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Hybridoma cell strain 2D3, monoclonal antibody to zearalenone secreted by same and application of monoclonal antibody

A hybridoma cell line and gibberellin monoclonal technology, applied in the field of anti-zearalenone monoclonal antibodies, can solve the problems of complicated operation, complicated processing process, uncomfortable and rapid detection, etc., and achieve the effect of high sensitivity

Active Publication Date: 2013-08-07
INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Thin-layer chromatography does not require special equipment for the detection of zearalenone, and it can be carried out in general laboratories, but the amount of reagents is large, the operation is cumbersome, the interference of other components is serious, and the accuracy is poor, so it cannot be accurately quantified. And it is harmful to the experimenters and the surrounding environment, so it is not suitable for on-site rapid detection
Precision instrument analysis methods include fluorescence spectrophotometry and high performance liquid chromatography, which have high sensitivity and good accuracy, but the instruments are expensive, requiring a high degree of purification of zearalenone samples, and the sample pretreatment process is cumbersome and time-consuming. The experimental environment has high requirements, and it is difficult to achieve rapid detection

Method used

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  • Hybridoma cell strain 2D3, monoclonal antibody to zearalenone secreted by same and application of monoclonal antibody
  • Hybridoma cell strain 2D3, monoclonal antibody to zearalenone secreted by same and application of monoclonal antibody
  • Hybridoma cell strain 2D3, monoclonal antibody to zearalenone secreted by same and application of monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: Screening of hybridoma cell line 2D3

[0021] 1. Animal Immunization

[0022] Six 6-week-old BALB / c mice were purchased and immunized with the commercially available zearalenone complete antigen ZEA-BSA. For the first immunization, the complete zearalenone antigen was emulsified with an equal volume of Freund's complete adjuvant, and then injected subcutaneously at multiple points on the back of the neck of the mice. The second immunization was carried out 4 weeks later, and the complete antigen was emulsified with an equal volume of Freund's incomplete adjuvant and zearalenone, and injected into mice intraperitoneally. The interval between the third immunization and the second immunization was 4 weeks, and the immunization method was the same. The fourth immunization was carried out 3 weeks after the third immunization. The immunization method was the same as the second immunization, which was also intraperitoneal injection. The four immunization doses wer...

Embodiment 2

[0027] Example 2: Determination of the variable region sequence of the anti-zearalenone monoclonal antibody hybridoma cell line 2D3 antibody

[0028] (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 2D3;

[0029] (2) cDNA synthesis: using the total RNA obtained in step 1 as a template, oligo(dT) 15 For primers, follow SuperScript TM -2II Reverse Transcriptase Instructions for reverse transcription to synthesize the first strand of cDNA; primer oligo(dT) 15 Purchased from Invitrogen;

[0030] (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. After the PCR product w...

Embodiment 3

[0032] Example 3: Preparation, purification, subtype and identification of anti-zearalenone monoclonal antibody

[0033] The anti-zearalenone monoclonal antibody hybridoma cell line 2D3 obtained in Example 1 was injected into BALB / c mice pre-treated with Freund's incomplete adjuvant, the ascites of the mice was collected, and octanoic acid-ammonium sulfate was used to The specific operation steps are: filter mouse ascites with double-layer filter paper, centrifuge at 12000r / min for 15min at 4°C, absorb the supernatant, mix the obtained ascites supernatant with 4 times the volume of acetate buffer, and stir Slowly add n-octanoic acid, the volume of n-octanoic acid required per milliliter of ascites is 33μL, mix at room temperature for 30min, let stand at 4°C for 2h, then centrifuge at 12000r / min for 30min at 4°C, discard the precipitate, and filter the obtained supernatant with double-layer filter paper After filtering, add 1 / 10 of the filtrate volume molar concentration is 0.1...

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Abstract

The invention provides a hybridoma cell strain 2D3, a monoclonal antibody to zearalenone secreted by the hybridoma cell strain 2D3 and application of the monoclonal antibody. The hybridoma cell strain 2D3 is preserved in China Center for Type Culture Collection with an accession number of CCTCC No. C201328 and can be used for preparation of a high-titer monoclonal antibody to zearalenone. According to detection results of enzyme linked immunosorbent assay (ELISA), the titer of the monoclonal antibody to zearalenone prepared through purification of mouse ascites can reach 1.5 * 10<5>. The monoclonal antibody to zearalenone has high sensitivity, half maximal inhibitory concentration IC50 of 20 pg / mL to zearalenone and cross reactivity of 4.9%, 3.3% and 3.2% with beta-zearalanel, alpha-zearalanel and beta-zeranol, respectively. The monoclonal antibody to zearalenone can be used for determination of the content of zearalenone.

Description

technical field [0001] The present invention relates to a hybridoma cell line 2D3, the anti-zearalenone monoclonal antibody produced by the hybridoma cell line and its application. Background technique [0002] Zearalenone (ZEA) is mainly produced by Fusarium graminearum and was first isolated from maize with scab. Zearalenone has estrogen-like effects, which can cause acute and chronic poisoning of animals, abnormal reproductive function and even death of animals, which can cause huge economic losses to livestock farms. Eating all kinds of pasta made from wheat flour containing scab can also cause symptoms of central nervous system poisoning, such as nausea, chills, headache, mental depression and ataxia. Zearalenone mainly contaminates grains such as corn, wheat, rice, barley, millet and oats. Among them, the positive detection rate of corn was 45%, and the positive detection rate of wheat was 20%. Zearalenone has residual properties and is not easy to be destroyed. If ...

Claims

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Application Information

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IPC IPC(8): C12R1/91C07K16/14G01N33/577C12N5/20
Inventor 李培武李鑫张奇何婷丁小霞张文李冉
Owner INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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