Benzoic acid artificial antigen and antibody, and preparation methods and applications thereof

An artificial antigen, benzoic acid technology, applied in chemical instruments and methods, animal/human protein, ovalbumin, etc., can solve the problems of poor repeatability and low titer of antibody preparation

Inactive Publication Date: 2013-10-16
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The technical problem to be solved in the present invention is to overcome the defects of low antibody preparation titer and poor repeatability in the traditional benzoic acid detection technology, and provide a benzoic acid artificial antigen and specific antibody

Method used

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  • Benzoic acid artificial antigen and antibody, and preparation methods and applications thereof
  • Benzoic acid artificial antigen and antibody, and preparation methods and applications thereof
  • Benzoic acid artificial antigen and antibody, and preparation methods and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Synthesis and Identification of Immunogens

[0028] S1. Weigh 13.7 mg of p-aminobenzoic acid and dissolve it in 3 mL of 1 mol / L hydrochloric acid solution, add 750 μL of 1 mol / L sodium nitrite solution, and react at 4°C for 30 min to obtain liquid A.

[0029] S2. Dissolve 10 mg of KLH in 0.15 mol / L, pH 9.6 carbonic acid buffer to obtain solution B.

[0030] S3. Add 1.2 mL of liquid A and 1.5 mL of 1 mol / L NaOH solution dropwise to the above liquid B, and stir at room temperature for 1 h. After the reaction, the supernatant was taken into a dialysis bag (molecular weight cut-off 10,000 Da), dialyzed with 0.1 mol / L, pH 7.4 PBS at 4 °C for three days, and the dialysate was changed every 12 h. Pack it into a small tube and store it at -20°C to obtain the artificial antigen (immunogen) with the structure of Formula 1.

[0031] Identification of the immunogen

[0032] The p-aminobenzoic acid, KLH, and the artificial antigen with the structure of Formula 1 were s...

Embodiment 2

[0033] Example 2: Synthesis and Identification of Envelope

[0034] S1. Weigh 13.7 mg of p-aminobenzoic acid and dissolve it in 3 mL of 1 mol / L hydrochloric acid solution, add 750 μL of 1 mol / L sodium nitrite solution, and react at 4°C for 30 min to obtain liquid A.

[0035] S2. 44 mg of OVA was dissolved in 0.15 mol / L, pH 9.6 carbonic acid buffer to obtain solution B.

[0036] S3. Add 1.2 mL of liquid A and 1.5 mL of 1 mol / L NaOH solution dropwise to the above liquid B, and stir at room temperature for 1 h. After the reaction, the supernatant was taken into a dialysis bag (molecular weight cut-off 10,000 Da), dialyzed with 0.1 mol / L, pH 7.4 PBS at 4 °C for three days, and the dialysate was changed every 12 h. Pack it into a small tube and store it at -20°C to obtain the artificial antigen (immunogen) with the structure of Formula 1.

[0037] Identification of the coater

[0038] The p-aminobenzoic acid, OVA, and the artificial antigen with the structure of Formula 1 were s...

Embodiment 3

[0039] Example 3 Preparation and Identification of Benzoic Acid Polyclonal Antibody

[0040] S1. Preparation of antiserum from immunized animals

[0041]The artificial antigen prepared in Example 1 was used as an immunogen to immunize male New Zealand white rabbits. For the first immunization, 0.5 mg of artificial antigen was emulsified with an equal volume of Freund's complete adjuvant, and multi-point immunization was performed on the back. Afterwards, 0.5 mg of artificial antigen was emulsified with the same amount of Freund's incomplete adjuvant, and the immunization was boosted at intervals of four weeks. One week after each booster immunization, blood was collected from ear veins, and the titer of antiserum was determined by ELISA method. After the titer is stable, the immunization is boosted again. Blood was collected from the heart 10 days after the last immunization, and the serum was retained by centrifugation.

[0042] S2. Antibody purification

[0043] Purify ...

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Abstract

The invention relates to the food safety field, and concretely relates to a benzoic acid artificial antigen and an antibody, and preparation methods and applications thereof. The molecular structure of the benzoic acid artificial antigen is represented by formula (I) shown in the specification. The benzoic acid artificial antigen is prepared through diazotization process coupling of p-aminobenzoic acid as a semiantigen and a carrier protein. The titer of an antiserum obtained by adopting the artificial antigen can reach 1:32000, the lowest detection limit is 0.71ng / mL, the 50% inhibiting concentration is 3.34ng / mL, and the generated antibody has the advantages of high specificity, high sensitivity and high accuracy, and lays a foundation for establishing a rapid, accurate and sensitive benzoic acid immunodetection method.

Description

technical field [0001] The invention relates to the field of food safety, in particular to a benzoic acid artificial antigen and antibody and a preparation method and application thereof. Background technique [0002] Benzoic acid, also known as benzoic acid, is a food preservative that is allowed to be used in my country and has a relatively long history. It is often used in the preservation of carbonated beverages, low-salt pickles, sauces, preserves, fruit wine, fudge, soy sauce, vinegar, and fruity beverages. Benzoic acid and its sodium salt are relatively safe. After entering the body, benzoic acid combines with glycine or glucuronic acid in the body to form hippuric acid, which is all excreted from the urine without accumulation in the body. However, it has been reported in recent years that benzoic acid and its sodium salt can cause allergic reactions, intestinal discomfort and other poisoning symptoms. Animal experiments have shown that feeding rats with feed contai...

Claims

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Application Information

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IPC IPC(8): C07K14/795C07K14/77C07K16/00G01N33/68
Inventor 孙远明沈玉栋王弘罗林徐振林杨金易雷红涛肖治理
Owner SOUTH CHINA AGRI UNIV
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