Gentamicin antibody and application thereof

A technology of gentamicin and single-chain antibody, which is applied in the direction of application, introduction of foreign genetic material using a vector, and cells modified by the introduction of foreign genetic material, etc., can solve the problems of complex production process, long consumption time, and difficult operation, etc. To achieve the effect of simple storage, high sensitivity and easy production

Inactive Publication Date: 2011-01-26
北京维德维康生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The preparation of monoclonal antibodies or polyclonal antibodies must be obtained through cell culture. The entire production process is complicated, time-consuming, expensive, and difficult to operate

Method used

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  • Gentamicin antibody and application thereof
  • Gentamicin antibody and application thereof
  • Gentamicin antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Embodiment 1, preparation and function test of antibody

[0056] 1. Preparation of gentamicin single-chain antibody

[0057] (1) Antibody screening

[0058] Six-month-old male Balb / c mice were taken, and the total RNA of splenocytes was extracted by Trizol one-step method, purified to obtain mRNA, and then reverse-transcribed to obtain cDNA. Using a complete set of primers, the heavy chain variable region (VH) and light chain variable region (VL) genes were respectively amplified using cDNA as a template. Splicing into a single-chain antibody gene (ScFv), then linking the ScFv with the vector pCANTAB5E to obtain pCANTAB5E / ScFv, and transforming Escherichia coli TG1 to obtain a mouse-derived non-immune single-chain antibody library. Phage particles carrying multiple specific single-chain antibodies against gentamycin were screened by ELISA, and their corresponding nucleotide sequences were obtained by sequencing.

[0059] The single-chain antibody consists of a heavy ...

Embodiment 2

[0089] Embodiment 2, the enzyme-linked immunosorbent assay kit that detects gentamicin and its preparation

[0090] 1. The enzyme-linked immunosorbent assay kit consists of the following materials:

[0091] 1. ELISA plate coated with gentamicin and carrier protein conjugate (GEN-BSA);

[0092] 2. Gentamicin antibody: the single-chain antibody described in Example 1. The concentration of the antibody working solution is 5.0 ng / mL, and the antibody working solution is obtained by diluting the purified antibody in Example 1 with the sample diluent;

[0093] 3. Gentamicin standard substance: gentamicin standard substance is gentamicin, and the concentration of the standard solution is 0μg / L, 0.5μg / L, 1.5μg / L, 4.5μg / L, 13.5μg / L , 40.5 μg / L; Gentamicin was purchased from Sigma-Aldrich Company in the United States; the product catalog number was G1397; it was diluted to the above-mentioned concentrations with sample diluent;

[0094] 4. Enzyme-labeled secondary antibody: horseradi...

Embodiment 3

[0142] Embodiment 3, the test paper that detects gentamicin and its preparation and application

[0143] 1. The structure of the test paper

[0144] The test paper is composed of a sample absorbent pad, a colloidal gold pad, a reaction film and a water absorbent pad;

[0145] The sample absorption pad, the colloidal gold pad, the reaction membrane and the water absorption pad are sequentially connected, the end of the sample absorption pad is connected to the beginning of the colloidal gold pad, the end of the colloid gold pad is connected to the beginning of the reaction membrane, and the end of the reaction membrane is connected to the beginning of the reaction membrane. The beginning of the absorbent pad is connected;

[0146] The colloidal gold pad is coated with a colloidal gold-labeled gentamicin antibody (protein shown in sequence 1 in the sequence listing);

[0147] Detection area and quality control area are arranged on described reaction film, and detection area (C...

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Abstract

The invention discloses a gentamicin antibody and application thereof. The antibody consists of a heavy chain variable region, a small peptide for connecting the heavy chain variable region and a light chain variable region, and the light chain variable region which are connected with each other in turn. The amino acid sequence of the heavy chain variable region is shown as the 11th to the 124th amino acid residues from an N terminal in a sequence 1; and the amino acid sequence of the light chain variable region is shown as the 140th to the 263th amino acid residues from the N terminal in thesequence 1. A kit and a colloidal gold strip card of the invention have the advantages of high sensitivity, high accuracy, high precision, low cost, simple operation, short detection time, suitability for various units to use, simple storage and long quality guarantee period, can quickly detect large quantities of samples at the same time, and can realize field high-throughput quick detection. Therefore, the antibody, the kit, colloidal gold test paper and a detection method of the invention of the invention can play major roles in the detection of the gentamicin.

Description

technical field [0001] The invention relates to a gentamicin antibody and its application. Background technique [0002] Gentamicin (GEN) is an aminoglycoside antibiotic, which plays an important role in the treatment of infectious diseases of Pseudomonas aeruginosa, Proteus and penicillin-resistant Staphylococcus aureus in livestock, but its residues are relatively toxic and side effects , is ototoxic and nephrotoxic. In order to strengthen the management of veterinary drug residues and ensure the quality and safety of animal products, the Ministry of Agriculture of my country revised the "Maximum Residue Limits of Veterinary Drugs in Animal Foods" in 2002, which stipulates that the maximum residue limit of gentamicin in milk is 200 μg / kg, which is 200 μg / kg. There are also corresponding limits for gentamicin residues in the muscle, fat, liver, and kidney of cattle, sheep, pigs, and chickens. [0003] The detection of gentamicin residues is currently mainly performed by hi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/44C12N15/13C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10G01N33/566G01N33/577G01N33/531
Inventor 沈建忠江海洋吴小平李杰超王战辉徐飞刘丹杨光李娜杨丽丽王富伟王世恩
Owner 北京维德维康生物技术有限公司
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