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Fab fragment of anti-R-ofloxacin (R-OFL) antibody as well as preparation and application of Fab fragment

A technology of dexofloxacin and fragments, which is applied to the Fab fragment of anti-dexofloxacin antibody and its preparation and application field, can solve the problem of no detection of dexofloxacin, Fab fragment purification method reports, There are no problems such as dextroofloxacin antibody, and the effect of high sensitivity, high sensitivity and simple operation is achieved

Active Publication Date: 2020-06-05
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Most of the current studies are focused on levofloxacin or racemic ofloxacin. There is no report on the purification method for the Fab fragment of the dexofloxacin antibody, and there is no specific detection of dexofloxacin based on the Fab fragment. Shaxing's report

Method used

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  • Fab fragment of anti-R-ofloxacin (R-OFL) antibody as well as preparation and application of Fab fragment
  • Fab fragment of anti-R-ofloxacin (R-OFL) antibody as well as preparation and application of Fab fragment
  • Fab fragment of anti-R-ofloxacin (R-OFL) antibody as well as preparation and application of Fab fragment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] This example provides the preparation and sequencing of anti-dexofloxacin antibody IgG.

[0045] 1. Experimental operation

[0046]1.1. Ascites preparation

[0047] 1.1.1, resuscitation can secrete the hybridoma cell line (South China Agricultural University, Institute of Food Science, key laboratory of Guangdong Province preparation) that can secrete anti-dexofloxacin antibody, use complete medium (Sigma basal medium adds 10% glutamine Amide dipeptide) culture, RNA extraction.

[0048] 1.1.2. When the cells grow to fill the bottom of the culture dish, remove the complete medium, use Sigma basal medium to suspend the cells, and suck up the suspended cells with a 1mL syringe.

[0049] 1.1.3. Female mice aged 8-12 weeks were selected as experimental mice, and 500 microliters of liquid paraffin were injected intraperitoneally into each mouse 7 days before intraperitoneal injection of cells.

[0050] 1.1.4. Each mouse was intraperitoneally injected with 500 microliters o...

Embodiment 2

[0060] This example provides enzyme cleavage of anti-dexofloxacin antibodies.

[0061] 1. Experimental operation

[0062] 1.1. Take 1 mL of IgG with a concentration of 10 mg / mL and thaw quickly.

[0063] 1.2. Take 0.125 mg of immobilized papain (purchased from Thermol Company), put it in a spin column, centrifuge at 4000 rpm for 2 min, and remove the preservation solution.

[0064] 1.3. Take 2 mL of activation solution, add 7 mg of cysteine ​​to make the final concentration of 20 mM in the activation solution, adjust the pH to 7, mix with papain on a shaker at 37°C, and activate for 15 minutes.

[0065] 1.4. After the activation is completed, centrifuge the mixture of enzyme and activation solution on the spin column at 4000rpm for 2min to leave the enzyme.

[0066] 1.5. Weigh 3.5 mg of cysteine ​​and add it to 1 mL of IgG with a concentration of 10 mg / mL, adjust the pH to 7, so that the final concentration of cysteine ​​in the antibody solution is 20 mM.

[0067] 1.6. Mix ...

Embodiment 3

[0071] This example provides the affinity purification (gel filtration chromatography) of the Fab fragment of an anti-dexofloxacin antibody.

[0072] 1. Experimental operation

[0073] 1.1. After recovering the enzymatic hydrolysis product, centrifuge at 4000rpm for 2min.

[0074] 1.2. Purify the enzymatic hydrolyzate after centrifugation directly with Superdex 200 (16 / 600) chromatographic column.

[0075] 1.3. After the sample is loaded on the column, wash 1 column volume with binding buffer (20mM PB+300mM NaCl) and 1 column volume with ultrapure water, set the flow rate to 0.8mL / min, and the system pressure to 0.3MPa.

[0076] 1.4. According to the UV detector to monitor the 280nm protein absorption peak, set the sample to be collected according to the peak collection method, and collect the flow-through protein fraction at a volume of 1 mL per tube.

[0077] 1.5. Referring to the SDS-PAGE electrophoresis method in Example 1, SDS-PAGE electrophoresis identifies the collect...

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Abstract

The invention provides a Fab fragment of an anti-R-ofloxacin (R-OFL) antibody as well as a preparation and application of the Fab fragment. The Fab fragment comprises a heavy chain, wherein the heavychain contains an amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence sharing at least 80% of homology with the SEQ ID NO: 2, or an amino acid sequence of the heavy chain is shown in SEQ ID NO: 2. The Fab fragment purification process is simple, simple to operate and high in yield, and the prepared anti-R-OFL Fab fragment has high sensitivity when used for Elisa detection.

Description

technical field [0001] The invention relates to the technical field of food safety detection, in particular to a Fab fragment of an anti-dexofloxacin antibody and its preparation and application. Background technique [0002] Fluoroquinolones (Fluoroquinolones, FQs) are clinically a class of broad-spectrum antibiotics. Among them, Ofloxacin (OFL), as a third-generation fluoroquinolone antibacterial drug, is one of the most commonly detected fluoroquinolones in aquatic environments. In order to ensure the quality and safety of animal food and public health safety, the Ministry of Agriculture Announcement No. 2292 prohibits the use of ofloxacin and other 4 kinds of veterinary drugs in food animals. Ofloxacin is a chiral drug, including two optically active enantiomers of levofloxacin (S-Ofloxacin, S-OFL) and dextroofloxacin (R-Ofloxacin, R-OFL) body, in which levofloxacin is two orders of magnitude higher than dextrofloxacin. Ofloxacin enantiomers have exactly the same chem...

Claims

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Application Information

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IPC IPC(8): C07K16/44C07K1/22C07K1/16C12N15/13C12P21/06G01N33/53
CPCC07K16/44C12P21/06G01N33/5308C07K2317/55
Inventor 沈兴鲍虹蕾雷红涛甘庆庆徐振林杨金易孙远明
Owner SOUTH CHINA AGRI UNIV
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