Marine shellfish toxin monoclonal antibody preparation method

A technology of shellfish toxin and marine shellfish, which is applied in biochemical equipment and methods, chemical instruments and methods, peptides, etc., can solve the problems of screening and consuming standard antigens, and achieve good antigen competitiveness, small antigen consumption, and simple and convenient preparation Effect

Inactive Publication Date: 2010-09-29
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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 synthesize

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, OA

[0021] Bovine Serum Albumin: Bovine Serum Albumi, BSA

[0022] The active ester method was used to prepare the immunogen and detection 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 dicycloethanecarbodiimide in 100 μL Mix well in N,N dimethylformamide (N,N-DMF), incubate at room temperature for 2h, add 4mg bovine serum albumin BSA (dissolved in 50μL 0.1mol / L N 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 was mixed wit...

Embodiment 2

[0033] 1. Preparation of OA-OVA antigen

[0034] Okadaic Acid, OA

[0035] Ovalbumin: Ovalbumin, OVA

[0036] The immunogen and detection original OA-OVA was prepared by the active ester method, and the operation method was as follows: 1 mg okadaic acid OA, 0.155 mg N-hydroxysuccinimide, 0.285 mg N, N dicycloethanecarbodiimide in 100 μL Mix well in N,N dimethylformamide (N,N-DMF), incubate at room temperature for 2h, add 2.5mg OVA (dissolved in 50μL 0.1mol / L N 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’s complete adjuvant an...

Embodiment 3

[0047] 1. Preparation of OA-hIgG antigen

[0048] Okadaic Acid, OA

[0049] Human immunoglobulin: human immunoglobulin G, hIgG

[0050] Prepare the immunogen and detect the original OA-hIgG by active ester method, the operation method is as follows: 1 mg okadaic acid OA, 0.155 mg N-hydroxysuccinimide, 0.285 mg N, N dicycloethane carbodiimide in 100 μL Mix well in N,N dimethylformamide (N,N-DMF), incubate at room temperature for 2h, add 4.0mg hIgG (dissolved in 50μL 0.1mol / L N 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 with the same amount of Freund’s compl...

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PUM

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Abstract

The invention relates to a preparation method of a marine shellfish toxin monoclonal antibody, which is characterized in that: the antigen which is used for preparing small molecule shellfish toxin monoclonal antibody for animal immunization and hybridoma screening is the same conjugate, thereby reducing the trouble and the waste in the different preparation of immunogen and a detection source in the preparation method of a conventional small molecule monoclonal antibody; and a screening method combined with a positive screening method and a negative screening method is adopted for monoclonal antibody hybridoma screening, so that the consumption of a large amount of standard antigen can be reduced and the antibody secreted by the screened hybridoma has better antigen competition activity. The marine shellfish toxin monoclonal antibody preparation method has the benefits of simple antigen preparation, small used amount of standard antigen, less cost and good antibody competition activity.

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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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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