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Anti-microcystin antibody hybridoma cell line and its secreted monoclonal antibody

A technology of hybridoma cell line and microcystin, which is applied in the fields of biotechnology and cell engineering, can solve the problems of limiting the number of conventional detections, taking a long time, and the accuracy is difficult to meet the requirements, etc., and the preparation method is simple and feasible, and the preparation method is simple , the effect of good immune response

Active Publication Date: 2020-04-24
TIANJIN AGRICULTURE COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] At present, HPLC-MS or LC-MS are mostly used for the detection of algal toxins in water bodies. Such detection techniques are often complex in operation, time-consuming, high in price, and difficult to meet the requirements of precision, which limits the number of routine detections. The detection technology has shown a good application prospect in the field of algal toxin detection, and has attracted the attention of researchers at home and abroad.

Method used

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  • Anti-microcystin antibody hybridoma cell line and its secreted monoclonal antibody
  • Anti-microcystin antibody hybridoma cell line and its secreted monoclonal antibody
  • Anti-microcystin antibody hybridoma cell line and its secreted monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0019] Preparation methods include:

[0020] 1) Preparation of immune antigen MC-LR-BSA

[0021] Prepare a solution of Microcystin-LR in dimethyl sulfoxide (DMSO) by adding N-hydroxysuccinamide (NHS) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC ) to activate MC-LR. Slowly add the above solution dropwise to bovine serum albumin (BSA) solution to couple microcystin to BSA, and dialyze the reaction product with phosphate buffered saline (PBS) to obtain a conjugate of microcystin and carrier protein .

[0022] 2) Immunization of mice

[0023] The present invention adopts a low-dose and long-period immunization scheme. 4-6 week-old female BALB / c mice were immunized using coupling protein MC-LR-BSA as an immunizing antigen. BALB / c mice were immunized 6 times at an immunization dose of 30 μg / mouse, and the spleens of the mice were removed three days after the last immunization for cell fusion.

[0024] 3) cell fusion

[0025] Take Sp2 / 0 myeloma cells and immunized BA...

Embodiment 1

[0042] Example 1 Preparation of immune antigen microcystin-BSA

[0043] Activation of microcystin: Dissolve 1mg of microcystin-LR in 0.05mL DMSO solution, measure NHS and EDC respectively and add to the above solution (molar ratio of microcystin: NSH:EDC is 1:2:2) , and shake vigorously for 1 h at room temperature in the dark. Coupling of microcystins: Slowly add the above solution dropwise to BSA solution (5mg protein dissolved in 1mL 0.1mol / L sodium bicarbonate solution), shake at room temperature for 2h in the dark; the reaction product is buffered in 0.01mol / L PBS The solution was dialyzed in the dark at 4°C for 72 hours, and the dialysate was changed every 6 hours to obtain a conjugate of microcystin and carrier protein.

Embodiment 2

[0044] The establishment of embodiment 2 hybridoma cell lines

[0045] 1) Immunization of mice

[0046] The present invention adopts a low-dose and long-period immunization scheme. 4-6 week-old female BALB / c mice were immunized using coupling protein MC-LR-BSA as an immunizing antigen. According to the immunization dose of 30 μg per mouse (according to the protein content), mix it with Freund’s complete adjuvant 1:1 (V / V), emulsify for 50 minutes, and reach the state of water-in-oil, insoluble in water or overnight at 4°C without stratification Considered effective emulsification. The emulsified complete antigen was injected subcutaneously at multiple points on the back of the neck to immunize BALB / c mice (30 μg / mouse); 2 weeks later, the same antigen and an equal volume of Freund’s incomplete adjuvant were emulsified, and 30 μg / mouse was boosted; After 6 times of immunization, booster immunization was performed by intraperitoneal injection at 100 μg / rat without adjuvant. ...

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Abstract

The invention discloses a hybridoma cell strain for an anti-microcystic toxin antibody and a monoclonal antibody secreted by the hybridoma cell strain, and the preservation number of the cell strain is CGMCC NO.13802. Microcystic toxin-LR-BSA coupled by BSA and microcystic toxin-LR is used for immunizing a BALB / c mouse, a spleen of the BALB / c mouse is taken, cell fusion is carried out on the spleen and Sp2 / 0 myeloma cells, and selective culture is carried out with an HAT culture medium. Antigen microcystic toxin-LR-OVA coupled by OVA and microcystic toxin-LR is taken as a coating antigen for enzyme-linked immunosorbent assay, an ELISA method is adopted for detecting antibodies in hybridoma cell culture supernatant, and screening is carried out, so that a hybridoma cell strain 6G7 stably secreting the anti-microcystic toxin antibody is obtained. The monoclonal antibody disclosed by the invention can specifically identify MC-LR, and 50% inhibiting concentration IC50 on MC-LR is 0.26ng / mL; and the monoclonal antibody can be used for study of bioactivity of microcystic toxin, establishment of an immunological detection method and preparation of a sample pretreatment reagent of the microcystic toxin.

Description

technical field [0001] The invention belongs to the field of biotechnology and cell engineering, and in particular relates to a hybridoma cell line secreting anti-microcystin antibody and the secreted monoclonal antibody. Background technique [0002] Microcystin (MC) is a secondary toxic metabolite produced by the explosive reproduction of cyanobacteria in water bodies. MC is a hepatotoxin that can accumulate in the digestive glands of mussels and scallops and enter fish, birds, and mammals along the food chain. Animals and humans and other advanced nutritional organisms, causing poisoning and even death. MC is a kind of biologically active cyclic heptapeptide compound. Due to the difference in the composition of two variable amino acids in the polypeptide, there are more than 60 isomers. Among them, MC-LR, MC-RR, and MC-YR are the most common and most abundant microcystins (L, R, and Y represent leucine, arginine, and tyrosine, respectively). The toxicity of MC is relate...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/14C12R1/91
CPCC07K16/14
Inventor 齐红莉王睿睿徐海龙王茜贾旭颖周文礼
Owner TIANJIN AGRICULTURE COLLEGE
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