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Anti-sterigmatocystin monoclonal antibody

A technology of Aspergillus versicolor and monoclonal antibody, applied in antifungal/algal/lichen immunoglobulins, measuring devices, instruments, etc., can solve the problems of consumer health threat, potential toxicity, and difficulty in identification, etc. The effect of fast inspection, stable inspection data and large inspection capacity

Inactive Publication Date: 2013-07-24
中国人民共和国台州出入境检验检疫局
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Natural mycotoxins in the form of natural components of food are not easy to identify, and due to their high potential toxicity, they pose a great threat to the health of consumers

Method used

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  • Anti-sterigmatocystin monoclonal antibody
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  • Anti-sterigmatocystin monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Construction of Anti-Varistoxin Monoclonal Antibody Hybridoma Cell Line

[0044] 1. Materials

[0045] 20% fetal bovine serum: product of Beijing Yuanheng Shengma Institute of Biotechnology; serum-free RPMI1640: product of Gibco; SP2 / 0 cells: imported from ATCC; Freund's complete adjuvant and Freund's incomplete adjuvant, chromogenic reagent TMB : Sigma company product; Balb / c and C57BL / 6 mice; aminopterin, hypoxanthine and thymidine, and other reagents are commercially available.

[0046] 2. Methods and Results

[0047] 1. Preparation

[0048] 1) Aminopterin (A) stock solution (100×):

[0049] Dissolve 1.8mgA in 90ml of triple distilled water, add 0.5ml of 1mM NaOH dropwise with magnetic stirring to dissolve, add 0.5ml of 1mM HCL to neutralize, dilute to 100ml by filtration, sterilize in 1ml aliquots, and store at -20°C.

[0050] 2) Hypoxanthine and thymidine (HT) stock solution (100×):

[0051] H136mg, T39mg, add 100ml of triple distilled water, mix wel...

Embodiment 2

[0095] Example 2 Screening and Identification of Hybridoma Cell Strains Secreting Anti-Varistoxin Monoclonal Antibody

[0096] 1. Materials

[0097] 20% fetal bovine serum: product of Beijing Yuanheng Shengma Institute of Biotechnology; serum-free RPMI1640: product of Gibco; SP2 / 0 cells: imported from ATCC; chromogenic reagent TMB: product of Sigma; secondary antibody: goat anti-mouse- HRP; other reagents were commercially available.

[0098] 2. Methods and Results

[0099] 1. Screening of hybridoma cells:

[0100] (1) After fusion, when the hybrid cell colony grows to a certain size, the antibody activity can be screened (detected every time the medium is changed).

[0101] (2) ELISA detection, take 100 mL of the supernatant for measurement. After two times of detection, it is confirmed that there is a positive result, and the subcloning is carried out immediately (the cell growth accounts for about 1 / 4-1 / 3 of the bottom area of ​​the well), and the subcloning is carried ...

Embodiment 3

[0129] Example 3 Fishing of the light and heavy chain genes of the anti-aspergillin monoclonal antibody ASH

[0130] 1. Materials

[0131] DNA fragment purification kit: product of OMEGA Biotechnology Company; T4DNA ligase: product of New England Biolabs; vector PGEM Teasy: product of Promega Company; competent bacteria JM109: purchased from Promega Company, the primers are as follows:

[0132] VLPD: GCGCCGTCTAGAATTAACACTCATTCCTGTTGAA

[0133] VHPD: GTTCTGACTAGTGGGCACTCTGGGCTC

[0134] VHPU5: AGGTCCAACTGCTCGAGTCTGG

[0135] VHPU6: AGGTCCAACTGCTCGAGTCAGG

[0136] VHPU7: AGGTCCAACTTCTCGAGTCTGG

[0137] VHPU8: AGGTCCAACTTCTCGAGTCAGG

[0138] VLPU5: CCAGATGTGAGCTCGTGATGACCCAGACTCCA

[0139] VLPU6: CCAGATGTGAGCTCGTCATGACCCAGTCTCCA

[0140]VLPU7: CCAGTTCCGAGCTCGTGATGACACAGTCTCCA

[0141] All the other are with embodiment two.

[0142] 2. Method results

[0143] (1) get the cell 5 * 10 of the logarithmic growth phase that embodiment 2 screens out 6 ~1×10 7 centrifuge to r...

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PUM

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Abstract

The invention discloses an anti-sterigmatocystin monoclonal antibody. The antibody comprises a light chain and a heavy chain, wherein an amino acid sequence of a variable region of the light chain is shown in a sequence table SEQ ID NO. 1, and an amino acid sequence of a variable region of the heavy chain is shown in a sequence table SEQ ID NO. 2. The invention further discloses a method for preparing the antibody. A detection kit for detecting sterigmatocystin is prepared by using the antibody disclosed by the invention. The sensitivity of the kit reaches 10 pgs per microliter, so that the content of the sterigmatocystin in a sample can be effectively detected. The kit is convenient to use and does not need valuable instruments, thereby facilitating large-scale popularization and application.

Description

technical field [0001] The present invention relates to a monoclonal antibody, in particular to an anti-Varitocin monoclonal antibody ASH, and also relates to the application of the monoclonal antibody in the preparation of a Versicolor detection kit. Background technique [0002] At present, there are about 100 kinds of mycotoxins known to contaminate grains. Mycotoxins are metabolic secondary organisms of molds. In addition to causing mycotoxin poisoning in animals, most of the mycotoxins in feed can also inhibit animal immunity and reduce resistance, thereby inducing other diseases. Animal experiments have shown that mycotoxins can cause symptoms such as slowed heart rate, rapid breathing, hair loss and abortion. Mycotoxins can also remain in animal products, indirectly endangering human health. [0003] Sterigmatocys (abbreviated as ST) is a heterocyclic compound with strong liver and kidney toxins and can induce various tumors. The high incidence of liver cancer in s...

Claims

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Application Information

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IPC IPC(8): C07K16/14G01N33/577
Inventor 江飞剑王海彬杨承刚韦国栋
Owner 中国人民共和国台州出入境检验检疫局
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