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A kind of preparation method of marine shellfish toxin monoclonal antibody

A technology for marine shellfish and shellfish toxins, applied in biochemical equipment and methods, chemical instruments and methods, microorganisms, etc., can solve the problems of screening and consuming standard antigens, achieve good antigen competitiveness, good antibody competition activity, and small antigen consumption Effect

Inactive Publication Date: 2016-08-03
JILIN UNIV
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  • Abstract
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  • Claims
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AI Technical Summary

Problems solved by technology

[0003] The purpose of the present invention is to provide a preparation method of marine shellfish toxin monoclonal antibody, which solves the problem of consuming a large amount of standard antigens in the screening of hybridomas in the process of monoclonal antibody preparation of expensive small molecule marine shellfish toxin, which improves the conventional small molecule monoclonal antibody preparation process In the use of two kinds of antigens and the screening method of antibody hybridoma cells, only one conjugate is needed for the immunization antigen and the detection antigen, which not only saves a large amount of antigen standards, but also has good binding activity between the obtained antibody and the antigen, and can be improved by using The monoclonal antibody of marine shellfish toxin okadaic acid prepared by the latter method, the conjugate of okadaicic acid and carrier protein was synthesized by the active ester method as the immunogen and detection agent, and the BALB / c mice were immunized, and conventional cell fusion Technology The immune spleen cells were fused with the mouse myeloma cell line SP2 / 0, and the antibodies secreted by the fused cells were screened using an improved method; after 3 times of cloning, a hybridoma cell line that could stably secrete OA was obtained. The antibody secreted by this strain of hybridoma belongs to the IgG1 subclass, with a relative molecular mass of about 160kD and an antibody titer of 2.56×10 6 , the antibody affinity was 9.0×10 8 L / mol, no cross-talk with non-diarrheal shellfish poisons such as domoic acid, gynotoxin 1 / 4, gynotoxin 2 / 3, saxitoxin, microcystin, nodularin, tetrodotoxin, etc. reaction, not only save a large amount of standard antigens, but also the obtained antibodies can be used in the establishment of immunological methods such as ELISA

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] 1. Preparation of OA-BSA antigen:

[0020] Okadaic Acid: OkadaicAcid, OA

[0021] Bovine Serum Albumin: BovineSerumAlbumi, BSA

[0022] The active ester method was used to prepare the immunogen and detect the original OA-BSA, and the operation method was as follows: 1 mg okadaic acid OA, 0.155 mg N-hydroxysuccinimide, 0.285 mg N, N dicycloethane carbodiimide in 100 μL N, N Mix well in dimethylformamide (N,N-DMF), incubate at room temperature for 2h, add 4mg bovine serum albumin BSA (dissolved in 50μL 0.1mol / LN a HCO 3 ), continue to incubate at room temperature for 2 h, remove unreacted substances by ultrafiltration and centrifugation, dissolve the conjugated antigen with 400 μL of PBS buffer (pH 7.4) to make the protein concentration 10 mg / mL, and store at -20 °C for later use.

[0023] 2. Animal immunization and titer determination

[0024] Healthy BALB / c female mice were immunized with a low-dose and short-cycle regimen. For the first immunization, 100 μg OA-BSA w...

Embodiment 2

[0033] 1. Preparation of OA-OVA antigen

[0034] Okadaic Acid: OkadaicAcid, OA

[0035] Ovalbumin: Ovalbumin, OVA

[0036] The active ester method was used to prepare the immunogen and detection original OA-OVA, and the operation method was as follows: 1 mg okadaic acid OA, 0.155 mg N-hydroxysuccinimide, 0.285 mg N, N dicycloethane carbodiimide in 100 μL N, N Mix well in dimethylformamide (N,N-DMF), incubate at room temperature for 2 hours, add 2.5mg OVA (dissolved in 50μL 0.1mol / LN a HCO 3 ), continue to incubate at room temperature for 2 h, remove unreacted substances by ultrafiltration and centrifugation, dissolve the conjugated antigen with 250 μL of PBS buffer (pH 7.4) to make the protein concentration 10 mg / mL, and store at -20 °C for later use.

[0037] 2. Animal immunization and titer determination

[0038] Healthy BALB / c female mice were immunized with a low-dose and short-cycle regimen. For the first immunization, mix 100 μg OA-OVA with the same amount of Freund...

Embodiment 3

[0047] 1. Preparation of OA-hIgG antigen

[0048] Okadaic Acid: OkadaicAcid, OA

[0049] Human immunoglobulin: humanimmunoglobulinG, hIgG

[0050] The active ester method was used to prepare the immunogen and detect the original OA-hIgG, and the operation method was as follows: 1 mg okadaic acid OA, 0.155 mg N-hydroxysuccinimide, 0.285 mg N, N dicycloethane carbodiimide in 100 μL N, N Mix well in dimethylformamide (N,N-DMF), incubate at room temperature for 2 hours, add 4.0 mghIgG (dissolved in 50 μL 0.1mol / LN a HCO 3 ), continue to incubate at room temperature for 2 h, remove unreacted substances by ultrafiltration and centrifugation, dissolve the conjugated antigen with 400 μL of PBS buffer (pH 7.4) to make the protein concentration 10 mg / mL, and store at -20 °C for later use.

[0051] 2. Animal immunization and titer determination

[0052]Healthy BALB / c female mice were immunized with a low-dose and short-cycle regimen. For the first immunization, mix 100 μg OA-hIgG wi...

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Abstract

The invention relates to a preparation method of marine shellfish toxin monoclonal antibody, which is characterized in that: the antigen used for preparing small molecule shellfish toxin monoclonal antibody for animal immunity and hybridoma cell screening is the same conjugate, which reduces the preparation of conventional small molecular substance monoclonal antibody In the method, the trouble and waste of different preparations of the immunogen and the detection original are used; and the screening method combined with the positive screening method and the negative screening method is used to screen the monoclonal antibody hybridoma cells, which can reduce the consumption of a large number of standard antigens, and the hybridomas screened by it Antibodies secreted by cells have better antigen competition activity. The positive effect of the invention is that the preparation of the antigen is simple, the dosage of the standard antigen is small, the cost is saved, and the competitive activity of the antibody is good.

Description

Technical field: [0001] The present invention relates to a preparation method of marine shellfish toxin monoclonal antibody, which is used for the screening of hybridoma cell lines in the preparation process of small molecular food pollutant monoclonal antibody, especially for the diarrhea shellfish toxin okadaic acid monoclonal in the marine shellfish toxin Clonal antibody preparation. Background technique: [0002] In recent years, global warming, frequent red tides, and marine shellfish toxins have caused increasingly serious seafood safety issues, which have become major issues worthy of attention in public health and import and export inspection and quarantine. The marine shellfish toxins that cause food-borne poisoning are mostly derived from certain algae. They accumulate in shellfish, snails and other seafood through the food chain, and the toxicity is amplified. Most of the toxins are very stable and cannot be destroyed by general processing. Incidents of poisoning...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/44C12N5/20
Inventor 卢士英任洪林李岩松柳增善周玉孟宪梅李兆辉于师宇林超闫东明王里奇李乐
Owner JILIN UNIV
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