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Colistin colloidal gold detection kit and application thereof

A technology for detection kits and colistin, which is applied in biological testing, measuring devices, material inspection products, etc., can solve the problems of high operation precision in incubation time, expensive instruments and equipment, and high requirements for sample preparation, so as to reduce the difficulty of operation , low cost and high detection sensitivity

Active Publication Date: 2019-02-22
北京纳百生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Traditional methods for detecting colistin residues include liquid chromatography, liquid chromatography-mass spectrometry, and enzyme-linked immunoassay (ELISA), among which the first two methods are instrumental methods. Quantitative detection by ultraviolet spectroscopy or mass spectrometry is cumbersome to operate, requires high sample preparation, high detection costs, and expensive equipment, which is unaffordable for grassroots enterprises; ELISA, as a rapid detection method based on antigen-antibody immune response, has Low cost, fast detection, etc., are also used in enterprises and grassroots laboratories, but they also have certain shortcomings, such as the need for a stable temperature environment during the operation process, long incubation time, and high requirements for operational precision. It is difficult to master, especially for occasions such as milk stations, milk tank cars, egg purchases, etc., it is difficult to be competent

Method used

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  • Colistin colloidal gold detection kit and application thereof
  • Colistin colloidal gold detection kit and application thereof
  • Colistin colloidal gold detection kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: Preparation of Colistin-Coupled Antigen

[0051] The colistin B hapten CL-HS was synthesized by the succinic anhydride method, and then coupled with bovine serum albumin, ovalbumin and other carrier proteins by the carbodiimide (EDC) method, and the coupled product was purified and then detected by ultraviolet light Concentration, ready for use after dialysis desalination, details are as follows:

[0052] Accurately weigh 140mg of colistin sulfate B and 20mg of succinic anhydride with an analytical balance, put them into a 10ml brown vial with a magnetic stirrer, add 3ml of pyridine to dissolve, put in a constant temperature oil bath at 80°C for 6 hours; The residue was fully washed 3 times, and there was a large amount of precipitation after standing still; the methanol was discarded, and the precipitation was freeze-dried with a freeze dryer, which was the hapten colistin-HS (CL-HS).

[0053] Take 41.6mg colistin-HS, dissolve in 2ml DMF (N-N dimethylformam...

Embodiment 2

[0056] Example 2: Preparation of colistin-specific monoclonal antibody

[0057] Dry Balb / C mice were immunized with hapten CL-HS conjugated antigen CL-HS-BSA and colistin B directly conjugated antigen CL-BSA, respectively. The adjuvant for the first immunization was Freund's complete adjuvant, and the adjuvant for subsequent immunization was Freund's incomplete adjuvant. The interval between immunizations was 2 weeks. After 3 immunizations, the serum titers were determined by ELISA, and the serum inhibitory titers and the cross-reactivity of the serum to colistin, colistin A, and colistin B were measured by competitive ELISA. Select mice with high serum titers and good responses to colistin A and B. Cell fusion is performed according to conventional methods, and hybridoma cell lines secreting specific monoclonal antibodies are screened.

[0058] (1) Serum titer detection:

[0059] Dilute the conjugated antigens CL-BSA, CL-HS-BSA, CL-OVA, CL-HS-OVA, etc. with 0.05M carbonate ...

Embodiment 3

[0071] Embodiment 3: the preparation of colloidal gold particle

[0072] Colloidal gold particles with a particle size of 30-40nm were prepared by the sodium citrate reduction method, and the specific operations were as follows:

[0073] (1) Take a 250 ml round bottom flask, measure 100 ml of distilled water and add 1 ml of 1% chloroauric acid solution, stir and heat to boil;

[0074] (2) Add 1.5ml of 1% sodium citrate aqueous solution to the above chloroauric acid aqueous solution, stir and mix, and keep boiling for 10 minutes, during which the color of the solution will change from transparent to black, then gradually turn red, and the final solution is wine red;

[0075] (3) Stop heating after 10 minutes, and after the solution cools down, add distilled water to make the volume to 100ml, which is the colloidal gold solution;

[0076] (4) Store the prepared colloidal gold solution at 2-8°C.

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Abstract

The invention discloses a colistin colloidal gold detection kit. The kit mainly comprises a colistin colloidal gold detection test strip, a lyophilized colloidal gold marked colistin specific monoclonal antibody microporous reagent, a microporous panel, a quantitative pipette and a sample diluting liquid, wherein the detection test strip is formed by a PVC back board, a water absorption pad, a sample pad, and a nitrocellulose film (NC film) which is sprayed with a detected antigen coated detection line (T line) and a goat-anti-mouse-anti antibody coated quality control line (C line). The kit is used for detecting colistin residue in eggs or milk without expensive equipment, is simple and convenient to operate, does not need special training, has high detection sensitivity, and is suitablefor wide application in the fields of local laboratories, field detection and the like.

Description

technical field [0001] The invention belongs to the technical field of rapid detection of veterinary drug residues, and relates to a colistin colloidal gold detection kit and an application thereof. Background technique [0002] Colistin sulfate is a basic polypeptide antibiotic, which is mainly used to prevent and control the infection of sensitive bacteria and promote the growth of livestock and poultry. Colistin sulfate can bind to the free phosphate of cell membrane lipoproteins, which reduces the surface tension of the cell membrane and increases the permeability, resulting in cytoplasmic outflow and cell death. Colistin sulfate has a strong inhibitory effect on Gram-negative bacteria (especially Escherichia coli, Salmonella, Pseudomonas aeruginosa, Proteus and Haemophilus, etc.), and has a strong inhibitory effect on Gram-positive bacteria (except Staphylococcus aureus and hemolytic streptococcus outside) and fungi have no effect. Colistin sulfate is difficult to abs...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/94G01N33/531G01N33/543
CPCG01N33/531G01N33/54306G01N33/9446
Inventor 杨春江于在江赵荣茂袁志波白民俊吴迪李月孙奇武秦丽丽李慧俐
Owner 北京纳百生物科技有限公司
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