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Enzyme-linked immunological kit for detecting neomycin drug and method

An enzyme-linked immunosorbent reagent and neomycin technology, applied in the field of immunological detection, can solve the problems of being unsuitable for on-site monitoring and screening of a large number of samples, complex instruments and equipment, cumbersome processes, etc., and achieve good preservation and stability, overcome The effect of technical problems

Active Publication Date: 2011-06-22
BEIJING WANGER BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The chemical methods for detecting neomycin residue mainly include thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), capillary electrophoresis (CE), etc., which are not suitable for on-site monitoring and monitoring due to complicated equipment and cumbersome processes. Large Sample Screening

Method used

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  • Enzyme-linked immunological kit for detecting neomycin drug and method
  • Enzyme-linked immunological kit for detecting neomycin drug and method
  • Enzyme-linked immunological kit for detecting neomycin drug and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Embodiment 1 Preparation of kit components

[0062] 1. Antigen Synthesis

[0063] a. Synthesis of neomycin immune antigen

[0064] The immunogen was obtained by coupling neomycin and bovine serum albumin by direct activation protein method.

[0065] The preparation process of the immunogen: take 18 mg of bovine serum albumin and dissolve it in 1 ml of water (I solution); take EDC 20 mg and dissolve it in 0.5 ml of water and add it to the solution (I solution), stir and react for 30 minutes; take 5 mg of neomycin sulfate and use 1.5 ml After dissolving in water, add it into the solution (II solution), stir and react overnight to obtain the immunogen.

[0066] b. Preparation of coated original neomycin-conjugated antigen

[0067] Coupling was obtained by coupling neomycin and thyroid protein by direct protein activation.

[0068] The preparation process of the original coating: take 18 mg of BCG protein and dissolve it in 1 ml of water (I solution); take 20 mg of EDC ...

Embodiment 2

[0087] Example 2 The formation of an enzyme-linked immunosorbent assay kit for detecting neomycin

[0088] Set up the ELISA kit for detection of neomycin, so that it contains the following components:

[0089] (1) ELISA plate coated with anti-neomycin antibody;

[0090] (2) Goat anti-mouse anti-antibody labeled with horseradish peroxidase;

[0091] (3) Neomycin monoclonal antibody working solution;

[0092] (4) 6 bottles of neomycin standard solution, the concentrations are 0 μg / L, 0.5 μg / L, 1.5 μg / L, 4.5 μg / L, 13.5 μg / L, 40.5 μg / L;

[0093] (5) The substrate chromogenic solution is composed of A liquid and B liquid, the substrate chromogenic liquid A liquid is carbamide peroxide, and the substrate chromogenic liquid B liquid is tetramethylbenzidine;

[0094] (6) The stop solution is 2mol / L hydrochloric acid;

[0095] (7) The concentrated washing solution is 0.01M, pH7.4, and contains 0.8-1.2% Tween 20 and 0.5‰ sodium azide preservative phosphate buffer;

[0096] (8) The ...

Embodiment 3

[0097] Example 3 Detection of Neomycin Residues in Samples

[0098] 1. Sample pretreatment

[0099] Animal tissue (chicken, chicken liver, pork, pork liver)

[0100] Weigh 2.0g of the homogenized tissue sample into a 50ml centrifuge tube, add 6ml of 0.1MPBS (PH=10-11) buffer solution, mix well up and down for 10min, put it in a 60°C water bath for 60min, take it out and cool it to room temperature and shake Homogenize, centrifuge at room temperature for 10 min at 6000 g or more, take the supernatant, dilute it at a volume ratio of 1:9, and take 20 μl for analysis.

[0101] 2. Detection with kit

[0102]Add 20 μl of serial standard solution or sample solution to the microwells of the enzyme-labeled plate coated with anti-neomycin antibody, then add 80 μl of enzyme-labeled antigen, and react at 25°C for 30 minutes. Pour out the liquid in the wells, add diluted washing solution to each well, pour out the liquid in the wells after 30 seconds, repeat this operation for a total o...

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Abstract

The invention provides an enzyme linked immune regent box to detect neomycin medicine. It includes: the enzyme labeled board which encrusts the encrusting substance, the enzyme marker, the special antibody for the neomycin, the neomycin standard solution, the substrate color solution, the stop solution, the condense washing solution, the condense recovery solution. The invention also discloses the method which includes the process: first to pre-treat the sample, then to detect by the regent box and last to analyze the detecting result. The invention is to detect the neomycin residue weight inthe animal food such as the chicken, chicken liver, pork, pork liver, milk powder, egg and feedstuff. The method is simple, low cost and has high sensitivity, also it is proper for detect the mass sample.

Description

technical field [0001] The invention relates to the field of immunological detection, in particular to an enzyme-linked immunoimmunoassay kit for detecting neomycin drugs in animal-derived foods such as chicken, chicken liver, pork, pig liver, milk powder, eggs and feed, and its Detection method. Background technique [0002] Neomycin has a broad antibacterial spectrum and has good inhibitory effects on Gram-positive bacteria and Mycobacterium tuberculosis. As a common veterinary drug, it is widely used in animal husbandry production. In veterinary medicine, neomycin is mainly used to treat bacterial enteritis in livestock and poultry. In livestock and poultry breeding, it can improve feed utilization and promote animal growth, but because of its high toxicity, residues in animal foods can cause greater potential harm to consumers and pose a threat to food hygiene and public health . [0003] The chemical methods for detecting neomycin residue mainly include thin-layer c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/543G01N33/569G01N33/535G01N33/577
Inventor 何方洋吴小平冯才伟万宇平郭勇赵正苗冯才茂朱亮汪善良刘平陈炜琳蒲小容冯月君余厚美罗贵昆屈晓丽
Owner BEIJING WANGER BIOTECH
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