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Mold toxin penicillic acid monoclone antibody and preparation method thereof

A technology of monoclonal antibody and penicillin, which is applied in the direction of biochemical equipment and methods, chemical instruments and methods, instruments, etc., can solve the problems of cumbersome operation, complicated processing, and expensive instruments, and achieve simple operation, little interference, The effect of high sensitivity

Inactive Publication Date: 2009-05-13
HUNAN AGRICULTURAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC), which are commonly used in instrumental analysis methods, are cumbersome to operate, complicated in sample pretreatment, expensive in equipment, and require specialized technicians during operation, so they are not suitable for popularization and application.

Method used

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  • Mold toxin penicillic acid monoclone antibody and preparation method thereof
  • Mold toxin penicillic acid monoclone antibody and preparation method thereof
  • Mold toxin penicillic acid monoclone antibody and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Animal Immunization Protocol

[0032] The complete antigens PA-BSA and PA-OVA were obtained by coupling penicillic acid (PA) with bovine serum albumin (BSA) and ovalbumin (OVA) respectively by carbodiimide method. Take 2g PA, 23g EDC, 9.2mg NHS, fully dissolve in 1mL DMF, and stir at 4°C for 6h. Dissolve 6.5 mg of BSA in 2.0 mL of 0.01 M phosphate buffer (PBS, pH 7.4), slowly add to the reaction solution, shake while adding, and react at room temperature in the dark for 12 hours. The reaction product was dialyzed with 0.01M phosphate buffer (pH7.4) at 4°C for 72 hours, the dialysate was changed every 4 hours on the first day, and the dialysate was changed every 12 hours thereafter. Store at -20°C after aliquoting. The same method was used to couple penicillin to ovalbumin (OVA), and 17 mg of OVA was used to replace BSA. The other steps were the same to obtain complete antigens PA-BSA and PA-OVA. Twelve 8-week-old BABL / C mice were selected for immunization. The immuno...

Embodiment 2

[0034] Cell fusion, selection and cloning protocols

[0035] Preparation of feeder cells: 1-2 days before fusion, 2 mice were taken to remove eyeballs for bloodletting, kill by neck dislocation, soak and disinfect in 75% alcohol for 10 minutes, and then fix the mice on the mouse board of the ultra-clean workbench. Open the abdominal skin to fully expose the peritoneum. Use a 5ml syringe to take 5ml of 1640 culture solution and inject it into the abdominal cavity. Without taking out the syringe, hold the alcohol cotton ball with tweezers and massage the abdomen on both sides for 1 minute, then suck out the culture solution and place it in a 50ml centrifuge tube. Carry out the same method three times. Centrifuge the liquid in the centrifuge tube at 1000 rpm for 10 minutes, discard the supernatant. Suspend the cells with 50ml of HAT culture medium, add dropwise to five 96-well cell culture plates, 37°C, 5% CO 2 cultivated in.

[0036] Preparation of SP2 / 0 myeloma cells: Expan...

Embodiment 3

[0041] Protocol for the preparation and purification of monoclonal antibody ascites

[0042] Get 6 BALB / c mice, intraperitoneally inject 0.5ml / mouse of sterilized liquid paraffin, and inoculate hybridoma cells (1-2×10 6 / ml), 0.5ml / mouse, after 7-12 days, when the abdomen of the mouse increases significantly, collect the ascites with a syringe, centrifuge at 10,000 rpm for 10 minutes at 4°C, remove the grease and precipitate, take the supernatant, and store it at -20°C for future use . The purification of ascites adopts the saturated ammonium sulfate method. The specific steps are: take 10ml of ascites sample, add 10ml of PBS (0.02M, pH7.0), slowly add 20ml of saturated ammonium sulfate solution on ice, stir well while adding, and stir for 10 minutes. overnight at 4°C. Centrifuge at 12,000 rpm for 10 minutes at 4°C, discard the supernatant, dissolve the precipitate with 12ml of PBS, add 8ml of saturated ammonium sulfate solution as above, stir for 10 minutes, and let stand a...

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Abstract

The invention provides a mycotoxin penicillic acid monoclonal antibody and a preparation method thereof. The preparation method comprises the following steps of coupling penicillic acid to carrier protein respectively so as to obtain complete antigen, immunizing a mouse with the complete antigen, adopting a hybridoma technique and obtaining the monoclonal antibody by cell fusion, screening, cloning, culture expansion and an animal in vivo induction ascites method. The obtained antibody has the titer of 1:2.05x10<5>, is of an IgG1 type, has no cross reaction rate with aflatoxin B1, zearalenone, T-2 toxin and Fumonisins, has the affinity constant of 1.54x10<8> L / mol, and can be used for developing ELISA kits for detecting the penicillic acid.

Description

technical field [0001] The invention belongs to the technical field of biological immunity, and relates to a mycotoxin penicillin monoclonal antibody and a preparation method thereof. Background technique [0002] In recent years, with the intensive production of the aquaculture industry, the feed industry has developed vigorously, and the problem of mildew in feed has attracted people's attention. It is very common for mold to pollute feed. After feed is contaminated by mold, in addition to causing deterioration of feed, what is more serious is that these molds can produce mycotoxins with teratogenic, carcinogenic, and mutagenic effects. Cause poisoning, cause huge economic loss to aquaculture. According to the World Food and Agriculture Organization report, about 25% of the world's crops are polluted by molds and toxins every year, causing direct and indirect economic losses of hundreds of billions of dollars. In addition, mycotoxins in feed may also cause harm to humans...

Claims

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Application Information

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IPC IPC(8): C07K16/44C12N5/20G01N33/577
Inventor 袁慧雷红宇邬静文利新李荣芳袁莉芸
Owner HUNAN AGRICULTURAL UNIV
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