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Tobramycin monoclonal antibody as well as preparation method and application of tobramycin monoclonal antibody

A monoclonal antibody, tobramycin technology, applied in biochemical equipment and methods, chemical instruments and methods, microorganism-based methods, etc., can solve the problems of time-consuming repeatability and specificity, and achieve good sensitivity, The effect of convenient and fast synthesis and simple preparation method

Active Publication Date: 2015-01-28
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Microbiological methods are time consuming and less reproducible and specific

Method used

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  • Tobramycin monoclonal antibody as well as preparation method and application of tobramycin monoclonal antibody
  • Tobramycin monoclonal antibody as well as preparation method and application of tobramycin monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Obtaining of hybridoma cell line 1A10 and identification of the monoclonal antibody against tobramycin produced by it

[0027] 1. Obtaining the hybridoma cell line 1A10

[0028] 1. Preparation of complete tobramycin antigen Tob-BSA / Tob-HSA

[0029] Dissolve 20mg of Tob (tobramycin) in 1mL of PBS (0.01M phosphate buffer saline) at pH 7.4, slowly add 0.5mL of freshly prepared 1% glutaraldehyde solution into the small molecule solution, stir at room temperature to activate 8min. Another 10 mg of BSA (HSA) was dissolved in 1 mL of 0.05 M, pH 9.6 CBS solution. The above-mentioned activated tobramycin solution was slowly added dropwise into the BSA (HSA) solution, and stirred at room temperature for 1 h. The solution was pre-cooled at 4° C., and a certain amount of sodium borohydride was weighed to make the final concentration of sodium borohydride in the reaction system 4 mg / mL, and stirred for 2 h. Dialyze at 4°C for three days, and store at -20°C.

[0030] ...

Embodiment 2

[0044] Example 2. The application of the indirect competition ELISA method established with the antibody secreted by the hybridoma cell line 1A10

[0045] The monoclonal antibody prepared by the hybridoma cell line 1A10 through ascites in vivo was applied to the tobramycin ELISA addition and recovery test, and the specific steps were as follows:

[0046] (1) Use 0.05 μg / mL Tob-OVA diluted with carbonate buffer solution (CBS) as the coating material to coat a 96-well microplate, 100 μL per well, coat at 37°C for 2 h, then wash with PBST Wash the plate three times, each time with 250 μL per well, each time for 3 min, and pat dry.

[0047] (2) Block with CBS containing 0.1% casein, 200 μL per well, block for 2 h at 37°C, wash the plate three times with PBST washing solution, 250 μL per well for 3 min each time, and pat dry.

[0048] (3) Prepare 0, 0.625, 1.25, 2.5, 5, 10, 20, 40 μg / L standard solutions of tobramycin in phosphate buffered saline (PBS). Add the standard solution ...

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Abstract

The invention relates to a tobramycin monoclonal antibody as well as a preparation method and application of the tobramycin monoclonal antibody and belongs to the field of food safety immunodetection. The monoclonal antibody is generated by mouse hybridoma cell line No. G (A10) with the preservation number of CGMCC No.9307. The preparation method comprises the following steps: (1) activating amino of tobramycin with a glutaraldehyde method, coupling the activated amino of the tobramycin with the amino of carrier protein, reducing C=N of Schiff base formed by glutaraldehyde and amino of the obtained conjugate into a stable C-N structure to obtain final stable conjugate serving as complete antigen; (2) immunizing, fusing and sieving the complete antigen obtained in the step (1), carrying out enlarged cultivation and mouse enterocoelia induction to generate ascites to obtain the monoclonal antibody used for specific detection of tobramycin; and (3) activating carboxyl of succinic acid with a DCC method, reacting activate fluid with ovalbumin amidogen to obtain carboxylation protein of ovalbumin-succinic acid, carrying out dialysis and condensing the tobramycin and the carboxyl of the protein by using an EDC method to prepare peridium of screened antibody.

Description

technical field [0001] A tobramycin monoclonal antibody and its preparation method and application relate to an anti-tobramycin specific monoclonal antibody produced by a hybridoma cell line 1A10 (monoclonal cell line No. G). The invention belongs to the field of food safety immunological detection. Background technique [0002] Tobramycin (Tob) is a second-generation aminoglycoside antibiotic. Compared with the first-generation aminoglycoside antibiotics, it has better curative effect and lower toxicity. It is usually used for injection to treat severe G bacteria infection. It has become one of the most widely used aminoglycoside antibiotics in the world. Tobramycin has also been widely used in the field of veterinary medicine, so it also exists in foods commonly used by people such as meat and milk. Since its safe therapeutic concentration is 4mg / L~8mg / L, a long-term blood concentration exceeding 8mg / L may cause severe nephrotoxicity and ototoxicity. Therefore, it is ne...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/44C07K16/02G01N33/577C12R1/91
Inventor 匡华曹珊珊胥传来徐丽广马伟刘丽强宋珊珊吴晓玲
Owner JIANGNAN UNIV
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