Method for preparing short and naked dinoflagellate toxin monoclone antibody

A monoclonal antibody, Gymnodon brevis technology, applied in the direction of antifungal/algal/lichen immunoglobulin, fermentation, etc., can solve the large demand for antigens, and it takes at least 3.5 months to produce and increase monoclonal antibodies. The cost of cloning antibodies and other issues, to achieve the effect of less antigen, time saving, and fast immunization

Inactive Publication Date: 2009-08-19
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the traditional method of preparing monoclonal antibodies requires at least 4 times of immunization before cell fusion can be performed, and it takes at least 3.5 months to produce monoclonal antibodies; at the same time, the traditional method has a large demand for antigens, generally 200 475 μg / mouse
This greatly increases the cost of monoclonal antibody preparation, especially for expensive marine biotoxins such as BTX. It takes tens of thousands of yuan to prepare a monoclonal antibody

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] [Example 1] Specific preparation of immune antigen (BTX-IgG) and detection antigen (BTX-BSA)

[0020] (1) Preparation of mixed solution: Take 0.5mg BTX, 0.08mg N-hydroxysuccinimide, 0.15mg N, N biscycloethane carbodiimide and mix them in 0.5mL DMF. Incubate for 2 hours to make a mixture, and divide the mixture into two equal parts; (2) Preparation of carrier protein reaction solution: Dissolve 0.4mg IgG in 0.5mL 0.1mol / L NaHCO 3 In the buffer solution, mix it as a carrier protein reaction solution; (3) add the carrier protein reaction solution prepared in step (2) to the mixed solution prepared in step (1), and incubate at 10-30°C for 2h, the obtained The product is the immune antigen (BTX-IgG), and the unreacted substances are removed by ultrafiltration and centrifugation, and stored at -20°C after aliquoting; (4) Take 0.4mg BSA and dissolve it in 0.5mL 0.1mol / L NaHCO 3 In the buffer solution, mix it as a carrier protein reaction solution; (5) add the carrier protein ...

Embodiment 2

[0022] 2. Animal immunization

[0023] (1) The immunized animals were 8-week-old male BALB / C mice, and blood was collected from the tail before immunization as negative serum;

[0024] (2) Prepare immune antigen standard solution 2 μg / μL. For the first immunization, the immune antigen was emulsified with complete Freund's adjuvant, the ratio of antigen to adjuvant was 1:1, and 100 μL / mouse was injected intraperitoneally. (3) After 18 days, intrasplenic injection was used, and the specific method was as follows: the mice were fasted for 24 hours, and each mouse was anesthetized with 100 μL of 2% sodium pentobarbital. Rats were fixed prone on a simple operating table, and the fur on the back was disinfected. A 1 cm long incision was cut down the midline of the body at a place 0.5 cm to the left of the midline of the back and 0.1 cm from the last rib, and the abdominal walls on both sides were gently pressed with hands to expose the spleen. Exit the abdominal cavity, use a 1mL ...

Embodiment 3

[0025] [Example 3] Preparation of reagent source and SP2 / 0 myeloma cell suspension

[0026]BTX is the product of Express Technology Co., Ltd.; human IgG is the product of Thermo; polyethylene glycol (polythylene glycol-4000, PEG), RPMI1640 medium, HAT selective medium, bovine serum albumin (BSA), pentadiene Aldehyde, β-mercaptoethylamine, Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FICA), o-phenylenediamine (OPD), N-hydroxysuccinimide (N-hydroxysuccinimide), N , N-dicycloethanecarbodiimide (N, N-dimethyformamide, DMF) etc. were purchased from Sigma; centrifugal ultrafiltration tubes were MILLPORE products; horseradish peroxidase-labeled goat anti-mouse IgG was purchased from Dingguo Biotechnology Co., Ltd. Company (Beijing), methanol, acetic acid, sodium carbonate, sodium bicarbonate, and chemical reagents used in ELISA are of domestic analytical grade.

[0027] SP2 / 0 myeloma cells were revived and expanded in culture 15 days before fusion. Take out the S...

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Abstract

A method for preparing a short-Gymnodinium toxin monoclonal antibody relates to the preparation of a monoclonal antibody in the immune safety detection technology, which is finished by the following steps: (1) the preparation of a complete antigen, comprising the preparation of an immune antigen (BTX-IgG) and a detection antigen (BTX-BSA); (2) animal immunization; (3) and the preparation of the monoclonal antibody by blending an immune mouse spleen cell with the serum titer greater than 10 and an SP2 / 0 myeloma cell according to a conventional method. The ascites titer of the monoclonal antibody which is prepared by the immune method can be up to 2.4*10, the affinity constant thereof is up to 0.6*10M, and the ascites titer and the affinity constant thereof are not lower than the ascites titer and the affinity constant of the antibody which is prepared by the traditional immune method; the immune speed is fast, thus the cost for preparing the monoclonal antibody is effectively reduced.

Description

[technical field] [0001] The invention relates to the preparation of a monoclonal antibody in immune safety detection technology, and further relates to the preparation of a monoclonal antibody that can be applied to the brevis dinodinogen in the water environment and aquatic products. [Background technique] [0002] Eutrophication of water body causes algae to proliferate abnormally and release biological toxins—AlgaeToxins. These secondary metabolites seriously endanger human water safety and the safety of other aquatic organisms. Among them, Brevetoxins (BTX), which cause paralysis of the nervous system, are highly toxic and harmful algae toxins. Toxins poison fish, shellfish and other aquatic animals through the food chain, and humans are poisoned by ingesting these animals. [0003] BTX is a ladder-type polyether compound with unique pharmacological functions produced by dinoflagellate of the genus Dinoflagellate. BTX is a neurotoxic shellfish poison, which binds to t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/08C07K16/14
Inventor 周玉张媛媛潘风光李岩松卢士英任洪林柳增善
Owner JILIN UNIV
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