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Aflatoxin nano-antibody immunosorbent, immunoaffinity column and preparation methods and application of aflatoxin nano-antibody immunosorbent and immunoaffinity column

一种黄曲霉毒素、免疫吸附剂的技术，应用在肽的制备方法、化学仪器和方法、抗真菌/藻类/地衣免疫球蛋白等方向，能够解决尚未有黄曲霉毒素免疫亲合柱报道等问题，达到成本低、制备方便、耐有机试剂的效果

Active Publication Date: 2014-06-18
OIL CROPS RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Nanobodies are heavy-chain antibodies that naturally occur in camelids. At present, there are no reports on aflatoxin nanobody immunosorbents and immunoaffinity columns.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1 Construction of aflatoxin nanobody gene library and preparation of nanobody

[0021] 1. Animal Immunization

[0022] A 2-year-old male alpaca was purchased and immunized with aflatoxin B1 antigen (AFB1-BSA, Sigma). After emulsifying 200 μg of aflatoxin B1 antigen with Freund's incomplete adjuvant, the alpaca was injected subcutaneously at multiple points. Immunize once every 2 weeks, 7-10 days after each immunization, blood was collected from the alpaca, and the serum titer was determined by indirect ELISA method. After the first immunization with the highest titer, 10 mL of blood was taken to extract total RNA.

[0023] 2. Construction of cDNA library

[0024] (1) Extraction of total RNA: select the first immunization with the highest titer of alpaca serum, and 7-10 days after immunization, take 10 mL of blood from the alpaca vein, and extract total RNA: use the LeukoLOCK Total RNA Isolation Kit of Life Technology Company to extract Total RNA in alpaca b...

Embodiment 2

[0069] Example 2 Preparation of aflatoxin nanobody immunoaffinity adsorbent and immunoaffinity column

[0070] The immunoaffinity adsorbent in this example includes a solid-phase carrier (silica gel microspheres) and aflatoxin B1 nanobody 2014AFB-G15 coupled to the solid-phase carrier. The specific preparation method is as follows: Weigh 1 g of acrylamide silica gel microspheres, Put it into an Erlenmeyer flask, wash the microspheres alternately with pure water and phosphate buffer with a pH of 6; measure 5 mL of phosphate buffer with a pH of 6 to dissolve the microspheres to obtain a microsphere solution, and transfer the microsphere solution to In the mixing cup, turn on the stirrer to suspend all the microspheres, dissolve 2mg of aflatoxin B1 nanobody 2014AFB-G15 with 1mL of phosphate buffer solution with a pH of 6, then add it dropwise to the above microsphere solution, and weigh 70mg EDC was quickly introduced into a stirring cup, and after stirring and reacting at 4°C...

Embodiment 3

[0072] Example 3 Preparation of aflatoxin nanobody immunoaffinity adsorbent and immunoaffinity column

[0073] The immunoaffinity adsorbent in this example includes a solid phase carrier (agarose) and aflatoxin B1 nanobody 2014AFB-G15 coupled to the solid phase carrier. The specific preparation method is as follows: Weigh 0.3g of agarose, put it into a cone Shaped flask, washed repeatedly with 1mM HCl solution for more than 15min, dissolved agarose in 5mL coupling buffer (0.1M NaCO 3 , 0.5M NaCl, pH8.3), then add 0.6mg aflatoxin B1 nanobody 2014AFB-G15, stir the reaction at room temperature at 150rpm for 1h to obtain an agarose gel solution, transfer the agarose gel solution to In a Sabouraud funnel, let the uncoupled antibody-containing solution flow out, then wash the agarose gel with a coupling buffer that is 5 times the volume of the agarose gel solution, and then add 2 times the volume of the agarose gel solution. Blocking buffer (0.1M Tris-HCl buffer, pH8.0) was react...

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PUM

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Abstract

The invention relates to an aflatoxin nano-antibody immunosorbent, an aflatoxin nano-antibody immunoaffinity column and preparation methods and application of the two. The immunosorbent comprises a solid phase carrier and an aflatoxin B1 nano-antibody 2014AFB-G15 coupled with the solid phase carrier, the half maximal inhibitory concentration (IC50) of the aflatoxin B1 nano-antibody 2014AFB-G15 to aflatoxin B1 is 0.66ng / mL, and the cross reaction rates of the aflatoxin B1 nano-antibody 2014AFB-G15 to aflatoxins B2, G1, G2 and M1 are 22.6%, 10.95%, 32.1% and 26%, respectively; an amino acid sequence of the aflatoxin B1 nano-antibody 2014AFB-G15 is represented by SEQIDNO:7, and a coding gene sequence of the aflatoxin B1 nano-antibody 2014AFB-G15 is represented by SEQIDNO:8. The aflatoxin nano-antibody immunoaffinity column disclosed by the invention can be used for purifying and concentrating a sample extracting solution before machinery detection and can be repeatedly used.

Description

technical field [0001] The invention relates to an aflatoxin nano-antibody immunoadsorbent, an immunoaffinity column and a preparation method and application thereof. Background technique [0002] Aflatoxins are mainly secondary metabolites secreted by Aspergillus flavus and Aspergillus parasiticus, and are natural toxic compounds that can cause various damages to humans and animals. More than 20 types of aflatoxins have been discovered, mainly including aflatoxin B1 (AFB 1 ), B2 (AFB 2 ), AFG and M1 (AFM 1 )wait. where AFB 1 Potassium is the most toxic, and its toxicity is 10 times that of potassium cyanide and 68 times that of arsenic. As early as 1993, aflatoxin B1 was designated as one of the strongest known carcinogenic chemicals by the Cancer Research Institute of the World Health Organization, that is, Class I carcinogens. my country is an area with heavy aflatoxin pollution, and aflatoxin pollution is likely to exist in various foods and agricultural products, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01J20/24B01J20/281B01J20/30C07K17/14C07K17/10G01N30/02
CPCC07K16/14C07K2317/22C07K2317/569C07K2317/92C07K2317/94G01N33/56961G01N2333/38C07K1/22C07K2317/565
Inventor 李培武张奇王妍入张兆威丁小霞
Owner OIL CROPS RES INST CHINESE ACAD OF AGRI SCI
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