. BALB/c mice were immunized 6 times at an immunization dose of 30. mu.g/mouse, and three days after the last immunization, spleens of the mice were harvested and used for cell fusion.

3) Cell fusion

Mixing Sp2/0 myeloma cells with spleen cells of immunized BALB/c mouse at a certain ratio, fusing with 50% polyethylene glycol 1500(PEG 1500), and culturing in DMEM medium containing 20% FCS and HAT at 5% CO₂After culturing in an incubator for 14 days, the cells are gradually cultured in DMEM medium containing 20% FCS and HT and DMEM medium containing 20% FCS, and when the fused cells grow to 1/7 in the bottom area of a 96-well plate, the supernatant is subjected to detection of monoclonal antibodies against microcystins.

4) Hybridoma selection

Using carbonate buffer solution of microcystin-ovalbumin (MC-LR-OVA) as coating antigen, 0.8% gelatin solution as sealing solution, horse radish peroxidase labeled goat anti-mouse IgG as second antibody diluted 1:12000, TMB-H₂O₂Taking a substrate, taking a 2M sulfuric acid solution as a stop solution, taking PBST as a washing solution, taking positive reference serum diluted by 1:500 and negative reference serum diluted by 1:500 as a control, performing indirect enzyme-linked immunosorbent assay (ELISA), detecting the secretion condition of the anti-microcystin antibody in the supernatant of the hybridoma cells, and screening the positive hybridoma cells secreting the anti-microcystin antibody. OD450nm values were measured by a microplate reader: to emptyAnd (3) zero adjustment is carried out on the white control, when the ratio of the OD450nm value of the positive reference serum to the OD450nm value of the negative reference serum is more than or equal to 2.1, the detection hole is judged to be positive, when the ratio of the OD450nm value of the positive reference serum to the OD450nm value of the negative reference serum is less than 1.5, the detection hole is judged to be negative, and the detection hole is judged to be suspicious when the ratio is in the middle.

5) Subcloning of hybridoma cells by limiting dilution method

Firstly, staining and counting positive well living cells by trypan blue, diluting the positive well living cells into 100 cells/15 mL culture medium by DMEM complete culture medium, adding diluted cell suspension into a 96-well cell culture plate to reach 1 cell per well, and carrying out 5% CO treatment at 37 DEG C₂Culturing in an incubator. And taking cell supernatant when culturing for 8-9 days, and timely carrying out ELISA detection. Selecting positive monoclonal cells, carrying out subcloning for more than 3 times until supernatant detection of all holes of the cells is positive and OD (optical density) values of all holes are relatively close, and carrying out expanded culture and cryopreservation on the positive cells after multiple times of subcloning culture.

6) Preparation of anti-microcystin monoclonal antibody ascites

BALB/c mice were intraperitoneally inoculated with liquid paraffin, 0.5mL per mouse. Two weeks later, the peritoneal cavity was inoculated with hybridoma cells diluted in serum-free medium, 5X 10 per mouse⁵0.2 mL. After 8 days, observing the ascites generation condition of the mice every day, when the ascites is as much as possible and the mice are dying, killing the mice, and sucking the ascites out under the aseptic condition. Standing at room temperature for 30min, centrifuging at 10000rpm for 10min, collecting supernatant, subpackaging, and freezing at-70 deg.C for use.

7) Purification of monoclonal antibody against microcystin

The purification scheme is an octanoic acid-ammonium sulfate precipitation method. Adding 2 parts of 0.06mol/L acetic acid buffer solution with pH4.0 into 1 part of pretreated ascites, and adjusting the pH to 4.5 by using 1mol/L HCl; adding 11 mu L of octanoic acid into diluted ascites per ml, dropwise adding octanoic acid under stirring at room temperature within 30min, standing at 4 deg.C for 2 hr, centrifuging at 12000rpm for 30min, and removing precipitate; filtering the supernatant with a nylon sieve (125 μm), adding 1/10 volumes of 0.1mol/L PBS, and adjusting pH to 7.2 with 1mol/L NaOH; adding saturated ammonium sulfate at 4 deg.C to 45% saturation, mixing gently for 30min, and standing for 1 hr; centrifuging at 12000rpm for 30min, and discarding the supernatant; dissolving the precipitate in a proper amount of PBS, dialyzing the PBS with 50-100 times of volume, and standing at 4 ℃ overnight; taking out, centrifuging at 12000rpm for 30min, removing insoluble precipitate, packaging, and freezing for use.

8) Determination of ascites titer of monoclonal antibody against microcystin

Diluting the monoclonal antibody ascites with PBS buffer solution at a ratio of 1: 100, then diluting by multiple times, adding an enzyme label plate coated with MC-LR-OVA, incubating at 37 ℃ for 30 min; PBST washing for 3 times, each time for 1min, and drying for the last time; adding HRP-labeled goat anti-mouse IgG diluted 1:12000 at a concentration of 100. mu.L/well, incubating at 37 ℃ for 30 min; PBST is washed for 5 times for 1min each time, and is dried by beating for the last time; adding substrate TMB-H₂O₂Developing at room temperature in dark at 100 μ L/well for 15 min; adding 50 mu L of 2mol/L sulfuric acid into each hole to terminate the reaction; OD450nm values were determined using a microplate reader. The ascites titer of the monoclonal antibody is determined to be positive by the negative OD450nm value being less than 0.2 and the ratio of the detection well to the negative OD450nm value being more than 2.1, and the maximum dilution of the positive well is used as the ascites titer of the monoclonal antibody.

9) Half-inhibitory concentration IC of anti-microcystis monoclonal antibody₅₀Measurement of (2)

The concentration of the coating antigen is 0.1 mug/mL, the dilution multiple of the anti-MC-LR monoclonal antibody is 65000 times, the concentration of the enzyme-labeled secondary antibody is 1:12000 for dilution, the antigen-antibody reaction time is 35min, the enzyme-labeled reaction time is 30min, the TMB substrate color development time is 15min, and the drug concentration setting range of the MC-LR standard substance is 0, 0.05, 0.1, 0.2, 0.4, 0.8 and 1.6 ng/mL. The reaction was stopped by adding 50. mu.L of 2mol/L sulfuric acid per well. OD450nm values were determined using a microplate reader. Performing statistics by enzyme immune software to calculate IC₅₀Value of

10) Identification of antibody subtypes

The anti-microcystin monoclonal antibody was sub-type tested using a commercial kit.

The invention can provide necessary components and technical means for the research of microcystin antigenicity, the establishment of a microcystin immunological detection method and the preparation of a pretreatment reagent of a detected sample by preparing the monoclonal antibody, and has important significance for exploring the biological functions of each structural region of MC and establishing a specific and sensitive detection method of MC.

The present invention will be described in further detail with reference to specific embodiments below:

EXAMPLE 1 preparation of immunoantigen microcystin-BSA

Activation of microcystins: dissolving 1mg of microcystin-LR in 0.05ml of MSO solution, respectively weighing NHS and EDC, adding into the above solution (molar ratio of microcystin: NSH: EDC is 1: 2), and shaking vigorously at room temperature in the dark for 1 h. Coupling of microcystins: the solution is slowly dripped into BSA solution (5mg protein is dissolved in 1mL of 0.1mol/L sodium bicarbonate solution), and is shaken for 2h at room temperature in the dark; and dialyzing the reaction product in 0.01mol/L PBS buffer solution at 4 ℃ in the dark for 72h, and replacing the dialyzate once every 6h to obtain the conjugate of the microcystin and the carrier protein.

EXAMPLE 2 establishment of hybridoma cell lines

1) Immunization of mice

The present invention employs a low dose long cycle immunization regimen. The coupling protein MC-LR-BSA is used as an immunizing antigen to immunize female BALB/c mice with the age of 4-6 weeks. Mixing the mixture with Freund's complete adjuvant 1: 1(V/V) at an immunization dose of 30 μ g/piece (based on protein content), emulsifying for 50min to obtain water-in-oil state, and effectively emulsifying without dissolving in water or layering overnight at 4 deg.C. The emulsified complete antigen, the neck and back, was injected subcutaneously at multiple points with immunized BALB/c mice (30. mu.g/mouse); after 2 weeks, emulsification with the same antigen and an equal volume of Freund's incomplete adjuvant was performed at 30. mu.g/booster; after 6 immunizations, 100. mu.g/mouse was boosted by intraperitoneal injection without adjuvant. From the third time, blood is collected from tail vein or eyeball after one week of each immunization, the mixture is kept stand at 37 ℃ for 1h, serum is separated out overnight at 4 ℃, the mixture is centrifuged at 5000rpm for 10min, and the serum is separated to test the serum titer. Three days after the last immunization, the spleen of the mouse was harvested and cell fusion was performed.

2) Cell fusion

Mixing Sp2/0 myeloma cells and immunized BALB/c mouse spleen cells in a ratio of 1: 5-1: 10 in a 50mL centrifuge tube, fully and uniformly mixing, centrifuging at 1000rpm for 10min, discarding supernatant, slightly pressing the bottom of the tube with palm to loosen and uniformly mix the cells, preheating in 38 ℃ water bath, and using a palm to preheat1mL of 50% PEG 1500 pre-warmed to 38 ℃ was added to a 1mL pipette over 60s with gentle shaking, 30mL of DMEM incomplete medium pre-warmed to 37 ℃ was added to 90s, the mixture was left at room temperature for 10min, centrifuged at 1000rpm for 10min, the supernatant was discarded, and 20mL of DMEM medium containing 20% FCS and HAT was added to the mixture to resuspend the mixture. Subpackaging the obtained mixture in 96-well plates with existing feeder cells in 5% CO₂Culturing in an incubator, observing the growth condition of the cells after 7d, and replacing 1/2 culture medium with DMEM culture medium containing 20% FCS and HT; after 14d, 20% FCS and HT DMEM medium were used for culture, and when the fused cells were grown to 1/7 in the bottom area of the well of the 96-well plate, the supernatant was taken for antibody detection.

3) Screening of anti-microcystin hybridoma

Coating the coupled coating antigen (MC-LR-OVA) on an ELISA plate by using a coating solution of 0.05mol/L pH 9.6 carbonate buffer solution in a dilution manner at a multiple ratio, coating the ELISA plate by 100 mu L/hole, and coating the ELISA plate at 4 ℃ overnight; PBST washing for 3 times, each time for 1min, and drying for the last time; sealing each hole with 0.8% gelatin solution at 300 μ L/hole, standing at 37 deg.C for 2 h; PBST washing for 3 times, each time for 1min, and drying for the last time; adding cell supernatant, positive reference serum diluted 1:500 and negative reference serum diluted 1:500 into corresponding wells, 100 μ L/well, and allowing reaction at 37 deg.C for 60 min; PBST washing for 3 times, each time for 1min, and drying for the last time; adding enzyme-labeled goat anti-mouse IgG diluted at a ratio of 1:12000, at a concentration of 100 μ L/well, standing at 37 deg.C for 60 min; PBST is washed for 5 times, each time for 5min, and is dried by beating for the last time; adding substrate TMB-H₂O₂Developing at room temperature in dark at 100 μ L/well for 15 min; the reaction was stopped by adding 50. mu.L of a 2mol/L sulfuric acid solution to each well. Measuring OD450nm value by an enzyme-labeling instrument: and (3) carrying out zero adjustment by using a blank control, judging that the detection hole is positive when the ratio of the OD450nm value of the positive reference serum to the OD450nm value of the negative reference serum is more than or equal to 2.1, and judging that the detection hole is negative when the ratio of the OD450nm value of the positive reference serum to the OD450nm value of the negative reference serum is less than 1.5, and judging that the detection hole is suspicious when the ratio is in the middle.

4) Subcloning of Positive hybridoma cells by limiting dilution method

And (3) carrying out subcloning on the screened positive hybridoma cells secreting the specific monoclonal antibody by adopting a limiting dilution method in time. Firstly, the first is yangViable cells in sex wells were stained and counted with trypan blue, diluted to 120 cells/15 mL medium with DMEM complete medium, the diluted cell suspension was added to 96 well cell culture plates, 0.15 mL/well, at 37 deg.C, 5% CO₂Culturing in an incubator. 4-5 days later, the formation of the cloned cells can be observed under a microscope, only single cloned growth hole is recorded, cell supernatant is taken at 8-9 days later, and ELISA detection is carried out in time. Selecting positive monoclonal cells, and performing subcloning for more than 3 times until all cell wells are positive in supernatant detection and the detection OD values of all wells are relatively close; the positive cells after multiple times of subclone culture are enlarged and frozen to obtain the hybridoma cell strain which stably secretes the microcystin monoclonal antibody and is named as 6G 7.

EXAMPLE 3 ascites in the production of monoclonal antibodies against microcystin

EXAMPLE 4 purification of monoclonal antibodies against microcystin (see FIG. 1)

The purification scheme is an octanoic acid-ammonium sulfate precipitation method. Adding 2 parts of 0.06mol/L acetic acid buffer solution with pH4.0 into 1 part of pretreated ascites, and adjusting the pH to 4.5 by using 1mol/L HCl; adding 11 mu L of octanoic acid into diluted ascites per ml, dropwise adding octanoic acid under stirring at room temperature within 30min, standing at 4 deg.C for 2 hr, centrifuging at 12000rpm for 30min, and removing precipitate; filtering the supernatant with a nylon sieve (125 μm), adding 1/10 volumes of 0.1mol/L PBS, and adjusting pH to 7.2 with 1mol/L NaOH; adding saturated ammonium sulfate at 4 deg.C to 45% saturation, mixing gently for 30min, and standing for 1 hr; centrifuging at 12000rpm for 30min, and discarding the supernatant; dissolving the precipitate in appropriate amount of PBS, dialyzing 50-100 times volume of PBS, and standing at 4 deg.C overnight; taking out, centrifuging at 12000rpm for 30min, removing insoluble precipitate, packaging, and freezing for use.

Example 5 determination of ascites titer of anti-microcystin monoclonal antibody

Diluting the monoclonal antibody ascites with PBS buffer solution at a ratio of 1: 100, then diluting by multiple times, adding an enzyme label plate coated with MC-LR-OVA, incubating at 37 ℃ for 30 min; PBST washing for 3 times, each time for 1min, and drying for the last time; adding HRP-labeled goat anti-mouse IgG diluted 1:12000 at a concentration of 100. mu.L/well, incubating at 37 ℃ for 30 min; PBST is washed for 5 times for 1min each time, and is dried by beating for the last time; adding substrate TMB-H₂O₂Developing at room temperature in dark at 100 μ L/well for 15 min; adding 50 mu L of 2mol/L sulfuric acid into each hole to terminate the reaction; OD450nm values were determined using a microplate reader. The ascites titer of the monoclonal antibody is determined to be positive by the negative OD450nm value being less than 0.2 and the ratio of the detection well to the negative OD450nm value being more than 2.1, and the maximum dilution of the positive well is used as the ascites titer of the monoclonal antibody. The results showed that the ascites indirect ELISA titer of the 6G7 cell line was 6.05X 10⁵。

Example 6 inhibitory concentration IC of anti-Microcystis monoclonal antibody₅₀Measurement of (2) and half inhibitory concentration Curve plotting (FIG. 2)

The concentration of the coating antigen is 0.1 mug/mL, the dilution multiple of the anti-MC-LR monoclonal antibody is 65000 times, the concentration of the enzyme-labeled secondary antibody is 1:12000 for dilution, the antigen-antibody reaction time is 35min, the enzyme-labeled reaction time is 30min, the TMB substrate color development time is 15min, and the drug concentration setting range of the MC-LR standard substance is 0, 0.05, 0.1, 0.2, 0.4, 0.8 and 1.6 ng/mL. The reaction was stopped by adding 50. mu.L of 2mol/L sulfuric acid per well. OD450nm values were determined using a microplate reader. Performing statistics by enzyme immune software to calculate IC₅₀Values and semi-inhibitory concentration curves were plotted.

Example 7 identification of antibody subtypes

The assay was performed using a commercial murine monoclonal antibody subtype kit. Firstly, diluting ascites with PBS until the concentration of the antibody is 0.1-1 mu g/mL, and adding 150 mu L of the diluted ascites into a test tube. And (3) shaking the vortex tube for 30s at 15-25 ℃ to enable the blue colloid in the tube to be fully suspended, inserting a test strip, and after 5-10 min, referring to a positive control, wherein the blue strip is the subtype type of the antibody. The monoclonal antibody has a subtype of IgG2b, and the light chain is a kappa chain.

The above-mentioned embodiments are only for illustrating the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and to carry out the same, and the present invention shall not be limited to the embodiments, i.e. the equivalent changes or modifications made within the spirit of the present invention shall fall within the scope of the present invention.

Claims

1. A hybridoma cell strain 6G7 for resisting a microcystin antibody is characterized in that the preservation number of the cell strain is CGMCC NO. 13802;

the hybridoma cell strain secretes an anti-microcystin monoclonal antibody;

the monoclonal antibody is a monoclonal antibody 6G7 hybridoma cell strain which takes Bovine Serum Albumin (BSA) as a carrier protein and is coupled with microcystins to prepare a complete antigen, immunizes a BALB/c mouse and utilizes a hybridoma technology to obtain the monoclonal antibody which can specifically identify the microcystins.

2. The monoclonal antibody against microcystin secreted by the hybridoma cell line of claim 1.