Unlock instant, AI-driven research and patent intelligence for your innovation.

A kind of f(ab) of anti-levofloxacin antibody 2 Fragment and its preparation method and application

A technology of levofloxacin and fragments, which is applied in the field of F2 fragments of anti-levofloxacin antibodies and its preparation, can solve the problems of non-detection of levofloxacin, difficulty in actual purification, reduction of protein yield in purification steps and Activity and other issues, to achieve the effect of high sensitivity, simple operation, simple purification process

Active Publication Date: 2022-05-03
SOUTH CHINA AGRI UNIV
View PDF7 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, different enzymatic hydrolysis conditions will produce antibody fragments of different sizes, which will bring different degrees of difficulty to the actual purification.
The increase in purification steps and improper strategies will reduce protein yield and activity
Patent CN108727502A discloses a method for protease digestion and two-step purification to prepare the Fab fragment of levofloxacin antibody, but there is no F(ab) for anti-levofloxacin antibody 2 There are no reports of fragment purification methods based on F(ab) 2 Fragment-specific detection of levofloxacin report

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A kind of f(ab) of anti-levofloxacin antibody <sub>2</sub> Fragment and its preparation method and application
  • A kind of f(ab) of anti-levofloxacin antibody <sub>2</sub> Fragment and its preparation method and application
  • A kind of f(ab) of anti-levofloxacin antibody <sub>2</sub> Fragment and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Preparation of Anti-Levofloxacin Antibody IgG and Variable Region Sequencing

[0042] 1. Experimental operation

[0043]1. Ascites preparation

[0044] (1) Resuscitate the levofloxacin-resistant hybridoma cell line (prepared by South China Agricultural University, School of Food Science, Guangdong Provincial Key Laboratory), use complete medium (gibco1640 basal medium with 6mL gibco double antibody, 6mL gibco glutamine , 100mL according to Kesai fetal bovine serum) culture;

[0045] (2) When the cells grow to fill the bottom of the culture dish, remove the complete medium, use the basal medium (gibco1640 basal culture) to suspend the cells, and absorb the suspended cells with a 1mL syringe;

[0046] (3) Select 8-12 week-old female mice as experimental mice, inject 500 microliters of liquid paraffin intraperitoneally into each mouse, and inject cell suspension into the abdominal cavity after 7 days;

[0047] (4) Each mouse was intraperitoneally injected with...

Embodiment 2

[0056] Embodiment 2 Enzymolysis of Anti-Levofloxacin Antibody

[0057] 1. Experimental operation

[0058] (1) Use pH 4.0 HCl solution to prepare pepsin into 4mg / mL stock solution and store at -20°C;

[0059] (2) According to the ratio of enzyme and antibody 1:10 (w / w), absorb pepsin and mix it with antibody, adjust the pH to (2.0, 2.4, 2.8) with 1M HCl solution, and place it on a constant temperature shaker at 37°C, (0.5 , 1.0, 1.5) h;

[0060] (3) Regulate the enzymolysis mixture to pH7 with NaOH solution, so that the enzymolysis reaction is terminated;

[0061] (4) The enzyme digestion reaction product and the original monoclonal antibody were identified by SDS-PAGE electrophoresis.

[0062] 2. Experimental results

[0063] The results of SDS-PAGE gel electrophoresis were as follows: image 3 Shown: more than 95% of the anti-levofloxacin antibody has been hydrolyzed by pepsin, and the main product of the hydrolysis is F(ab) 2 , Fab fragment. By calculating the concent...

Embodiment 3

[0064] Embodiment 3 anti-levofloxacin antibody F (ab) 2 Fragment Gel Filtration Chromatography

[0065] 1. Experimental operation

[0066] In order to convert the F(ab) in the enzymatic hydrolysis product 2 Fragments are separated from other impurity fragments according to molecular weight, and the method of purification using gel filtration chromatography is as follows:

[0067] (1) Use binding buffer PBS (20mM PB, 150mM NaCl) to equilibrate the chromatographic column (Superdex 200, GE, USA) with 1.5 column volumes, use a lower flow rate to make the separation of different components more thorough, set the flow rate to 0.8mL / min, system pressure 0.3MPa;

[0068] (2) Pass the enzymatic hydrolyzate of Example 2 through the chromatographic column, monitor the 280nm protein absorption peak according to the ultraviolet detector, collect samples according to the peak mode, and collect the protein components flowing through in a volume of 1 mL per tube;

[0069] (3) SDS-PAGE el...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a F(ab) of an anti-levofloxacin antibody 2 Fragment, the F(ab) 2 The fragment has two Fab parts connected together by a disulfide bond, each Fab part comprises a light chain and a heavy chain Fd part, the amino acid sequence of the light chain is shown in SEQ ID NO: 3, and the heavy chain Fd part As shown in SEQ ID NO:4. The present invention uses pepsin enzymolysis and adopts a two-step purification method to purify the enzymolysis product, and obtains the anti-levofloxacin antibody F(ab) for the first time 2 . The method of the present invention has simple flow, simple operation and high output, and the F(ab) of the anti-levofloxacin antibody is 2 The half-inhibitory concentration of the fragment to levofloxacin is 0.33ng / mL, the detection limit is 0.04ng / mL, and the detection range is 0.05-2.15ng / mL, which has great application prospects.

Description

technical field [0001] The present invention relates to the technical field of food safety detection, more specifically, relate to a kind of F (ab) of anti-levofloxacin antibody 2 Fragment and its preparation method and application. Background technique [0002] Ofloxacin (OFL) is a third-generation artificially synthesized broad-spectrum fluoroquinolone antibacterial drug. It targets bacterial deoxyribonucleic acid gyrase, inhibits its synthesis, and blocks DNA replication, thereby inhibiting its synthesis. Gram-positive bacteria and most Gram-negative bacteria cause irreversible damage and are among the most commonly detected fluoroquinolones in our aquatic environment. In order to ensure the quality and safety of animal food and public health safety, the Ministry of Agriculture Announcement No. 2292 stipulates that the use of 4 kinds of veterinary drugs including ofloxacin in food animals should be stopped. Among the existing detection methods, the immunoassay method ha...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/44C12N15/13C07K1/22C07K1/16C07K1/14C12P21/06G01N33/53
CPCC07K16/44C12P21/06G01N33/53C07K2317/54C07K2317/51C07K2317/515
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