Antibody chip kit and method for detecting residual cephalosporin antibiotics in food

An antibody chip and cephalosporin technology, which is applied in the field of antibody chip kits for the detection of cephalosporin antibiotic residues in food, can solve the problems of low sensitivity and specificity of microbial methods, single detection components of Elisa, and qualitative detection of colloidal gold technology, etc. problem, to achieve the effect of long signal duration, good market application value and high accuracy

Active Publication Date: 2015-04-22
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There are deficiencies in the current detection methods of veterinary drug residues, such as complex operation of instrument methods, high degree of instrumentation and high price, low sensitivity and specificity of microbial methods, single detection components of Elisa, and colloidal gold technology can only be used for qualitative detection

Method used

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  • Antibody chip kit and method for detecting residual cephalosporin antibiotics in food
  • Antibody chip kit and method for detecting residual cephalosporin antibiotics in food
  • Antibody chip kit and method for detecting residual cephalosporin antibiotics in food

Examples

Experimental program
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Effect test

Embodiment 1

[0037] The preparation of the monoclonal antibody of embodiment 1 anti-cephalexin, cefadroxil and cephradine

[0038] 1.1 Preparation of immunogen

[0039] Accurately weigh 36.5 mg of cephalexin and completely dissolve it in 4 mL of PBS to obtain solution A. Accurately weigh 68mg of BSA and fully dissolve it in 6mL of PBS to form B solution. Another 120.0 mg of carbodiimide (EDC) and 60 mg of N-hydroxysuccinimide (NHS) were dissolved in 1 mL of LPBS to obtain solution C. In ice bath, add liquid C dropwise to liquid B under magnetic stirring, react in ice bath for 4h, then add the mixed solution dropwise into liquid A, and react in ice bath for 12h. After the reaction is completed, transfer the reaction solution into a dialysis bag, dialyze in PBS at 4°C for 3 days, change the dialysate every 4 to 6 hours, and freeze-dry the sample after the dialysis to obtain the conjugate cephalexin-BSA, and place it at -20 Store at ℃.

[0040] 1.2 Preparation of coating agent

[0041]Di...

Embodiment 2

[0048] Example 2 Preparation of monoclonal antibodies against ceftiofur, desfuroyl ceftiofur, ceftriaxone, cefotaxime and cefquinome

[0049] 2.1 Preparation of immunogen

[0050] Dissolve 50 mg of ceftiofur in 1 mL of dimethylformamide (DMF) to obtain liquid A. Weigh 66mg of HSA and dissolve it in 10mL of PBS to make solution B. Add 20mg of N,N'-dicyclohexylcarbodiimide (DCC) and 10mg of N-hydroxysuccinimide (NHS) to solution A respectively, and react at room temperature for 24 hours. After the reaction is completed, centrifuge to remove the precipitate. Drop into solution B, and react in ice bath for 24h. After the reaction is completed, transfer the reaction solution into a dialysis bag, dialyze in PBS at 4°C for 3 days, replace the dialysate every 4-6 hours, and freeze-dry the sample after the dialysis to obtain the conjugate ceftiofur-HSA. Store at 20°C.

[0051] 2.2 Preparation of coating agent

[0052] Dissolve 120 mg of OVA in 8 mL of carbonate buffered saline (CB...

Embodiment 3

[0059] The selection of embodiment 3 chip parameters

[0060] 1) Selection of substrates: Spot 0.5 μg / mL Cy3-OVA on polylysine slides, positively charged slides, core polymer substrate G, aldehyde-based slides, and agarose-modified slides respectively, and use The InnoScan 700A scanner scans and stores the data, the results are shown in the attachment figure 2 ,from diagram 2-1 It can be seen that the relative fluorescence signal intensity of the core polymer substrate G is the highest, indicating that the core polymer substrate G has the best adsorption to the sample. from Figure 2-2 It can be seen that the background value is lower than that of the aldehyde-based substrate but significantly higher than that of the other three substrates. Considering the two factors of adsorption and background value comprehensively, the crystal core polymer substrate G was selected as the substrate.

[0061]2) Selection of sampling order: according to the following four ways: 2 is fro...

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Abstract

The invention discloses an antibody chip kit for detecting residual cephalosporin antibiotics in food, and belongs to the technical field of the drug residue detection. The kit comprises a chip, an antibody, a second antibody marked by Cy3, and an extraction reagent, the antibody is composed of a cefalexin monoclonal antibody and a ceftiofur monoclonal antibody, and the cefalexin monoclonal antibody is secreted by hybridoma 3A6 with a preservation number of CCTCC NO: C201340; the ceftiofur monoclonal antibody is secreted by hybridoma 4D5 with a preservation number of CCTCC NO: C201341, and the chip fixes cefalexin coating antigen and cefalexin coating antigen. The invention further discloses an antibody chip method for detecting residual cephalosporin antibiotics in food, the method can be used for simultaneously detecting 8 kinds of cephalosporins, and has the advantages of high accuracy, high precision, high efficiency, etc.

Description

technical field [0001] The invention belongs to the technical field of biochip and drug residue detection, and in particular relates to an antibody chip kit and a detection method for detecting cephalosporin antibiotic residues in food. Background technique [0002] Cephalosporin antibiotics belong to β-lactam drugs, which have the advantages of broad antibacterial spectrum, strong antibacterial effect, and resistance to penicillinase. It is commonly used in the treatment of sepsis, meningitis and endocarditis in animal husbandry. Irrational use causes these drugs to accumulate in tissues of animal origin. After people eat these animal-derived foods, allergic reactions and even shocks occur, which pose a serious threat to human health. The abuse of a large number of drugs will also lead to the emergence of drug-resistant strains and destroy the ecological environment. In order to ensure the safety of human food and the stability of the ecological environment, my country h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/02G01N33/577
Inventor 袁宗辉彭大鹏魏娜娜王玉莲陈冬梅陶燕飞刘振利
Owner HUAZHONG AGRI UNIV
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