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Preparation method of citrinin immunoaffinity column

A technology of citrinin and immunoaffinity, applied in the field of preparation of citrinin immunoaffinity column, can solve the problems of low accuracy, lack of selectivity, cumbersome steps, etc.

Inactive Publication Date: 2013-06-19
SHANDONG NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These instrumental analysis methods have high requirements on sample purity and require certain pretreatment. However, traditional sample pretreatment techniques, such as liquid-liquid extraction, solid-phase extraction, and solid-phase microextraction, have cumbersome steps and lack of selectivity. Easily interfered by other components, low accuracy

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] (1) Preparation of immunogen

[0023] Dissolve 10.0mg bovine serum albumin in 2.5mL0.1mmolL -1 , prepare bovine serum albumin-sodium acetate solution in the sodium acetate solution of pH5, dissolve 1.0mg citrinin in 0.2mL methanol to prepare citrinin-methanol solution, mix citrinin-methanol solution with 320mL37% volume The specific formaldehyde-water solution was added to the above-mentioned bovine serum albumin-sodium acetate solution, placed at 37°C for 24 hours, and the mixture was dialyzed at 4°C with a phosphate buffer solution of pH 7.4 and 0.01mol / L for three days;

[0024] (2) Preparation and purification of anti-citrinin antibody

[0025] Add appropriate amount of normal saline and the same amount of Freund's complete or incomplete adjuvant to 1.5mL of immunogen, use Freund's complete adjuvant for the first immunization, and use Freund's incomplete adjuvant for subsequent immunizations, emulsify, and use back subcutaneous injection and leg muscle Combination...

Embodiment 2

[0033] (1) Preparation of immunogen

[0034] Dissolve 20.0mg hemocyanin in 5.0mL0.1mmolL -1 , prepare hemocyanin-sodium acetate solution in the sodium acetate solution of pH5, dissolve 2.0mg citrinin in 0.4mL methanol and prepare citrinin-methanol solution, and citrinin-methanol solution and 640mL37% volume ratio The formaldehyde-water solution was added to the above-mentioned hemocyanin-sodium acetate solution, placed at 37°C for 24 hours, and the mixture was dialyzed at 4°C with pH 7.4, 0.01mol / L phosphate buffer solution for three days;

[0035] (2) Preparation and purification of anti-citrinin antibody

[0036] 3.0 mL of immunogen was added with appropriate amount of normal saline and the same amount of Freund’s complete or incomplete adjuvant. Freund’s complete adjuvant was used for the first immunization, and Freund’s incomplete adjuvant was used for subsequent immunizations, emulsified, and injected subcutaneously on the back and into the leg muscles. Combination of i...

Embodiment 3

[0044] (1) Preparation of immunogen

[0045] Dissolve 40.0mg ovalbumin in 10.0mL0.1mmolL -1 , prepare ovalbumin-sodium acetate solution in the sodium acetate solution of pH5, dissolve 4.0mg citrinin in 0.8mL methanol and prepare citrinin-methanol solution, and citrinin-methanol solution and 1280mL37% volume ratio The formaldehyde-water solution was added to the above-mentioned ovalbumin-sodium acetate solution, left at 37°C for 24 hours, and the mixture was dialyzed at 4°C with a phosphate buffer solution of pH 7.4 and 0.01mol / L for three days;

[0046] (2) Preparation and purification of anti-citrinin antibody

[0047] Add appropriate amount of normal saline and the same amount of Freund's complete or incomplete adjuvant to 6.0 mL of immunogen. Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for subsequent immunizations, emulsified, and injected subcutaneously on the back and into the leg muscles. Combination of inje...

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Abstract

The invention discloses a preparation method of a citrinin immunoaffinity column, belongs to the technical field of immunoaffinity chromatography and biotoxin detection, and discloses the preparation method of the citrinin immunoaffinity column. Immunogen is prepared by connecting citrinin and carrier protein through a formaldehyde condensation method. An immune animal gets an anti-citrinin antibody. Cupric embedded ionic polymer is prepared in a mass polymerization method. Immunoaffinity adsorbent which is prepared by adsorbing the polymer to the anti-citrinin antibody is filled into the citrinin immunoaffinity column which is prepared in a solid phase extraction pipe. The citrinin immunoaffinity column which is prepared in the preparation method of the citrinin immunoaffinity column is capable of combining with the citrinin in a specific mode, and cirtrinin solution with high purity and concentration is obtained through purification and enrichment. The maximum combination volume of the citrinin immunoaffinity column which is prepared in the preparation method of the citrinin immunoaffinity column is 597 ng, citrinin fortified recovery in a sample is 83.7% to 93.2%, and the recovery rate is no lower than 80% when the citrinin immunoaffinity column is repeatedly used for three times. The preparation method of the citrinin immunoaffinity column can be applied to rapid detection of the citrinin in fermented food.

Description

technical field [0001] The invention relates to the technical field of immunoaffinity chromatography and biological toxin detection, in particular to a preparation method of a citrinin immunoaffinity column. Background technique [0002] Red yeast rice has long been widely used as a food preservative and flavor enhancer in various traditional foods. Citrinin is a secondary metabolite produced by a fungus in red yeast rice food, which has obvious nephrotoxicity, and an excessive dose will cause animal death. At present, the commonly used detection methods for citrinin are mostly instrumental analysis methods, such as high performance liquid chromatography, liquid chromatography mass spectrometry and high performance capillary electrophoresis. These instrumental analysis methods have high requirements on sample purity and require certain pretreatment. However, traditional sample pretreatment techniques, such as liquid-liquid extraction, solid-phase extraction, and solid-phase...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01D15/38B01J20/281G01N1/34C07K14/765C07K14/77C07K14/795C07K16/06C08F220/56C08F220/06C08F222/14C08F2/02
Inventor 张鸿雁邴欣周建华王瑶
Owner SHANDONG NORMAL UNIV
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