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Aspergillus flavus toxin immuno-affinity column preparation method

A technology of aflatoxin and immunoaffinity, which is applied in the field of preparation of immunoaffinity columns, can solve the problems of increasing non-specific adsorption, cyanogen bromide is highly toxic, and cross-linked antibodies are uneconomical, so as to avoid non-specific adsorption, good active effect

Inactive Publication Date: 2008-08-13
SHANGHAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the coupling efficiency of cyanogen bromide is high, but the pKa=10.4 of the amino group in the isourea derivatives generated after the coupling of the matrix and the ligand, so it usually has a certain positive charge, so that the matrix may have an anion ion exchange effect, increasing Larger non-specific adsorption, in addition, the matrix activated by cyanogen bromide is not stable enough to bind to the ligand, and the ligand may fall off, and cyanogen bromide is highly toxic and volatile, so it is inconvenient to operate
When the epichlorohydrin method is used to couple macromolecules, the coupling efficiency is low
The protein A method is used to couple antibodies, which can connect the non-binding active region Fc of the antibody to the carrier to better maintain the activity of the antibody, but protein A is more expensive and uneconomical for cross-linking antibodies

Method used

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  • Aspergillus flavus toxin immuno-affinity column preparation method
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Experimental program
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Embodiment 1

[0030] Embodiment one: the specific steps of a preferred embodiment of the present invention are:

[0031] 1) Preparation of hydrazide antibody

[0032] Precipitate the ascites (aflatoxin antibody) with ammonium sulfate, centrifuge, remove the supernatant, dissolve the precipitate with distilled water to make the antibody concentration greater than 25mg / ml, and dialyze against pH 5.8 0.2M sodium phosphate buffer. Add one-tenth volume of 20-40 mg / ml sodium periodate aqueous solution to the antibody solution, stir and react at room temperature in the dark for 1 hour, and separate the antibody through Sephadex G25. Add excess adipate dihydrazide aqueous solution (pH 5.8) to the above-mentioned sodium periodate oxidized antibody (add 0.5-2 mg adipate dihydrazide per mg of antibody), react overnight at 4°C, pH 5.8 for 0.2M sodium phosphate Dialysis. 2) Preparation of aldylated solid phase matrix (Sepharose 4B)

[0033] Take an appropriate amount of Sepharose 4B and drain, weigh ...

Embodiment 2

[0042] Embodiment 2 Immunoaffinity Column Fluorophotometry Determination of Aflatoxins

[0043] In the blank sample (Canadian wheat) without AFT, add 1, 5, 20, 50ppb aflatoxin mixed standard (AFB 1 , AFB 2 , AFG 1 , AFG 2 ), the sample extract was purified with the affinity column prepared in Example 1, and the concentration was measured with a fluorophotometer. The specific operation method refers to the national standard GB / T 18979-2003. The results are shown in Table 1 as follows:

[0044]Table 1 adopts the immunoaffinity column prepared by the present invention and the result of measuring aflatoxin by fluorescence photometry

[0045]

[0046] As can be seen from the above data, in the blank sample Canadian wheat, 4 levels of concentration addition recovery experiments are carried out to AFT, the average recovery rate is 82.7~99.3%, and the coefficient of variation is 5.3~9.3%, which shows that the aflatoxin prepared by the present invention The immunoaffinity colu...

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Abstract

The present invention relates to preparation method of aflatoxin immuno-affinity column, including steps of: preparation of hydrazine antibody: employing sodium periodate oxidating unconjugated active area Fc hydroxoy of antibody to aldehyde group, and reacting with adipic dihydrazide to gain hydrazine antibody; preparation of aldehyde solid substrate: oxidating solid substrate containing continued diol structure by sodium periodate to gain aldehyde solid substrate; coupling of antibody and solid substrate: mixing hydrazine antibody and aldehyde solid substrate, adding sodium borohydride to seal unreacted aldehyde group on solid substrate after oscillating reaction to gain coupling antibody solid substrate; column packed. The present invention couples the unconjugated active area Fc end of antibody to solid substrate to remain activity of antibody better; hydrazide derived on antibody instead of solid substrate to avoid nonspecific adsorption of hydrazide group; the combined affinity column not introducing new groups ( such as carboxyl, amido and so on) except coupling antibody to avoid nonspecific adsorption from them.

Description

Technical field: [0001] The invention relates to a preparation method of an immunoaffinity column, in particular to a preparation method of an aflatoxin immunoaffinity column. Background technique: [0002] Aflatoxins (AFT) are secondary metabolic toxic products of a group of fungi. AFT pollution is serious all over the world. In 1993, AFT was classified as a Class I carcinogen by the Cancer Research Institute of the World Health Organization (WHO). Aflatoxin B in naturally contaminated food and feed 1 (AFB 1 ) is the most common and is the main component of the toxin produced by Aspergillus flavus. It is also the most toxic and carcinogenic, which not only brings significant economic losses to the society, but also seriously threatens the health of consumers. All countries and regions in the world have established strict AFT limit standards, and the limit requirements are becoming increasingly stringent. At present, the detection methods of aflatoxin include thin layer...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/531G01N33/558
Inventor 陈宇光戴小峰田汉莉沈彦萍顾鸣黎双华
Owner SHANGHAI UNIV
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