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Hybridoma cell strain ST03, anti-aflatoxin biosynthesis precursor ST monoclonal antibody and application thereof

一种杂交瘤细胞株、黄曲霉毒素的技术，应用在微生物、生物材料分析、生物化学设备和方法等方向，能够解决抗体灵敏度不高等问题

Active Publication Date: 2014-06-11
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

At present, ST artificial antigen, the biosynthetic precursor of aflatoxin, is mainly prepared by the sodium borohydride reduction method, and the sensitivity of the antibody prepared by the synthetic antigen is often not high

Method used

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  • Hybridoma cell strain ST03, anti-aflatoxin biosynthesis precursor ST monoclonal antibody and application thereof
  • Hybridoma cell strain ST03, anti-aflatoxin biosynthesis precursor ST monoclonal antibody and application thereof
  • Hybridoma cell strain ST03, anti-aflatoxin biosynthesis precursor ST monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Synthesis of Artificial Antigen of Aflatoxin Biosynthesis Precursor ST

[0022] (1) Dissolve 0.1 g of commercially available glycolic acid (moisture ~ 1%) in 0.4 mL of trifluoroacetic acid; weigh 1 mg of aflatoxin biosynthesis precursor ST and dissolve in 0.4 mL of acetonitrile; use a syringe to aspirate Aspergillus aflatoxin Toxin biosynthesis precursor ST acetonitrile solution was gently blown into the mixture of glycolic acid / trifluoroacetic acid, and the reaction was carried out under magnetic stirring at room temperature (20°C~30°C) for 4 h; the reaction solution was rotatably evaporated to obtain a light yellow The green oil is ST hapten, the precursor of aflatoxin biosynthesis;

[0023] (2) Take 1 mg of aflatoxin biosynthetic precursor ST hapten and 4 mg of N-hydroxysuccinimide in a reaction bottle, and react with magnetic stirring at room temperature for 1 h; weigh 7 mg of carbodiimide and dissolve in 0.2 In mL of 1,4-dioxane; slowly drop the 1,4-diox...

Embodiment 2

[0041] Embodiment 2 Preparation of hybridoma cell line ST03

[0042] 1. Animal immunization

[0043] Eight 6-week-old Balb / c mice were purchased and immunized with the artificial antigen of aflatoxin biosynthesis precursor ST synthesized in Example 1: aflatoxin biosynthesis precursor ST-BSA. For the first immunization, the aflatoxin biosynthetic precursor ST-BSA was emulsified with an equal volume of Freund's complete adjuvant, and then injected subcutaneously at multiple points on the back of the mouse. The second immunization was carried out 3 weeks later, using Freund's incomplete adjuvant to emulsify with an equal volume of aflatoxin biosynthetic precursor ST-BSA, and intraperitoneally injecting into mice. The interval between the third immunization and the second immunization was 3 weeks, and the immunization method was the same as that. The fourth immunization was carried out 3 weeks after the third immunization, and the immunization method was the same as the th...

Embodiment 3

[0048] Example 3 Determination of the variable region sequence of the anti-aflatoxin biosynthetic precursor ST monoclonal antibody hybridoma cell line ST03

[0049] (1) Extraction of total RNA: use the total RNA extraction kit of Tiangen Company and follow the instructions to extract the total RNA of hybridoma cell line ST03;

[0050] (2) cDNA synthesis: using the total RNA obtained in step 1 as a template, oligo (dT) 15 For primers, follow SuperScript TM -2 II Reverse Transcriptase Instructions for reverse transcription to synthesize the first strand of cDNA; primer oligo (dT) 15 Purchased from Invitrogen;

[0051] (3) Cloning of variable region genes by PCR method: Design primers according to the conserved sites of mouse antibody gene sequences in GENEBANK, and use cDNA as a template to amplify antibody light and heavy chain variable region genes. The PCR program was: 94°C for 30s, 58°C for 1min, 72°C for 1min, 30 cycles of amplification, and finally 72°C for 10min. Aft...

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Abstract

The invention relates to a hybridoma cell strain ST03, an anti-aflatoxin biosynthesis precursor ST monoclonal antibody and application thereof. The hybridoma cell strain ST03 with the preservation number of CCTCCNO.C2013187 can be used for preparing high-valence anti-aflatoxin biosynthesis precursor ST monoclonal antibody, wherein valence measured by an enzyme linked immunosorbent assay (ELISA) method of the anti-aflatoxin biosynthesis precursor ST mouse ascites antibody can reach 6.4*10<5>. The anti-aflatoxin biosynthesis precursor ST monoclonal antibody is high in sensitivity, has 50% inhibition concentration IC50 of 0.36 ng / mL on the aflatoxin biosynthesis precursor ST, has no cross reaction with the aflatoxin B1, aflatoxin B2, aflatoxin G1 and G2, and can be applied to measuring content of the aflatoxin biosynthesis precursor ST.

Description

technical field [0001] The invention relates to a hybridoma cell line ST03, an anti-aflatoxin biosynthetic precursor ST monoclonal antibody and applications thereof. Background technique [0002] Aflatoxin intermediate Aspergillus versicolor or ST is the early intermediate of aflatoxin synthesis, mainly produced by fungi such as Aspergillus versicolor, Aspergillus flavus, Aspergillus nidulans, Aspergillus rugosa, and can pollute most food and feed Grass, especially the pollution of wheat, corn, peanut and forage grass is more serious. The basic structure of the biosynthetic precursor ST of aflatoxin is composed of difuran ring and xanthone, which is similar in structure to aflatoxin, and its toxicity is second only to aflatoxin. Genetic toxicity and strong carcinogenicity, after it pollutes food and feed, it directly or indirectly enters the human food chain, threatening human health and life safety, and the degree of harm is positively correlated with the intake of aflatox...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/14G01N33/577
CPCC07K2317/92C07K16/14G01N33/56961G01N2333/38G01N2469/10
Inventor 李培武李敏张奇张兆威丁小霞
Owner OIL CROPS RES INST CHINESE ACAD OF AGRI SCI
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