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Synthetic method for artificially coupling antigen through aflatoxin B1-carrier proteins

A technology of aflatoxin and carrier protein, which is applied in the field of synthesis of aflatoxin B1-carrier protein artificially coupled antigen by a two-step method, which can solve the problem of low purity requirements of the sample to be tested, the inability to achieve trace detection, and the limitation Problems such as popularization and application, to achieve the effect of easy popularization and application, mild coupling reaction and good immunogenicity

Inactive Publication Date: 2015-11-04
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the TLC method has the advantages of simple operation and does not require complex and sophisticated instruments and equipment. The disadvantage is that the sensitivity is low and trace detection cannot be achieved; although the sensitivity of HPLC meets the requirements, the sample pretreatment is cumbersome, the equipment is expensive, and strict operating environment is required. Professional operators, thus limiting the promotion and application of this method in clinical testing; enzyme-linked immunosorbent method has high sensitivity and specificity, the purity of the sample to be tested is not high, the operation is simple, and only the conventional enzyme standard is needed It is especially suitable for the detection of large quantities of samples and is convenient for the promotion and use of grassroots. In recent years, it has been widely used in aflatoxin B 1 rapid quantitative detection; this method is simple to operate, has high sensitivity and can directly observe the results without any instrument readings, and can be used for preliminary screening of the total amount of aflatoxins in food and agricultural products

Method used

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  • Synthetic method for artificially coupling antigen through aflatoxin B1-carrier proteins
  • Synthetic method for artificially coupling antigen through aflatoxin B1-carrier proteins
  • Synthetic method for artificially coupling antigen through aflatoxin B1-carrier proteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Aflatoxin B 1 - Preparation and identification of bovine serum albumin (BSA) artificial conjugated antigen

[0036] 1. Weigh 2mg of aflatoxin B 1 Dissolve the standard product in 4ml of solvent with a volume ratio of methanol and pyridine of 1:1, and add 5m oximating agent carboxymethylhydroxylamine hemihydrochloride (CMO); after stirring for 2 hours at 70°C, place the resulting product in a fume hood Naturally evaporate to dryness; add 1ml of distilled water to dissolve, adjust the pH to 8.0 with sodium hydroxide (1mol / L), and extract unreacted AFB in the system three times with 5ml of benzene 1 , hydrochloric acid (0.2mol / L) to adjust the pH to 3.0, and finally use 5ml of ethyl acetate to extract the precipitate three times and dry it naturally under the fume hood. The obtained dry product is AFB 1 O; the resulting AFB 1 O was dissolved in 2ml of dimethylformamide (DMF), and the activation reagents, namely 8.9mg of dicyclohexylcarbodiimide (DCC) and 5.0m...

Embodiment 2

[0038] Example 2: Aflatoxin B 1 - Preparation and identification of artificial conjugated antigen from ovalbumin (OVA)

[0039] 1. Weigh 2mg of aflatoxin B 1 The standard product was dissolved in 4ml of methanol and pyridine in a solvent with a volume ratio of 1:1, and 5mg of the oximating agent carboxymethylhydroxylamine hemihydrochloride (CMO) was added, stirred at 70°C for 2 hours, and the resulting product was placed in a ventilated Evaporate naturally under the cabinet; add 1ml of distilled water to dissolve, adjust the pH to 8.0 with sodium hydroxide (1mol / L), and extract the unreacted AFB in the system three times with 5ml of benzene 1 , 0.2mol / L hydrochloric acid to adjust the pH to 3.0, and finally use 5ml ethyl acetate to extract the precipitate three times and dry it naturally under the fume hood. The obtained dry product is AFB 1 O; the resulting AFB 1 O was dissolved in 2ml of dimethylformamide (DMF), and after adding the activating reagent, that is, 8.9mg of D...

Embodiment 3

[0040] Example 3: Anti-aflatoxin B 1 Preparation and identification of polyclonal antibodies

[0041] 1. Animal experiment: the prepared aflatoxin B 1 -BSA artificially conjugated antigen (4mg / ml) was diluted with phosphate buffered saline (PBS, 0.01M, pH=7.4), mixed with an equal volume of Freund's complete adjuvant, fully emulsified, and then immunized with Balb / C mice (6-8 weeks old), multiple subcutaneous injections on the back, 100 μg protein per mouse, two weeks later, emulsify the artificial antigen with Freund’s incomplete adjuvant, and perform secondary immunization, 100 μg protein per child, two weeks later, use it with The third immunization was carried out with the same dose as the second immunization, and blood was collected from the tail of the mice 7 days after the third immunization to prepare antiserum.

[0042] 2. Indirect ELISA detection antibody titer test: to determine whether the antibody produced by the animal body is specific for aflatoxin B 1 Aflato...

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Abstract

The invention relates to the technology of artificial antigen synthesis, and aims to provide a synthetic method for artificially coupling antigen through aflatoxin B1-carrier proteins. The method comprises two steps: firstly, forming aflatoxin B1 oxime with an active group through the effect with carboxymethoxylamine hemihydrochloride, and preparing an artificial antigen conjugate of aflatoxin B1 and carrier proteins through a carbodiimide synthesis method, carrying out covalent coupling on the aflatoxin B1 oxime and the carrier proteins, and finally performing dialysis and freeze drying on the product to obtain the aflatoxin B1 artificial coupling antigen. The method is mild in reaction condition, simple, practicable, short in coupling time consumption and low in cost, and is easy for large-scale production and popularization and application; the synthetic product is good in stability and appropriate in coupling ratio, and is unlikely to be degraded for long-term storage; and the synthetic product has higher immunogenicity, can prepare a monoclonal or polyclonal antibody for specifically recognizing aflatoxin B1, and has important meanings for establishing aflatoxin B associated immunological detection methods.

Description

technical field [0001] The present invention relates to the synthetic technology of artificial antigen, specifically two-step method for preparing aflatoxin B 1 - A synthetic method for artificially coupling antigens to carrier proteins. Background technique [0002] Aflatoxin is a class of secondary metabolites produced by Aspergillus flavus and Aspergillus parasiticus with similar chemical structure. It is often found in soil, animals and plants, and various nuts. It is also often found in products. There are more than 20 kinds of aflatoxins and their derivatives, and their basic structures all contain a dihydrofuran and oxinone (coumarin), among which dihydrofuran is related to basic toxicity, and coumarin is related to carcinogenicity . [0003] Aflatoxin B in Naturally Contaminated Food 1 The pollution is the most serious, the most common and the most toxic. It is also one of the most carcinogenic substances found so far. Stomach cancer, colon cancer, breast cancer...

Claims

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Application Information

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IPC IPC(8): C07K14/765C07K14/795C07K14/77C07K1/14C07K1/10
CPCC07K14/765C07K14/77C07K14/795
Inventor 方维焕章先李肖梁谢珲王歆孙孟娇
Owner ZHEJIANG UNIV
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