Colloidal gold test paper for jointly detecting abrin and ricin and special monoclonal antibody

A technology of abrin toxin and monoclonal antibody, which is applied in measuring devices, anti-animal/human immunoglobulins, instruments, etc., can solve problems such as unsatisfactory, and achieve high accuracy, high specificity, and simple methods

Inactive Publication Date: 2011-04-20
MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For the detection and identification of acacia and ricin, traditional methods can no longer meet the requirements of the current anti-terrorism situation

Method used

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  • Colloidal gold test paper for jointly detecting abrin and ricin and special monoclonal antibody
  • Colloidal gold test paper for jointly detecting abrin and ricin and special monoclonal antibody
  • Colloidal gold test paper for jointly detecting abrin and ricin and special monoclonal antibody

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1, preparation of abrin toxin monoclonal antibody and polyclonal antibody

[0031] 1. Preparation of hybridoma cells

[0032] (1) Antigen preparation

[0033] Recombinant plasmid pET-HisA is disclosed in literature (Abrin-aAchainexpressedassolubleforminEscherichiacolifromaPCR-synthesizedgenescatalyticallyandfunctionallyactiveLi-ChunWanga, b, LinKanga, Ting-MaoHub, Jing-LinWanga, *Biochimie86(2004)327-333), the public can be obtained from the Chinese People's Liberation Army obtained from the Institute of Microbial Epidemiology. The gene encoding the abrin toxin A chain is contained in the plasmid pET-HisA.

[0034] 1. Expression of antigenic protein by abrin toxin A chain recombinant genetically engineered bacteria

[0035] The recombinant plasmid pET-HisA was transferred into E.coliBL21, screened for Amp resistance, sequenced and identified, and a positive recombinant bacterium was obtained, which was designated as BL21-pET-HisA. The recombinant engineer...

Embodiment 2

[0074] Embodiment 2, the preparation of multiple antibodies against ricin

[0075] 1. Preparation of detoxified ricin

[0076] Castor seeds were purchased from Beijing Xiuhe Seed Co., Ltd., and the product catalog number is Castor No. 1.

[0077] Crude extraction: remove the castor seeds and weigh 100g, soak them in PBS (0.01M, pH7.2) for 24 hours until the seeds swell; remove the shells and wash them, and place the washed seeds in 500mL PBS (0.01M, pH7.2) .2) In the buffer solution, homogenate in a homogenizer, and extract at 4°C overnight for 24 hours (placed for 24 hours); the next day, filter the extract with degreasing gauze to remove the residue, and place at 4°C with Centrifuge at 12000rpm for 20min, take the supernatant, filter it with a 0.45μm filter, adjust the supernatant to pH7.2; slowly add saturated ammonium sulfate solution (pH7.4) to the supernatant until the saturation is 30%, and stir magnetically at 4°C for 1h After resting for 1 hour, centrifuge at 12000r...

Embodiment 3

[0086] Embodiment 3, preparation and performance detection of colloidal gold test paper

[0087] 1. Preparation

[0088] (1) Preparation of sample pads

[0089] The material of the sample pad is glass fiber membrane;

[0090] The glass fiber membrane was treated as follows to effectively remove the non-specific phenomenon of colloidal gold test paper and increase its sensitivity: soak the sample pad in 0.1mol / L carbonate buffer solution containing mouse serum albumin and pH 9.9 for 2 hours, Bake at 37° C. for 2 hours for later use; the final concentration of mouse serum albumin in the buffer is 0.4% (volume percentage). The blocking agent can block too many binding sites, and increasing the pH value can make the protein in the sample easier to bind.

[0091] (2) Preparation of colloidal gold pads

[0092] 1. Determine the determination of the minimum conjugated antibody concentration:

[0093] (1) Take 11 clean test tubes, numbered 1, 2, 3, ... 7 respectively, and add 1 m...

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Abstract

The invention discloses a colloidal gold for jointly detecting abrin and ricin and a special monoclonal antibody. The monoclonal antibody of the abrin is secreted by Abrin-a A chain monoclonal antibody cell strains having a preservation number of CGMCC No. 4062. The colloidal gold disclosed by the invention has the advantages of high sensitiveness, high specificity, high precision, high degree of accuracy, low cost, short detection time and long shelf life, is simple to operate and store, and is suitable for use in various enterprises. The method for detecting the abrin by using the test paper in the invention is simple, rapid, visual and correct, the scope of application is wide and is easy to popularize and apply, and the cost is low.

Description

technical field [0001] The invention relates to a colloidal gold test paper and a special monoclonal antibody for combined detection of abrin toxin and ricin. Background technique [0002] Abrin and Ricin are highly toxic plant toxins isolated from plant seeds, wherein the molecular weight of abrin toxin is about 62KD, pI6.9; the molecular weight of ricin is about 65KD, pI7. 3. These two toxins have the same AB chain structure and the same pathogenic mechanism, and their molecular weights are also similar, showing high homology. [0003] Since the 9.11 terrorist attack on the United States and the anthrax spore incident, countries around the world have begun to pay close attention to bioterrorism attacks. Since these two toxins are rich in natural resources, they are relatively easy to prepare, and with the help of today's highly developed bioengineering techniques, they can also be produced in large quantities. There are no specific symptoms after toxin poisoning. When t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18G01N33/558G01N33/577
Inventor 王景林高姗聂聪王俊虹王利春康琳周阳
Owner MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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