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Preparation method and application of bacitracin antigen

A bacitracin and antigen technology, applied in the field of monoclonal antibody preparation and bacitracin antigen, can solve the problems of poor reproducibility, cumbersome operation, low sensitivity, etc., and achieve the effects of low cost, large sample volume and high sensitivity

Inactive Publication Date: 2014-12-31
BEIJING KWINBON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, foreign reports on the detection methods of colistin and bacitracin drug residues in animal foods include microbiological methods, thin-layer chromatography, immunological methods, capillary electrophoresis, high-performance liquid chromatography, etc. The methods reported in China are mainly It is a microbial method, but its operation is cumbersome, low sensitivity, and poor reproducibility

Method used

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  • Preparation method and application of bacitracin antigen
  • Preparation method and application of bacitracin antigen
  • Preparation method and application of bacitracin antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1, Preparation and Identification of Bacitracin Artificial Antigen

[0025] 1. Synthesis of bacitracin immune antigen

[0026] (1) Dissolve 45 mg of bacitracin in 1.5 mL of water.

[0027] (2) Add 10 μL of 50% 1,5-glutaraldehyde (GA) into the bacitracin solution, and stir for 18 hours at room temperature.

[0028] (3) Dilute 100 mg of bovine serum albumin (BSA) with 1.5 mL of water, add it to the bacitracin solution, and react for 24 hours.

[0029] (4) Add 24mg NaBH 4 Reaction 3h.

[0030] (5) Dialyze with triple distilled water for 48 hours to obtain the immunogen.

[0031] 2. Synthesis of bacitracin-coated antigen

[0032] (1) Dissolve 45 mg of bacitracin in 1.5 mL of water.

[0033] (2) Add 10 μL of 50% 1,5-glutaraldehyde (GA) into the bacitracin solution, and stir for 18 hours at room temperature.

[0034] (3) Take 60 mg of ovalbumin (OVA) and dilute it with 1.5 mL of water, add it to the bacitracin solution, and react for 24 hours.

[0035] (4) Add...

Embodiment 2

[0039] Example 2, Preparation and Specific Identification of Antibodies

[0040] 1. Preparation of anti-bacitracin monoclonal antibody

[0041] (1) Use the immunogen prepared above at 100 μg / mouse, dissolve the immunogen in normal saline and mix it with Freund’s complete adjuvant in an equal volume, and immunize 6-8 week-old Balb / c female mice subcutaneously on the back of the neck. On the 7th, 14th, and 28th day afterward, the immunogen and Freund's incomplete adjuvant were mixed in equal volumes, and each booster immunized once, and the immune complex was 100 μg per mouse 3 days before the fusion, without Freund's adjuvant, and then boosted immunization again .

[0042] (2) According to the conventional method, the splenocytes of the immunized mice were mixed with the mouse myeloma cells (SP2 / 0) in the logarithmic growth phase, and then slowly added the preheated fusion agent (PEG4000) within 45s for fusion , suspend evenly with HAT medium, then add appropriate amount of f...

Embodiment 3

[0059] Example 3, Preparation of Enzyme-labeled Secondary Antibody

[0060] 1. Preparation of goat anti-mouse anti-antibodies

[0061] Goats were used as immunized animals, and pathogen-free sheep were immunized with mouse antibodies as immunogens to obtain goat anti-mouse antibodies.

[0062] 2. Preparation of enzyme-labeled secondary antibody

[0063] (1) Weigh 2 mg of horseradish peroxidase (HRP) and dissolve in 0.5 mL of water, add 0.5 mL of 0.06 mol / L NaIO 4 solution, at 4°C in the dark for 30 min;

[0064] (2) Add 0.5 mL of 160 mmol / L ethylene glycol, and let it react for 30 min at room temperature;

[0065] (3) Add 2 mg of goat anti-mouse anti-antibody prepared in step 1, mix well, put it into a treated dialysis bag, dialyze in 1000 mL of 0.05 mmol / L sodium carbonate buffer, and overnight at 4°C;

[0066] (4) Aspirate the dialysate into a 10 mL centrifuge tube, add 0.25 mL of 5g / L NaBH 4 solution, mix well and place at 4°C for 2 h;

[0067] (5) Add an equal volume...

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PUM

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Abstract

The invention discloses a bacitracin artificial antigen, a corresponding monoclonal antibody, a preparation method of the bacitracin artificial antigen, a preparation method of the corresponding monoclonal antibody, and applications of the antigen and the antibody. The bacitracin antigen can be applied to prepare the bacitracin specific antibody. The bacitracin artificial antigen can generate the bacitracin specific antibody through an immune animal, can be used for preparing an enzyme-linked immune detection kit for detecting the bacitracin residual, and has the advantages of simplicity, rapidness, large sample throughput, high sensitivity, strong specificity and the like.

Description

technical field [0001] The invention belongs to the technical field of food safety detection, and in particular relates to a method for preparing a bacitracin antigen and a monoclonal antibody and an application thereof. Background technique [0002] Bacitracin, also known as subtilisin, is isolated from the culture fluid of Bacillus licheniformis or Bacillus subtilis variants. Bacitracin has a strong inhibitory effect on Gram-positive bacteria, and its antibacterial spectrum is similar to that of penicillin G. It is mainly used for the treatment of penicillin-resistant Staphylococcus infection, and can also be used as a feed additive. It is currently widely used in feed at home and abroad. A new type of antibiotic. It is composed of a variety of amino acids, including bacitracin A, B, C, D, E and other isomers, of which the highest content and the most effective is bacitracin A, soluble in ethanol, acetic acid, pyridine, slightly soluble In acetone, insoluble in water, et...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/32C07K16/12G01N33/68
CPCC07K14/32C07K16/1278G01N33/6854
Inventor 冯才伟罗晓琴陶光灿徐念琴韩京朋韩雪倩吴小胜余厚美
Owner BEIJING KWINBON BIOTECH
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