Brevetoxin B hapten, artificial antigen, monoclonal antibody, preparation method of brevetoxin B hapten, preparation method of artificial antigen and application of artificial antigen or monoclonal antibody

A monoclonal antibody and artificial antigen technology, applied in the biological field, can solve the problems of high requirements for professional and technical personnel, expensive equipment, interference, etc., and achieve the effects of good immune effect, rapid method and reduced detection cost.

Active Publication Date: 2020-05-12
NATIONAL MARINE ENVIRONMENTAL MONITORING CENTRE
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  • Application Information

AI Technical Summary

Problems solved by technology

Due to the background interference of the co-elution in the HPLC method, there are problems in the sensitivity and selectivity of the ultraviolet detection, and the detection of the mass spectrometry (LC-MS) can improve the selectivity and sensitivity, but the equipment is expensive and requires high professional technical personnel.
Immunotechnical detection is based on the principle of antigen-antibody reaction, but antibodies are often only established against a certain toxin, so they often show low cross-reactivity to other shellfish toxins, and cannot detect all shellfish toxin components

Method used

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  • Brevetoxin B hapten, artificial antigen, monoclonal antibody, preparation method of brevetoxin B hapten, preparation method of artificial antigen and application of artificial antigen or monoclonal antibody
  • Brevetoxin B hapten, artificial antigen, monoclonal antibody, preparation method of brevetoxin B hapten, preparation method of artificial antigen and application of artificial antigen or monoclonal antibody
  • Brevetoxin B hapten, artificial antigen, monoclonal antibody, preparation method of brevetoxin B hapten, preparation method of artificial antigen and application of artificial antigen or monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Synthesis of brevetoxin B group hapten

[0030] 50 mg, brevetoxin-3 (PbTx-3) (purchased from China Taiwan Algae Research Co., Ltd., BC-M-001), dissolved in 2 mL of acetone, added 16 mg carbonyldiimidazole (CDI) (purchased from sigma company, catalog number 115533) in the above solution, stirred by magnetic force, and reacted at 37°C for 2h. Afterwards, 0.1 mL of double distilled water was added, reacted for 10 min, and dried under nitrogen. Dissolve in 10 mL of ethyl acetate, wash with 1 mM dilute hydrochloric acid twice, dry over sodium sulfate, and blow dry under nitrogen to obtain the brevis group B hapten.

Embodiment 2

[0031] Example 2: Identification of brevetoxin B group haptens

[0032] The brevedinin B group hapten obtained in Example 1 was sent to Beijing Zhongkehuiren Technology Co., Ltd. for NMR detection.

[0033] The result is as figure 1 as shown, 1H NMR: δ1.02(3H,d,J=7.0Hz),1.10(3H,s),1.21(3H,s),1.25(3H,s),1.27(3H,s),1.29(3H,s ),1.61-1.83(2H,1.76(ddd,J=13.2,10.2,2.7Hz),1.69(ddd,J=13.6,10.2,8.0Hz)),1.90-2.52(23H,2.14(dddd,J=13.8 ,3.2,2.9,2.7Hz),2.16(ddd,J=14.6,9.2,7.6Hz),2.20(ddd,J=14.5,4.6,4.4Hz),2.16(ddd,J=14.5,1.9,1.8Hz) ,2.11(dd,J=14.8,10.2Hz),2.18(dd,J=14.8,3.4Hz),2.23(ddd,J=14.6,6.8,5.7Hz),2.38(ddd,J=14.6,8.7,7.1 Hz), 2.37(dd, J=12.9, 2.8Hz), 2.47(dd, J=12.9, 2.2Hz), 2.19(ddqd, J=10.2, 8.0, 7.0, 4.2Hz), 2.04(ddd, J=13.6 ,4.2,2.7Hz),2.22(s),2.23(dd,J=14.6,7.3Hz),2.28(dd,J=14.6,6.4Hz),2.07(ddd,J=13.2,3.2,3.0Hz), 2.22(ddd, J=14.8,10.1,9.7Hz),2.17(ddd,J=14.8,4.4,3.5Hz),2.19(ddd,J=14.8,3.5,3.2Hz),2.42(ddd,J=14.8, 10.2, 10.2Hz), 2.00 (dddd, J=13.8, 10.2, 10.1, 3.0Hz)), 2.17 ...

Embodiment 3

[0034] Example 3: Preparation of brevetoxin B group artificial antigen

[0035] (a) Take brevedinin B group hapten 5mg prepared in Example 1, 0.2mL, dissolve in DMSO to obtain solution a;

[0036] (b) 0.01 M, pH 9.6 carbonate buffer solution dissolves 25 mg of carrier protein (KLH, purchased from sigma company, product number H7017; OVA, purchased from sigma company, product number A5503), the concentration is 20 mg / mL, to obtain solution b ;

[0037] (c) Add solution a to solution b dropwise, stir, react at room temperature for 3 hours, then transfer to a refrigerator at 2-8°C, and continue to react for 8-12 hours. After the reaction, 0.01M, pH 7.4, PBS dialysis for three days, Aliquot and store at -20°C to obtain the coupling protein brevedinin-3-KLH and the coupling protein brevedinin-3-OVA.

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Abstract

The invention discloses a brevetoxin B hapten, an artificial antigen, a monoclonal antibody, a preparation method of the brevetoxin B hapten, a preparation method of the artificial antigen and an application of the artificial antigen or the monoclonal antibody. The brevetoxin B hapten retains a basic parent nucleus chemical structure of the brevetoxin B. Hydroxyl groups of brevetoxin-3 react withcarbonyl diimidazole to convert the carbonyl diimidazole into ester-based imidazole, and an introduced active group is easily coupled with amino groups of a carrier protein. The synthesis method is simple, and the product yield is high. The universal hapten is used as a raw material to prepare an artificial antigen system suitable for animal immunization. The brevetoxin-3 monoclonal antibody obtained by a monoclonal antibody technology has high cross reactivity with the brevetoxin-3 B, has strong affinity, high sensitivity and good antibody titer, and is a broad-spectrum monoclonal antibody ofthe brevetoxin-3 B.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a brevetoxin B group hapten, an artificial antigen, a monoclonal antibody and a preparation method and application thereof. Background technique [0002] Neurotoxic shellfish poisoning toxins (NSP toxins) mainly refer to a class of toxins isolated from a red tide organism, Ptychodiscus breve - brevetoxins (PbTx). The physicochemical properties and pharmacological properties are similar to that of ciga toxin (CTX). PbTx can cause massive death of fish, molluscs, crustaceans, sponges, echinoderms and benthic algae. After the poisonous Gymnodinosa brevis is ingested by shellfish, its toxins accumulate in the body and are transmitted to humans through the food chain, causing food poisoning. The main symptoms of poisoning are dilated pupils, irregular body temperature, nausea, vomiting, diarrhea, and abnormal movements, but there is no sense of paralysis. Therefore, to dist...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D493/22C07K14/765C07K14/77C07K14/795C07K1/107C07K16/44G01N33/577
CPCC07D493/22C07K14/795C07K14/765C07K14/77C07K16/44G01N33/577G01N2333/405Y02A50/30
Inventor 王睿睿
Owner NATIONAL MARINE ENVIRONMENTAL MONITORING CENTRE
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