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Monoclonal antibody specifically combined with ricin and application thereof

A ricin, specific technology, applied in the field of immunity, can solve the problem of high toxicity

Active Publication Date: 2022-05-31
北京弘进久安生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] RT has a wide range of sources, easy extraction, stable properties, high toxicity, and is easy to be used as a chemical weapon, so it is very easy to be used by criminals or even terrorist organizations, and it is extremely dangerous and threatening

Method used

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  • Monoclonal antibody specifically combined with ricin and application thereof
  • Monoclonal antibody specifically combined with ricin and application thereof
  • Monoclonal antibody specifically combined with ricin and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] The extraction of embodiment 1.RT crude poison

[0033] Take 50g of castor bean and shell it, put it in a pulverizer, add liquid nitrogen to pulverize it, and take it out. Then add 100 mL of 10 mM phosphate buffer (pH 7.2), leaching at 4 °C for 24 h, centrifugation at 17000 g for 40 min at 4 °C, to remove the precipitate and supernatant, collect the supernatant and filter it with three layers of filter paper. Then add saturated ammonium sulfate solution, set at 4°C overnight, centrifuge at 17000g for 40min at 4°C, and collect the precipitate. The obtained precipitate was dissolved in 10 mM phosphate buffer (pH 7.2), and dialyzed with 10 mM phosphate buffer (pH 7.2) overnight, centrifuged at 17,000 g at 4°C for 15 min, and the supernatant obtained was crude ricin.

Embodiment 2

[0034] Example 2. Purification of RT crude virus

[0035] (1) Affinity chromatography

[0036] Add an appropriate amount of crude ricin into the balanced Sepharose 4B medium, mix and absorb it for 1.5 hours, put the medium into the gravity column, connect the constant flow pump and control the flow rate to 70r / min, and use the HD-A chromatogram to collect and analyze The chromatogram was recorded by the instrument. The impurity protein was washed with 10 mM phosphate buffer (pH 7.2), and after the UV curve was flattened, 150 mM galactose-PBS (pH 7.2) was added to elute the protein, and the elution peaks were collected.

[0037] (2) Molecular sieve chromatography

[0038] The collected solution after affinity was loaded onto a Sephacryl S-100HR prepacked column at a flow rate of 0.5 mL / min, eluted with 10 mM phosphate buffer (pH 7.2) buffer, and the elution peaks were collected.

Embodiment 3

[0039] Example 3. RT identification and protein concentration determination

[0040] The collected RT proteins were boiled with β-mercaptoethanol-containing and β-mercaptoethanol-free loading buffers, respectively, and run for SDS-PAGE electrophoresis. It was stained with Coomassie brilliant blue R-250, and the destaining solution was decolorized on a shaking table, and the relative molecular weights of RT, RTA and RTB were determined by analyzing the gel image. The concentration of RT was determined by the BCA method.

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Abstract

The invention discloses an antibody specifically combined with ricin or an active fragment of the antibody, colloidal gold immunochromatography test paper or a kit comprising the antibody and application of the antibody and the colloidal gold immunochromatography test paper or the kit. The test paper or the kit is convenient to produce, stable in effect, free of non-specific reaction, low in coating amount and beneficial to mass production. The method disclosed by the invention is simple, convenient and rapid to operate, good in reproducibility and extremely convenient to use on site, and the establishment of the method not only enriches the existing ricin detection method, but also lays a foundation for the treatment and emergency prevention of ricin poisoning.

Description

technical field [0001] The invention relates to the field of immunity, in particular to an antibody that specifically binds to ricin, and a detection test paper and a kit for detecting ricin. Background technique [0002] Ricin Toxin (RT) is derived from the seeds of the Euphorbiaceae plant Ricinus communis. It is a deadly and highly toxic biological toxin that can cause accidental poisoning and can also be used in criminal attempts. Once a molecule of RT enters the cell, it is enough to stop the whole cell protein synthesis and die, and its toxicity is 6000 times that of cyanide. The toxicity of castor seeds was studied at the end of the 19th century in Schmiedeberg's laboratory in Strasbourg, and subsequently at the Faculty of Medicine in Dorpat (now Tartu), purified from castor bean or filter cake. highly toxic protein and named RT. The molecular weight of RT is about 60kDa. It is a type II ribosome inactivating protein (RIP) composed of A chain (RTA) and B chain (RTB) ...

Claims

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Application Information

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IPC IPC(8): C07K16/16G01N33/543G01N33/558G01N33/68C07K14/415C07K1/22C07K1/30C07K1/14
CPCC07K16/16G01N33/54313G01N33/558G01N33/68C07K14/415C07K2317/56C07K2317/565G01N2333/415Y02A50/30
Inventor 许宏瑞王德鸿魏颖由赛男王福崔贝贝邓鹏应天翼李岩松余涛
Owner 北京弘进久安生物科技有限公司
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