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Antibody chip kit and method for detecting aminoglycoside antibiotic residues in food

A food and neomycin technology, which is applied in the direction of testing food, material inspection products, measuring devices, etc., can solve the problems of low detection sensitivity and inability to detect aminoglycoside antibiotics at the same time, and achieve high detection efficiency and good market application value , good precision effect

Inactive Publication Date: 2016-11-23
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] CN 102585006A discloses a monoclonal antibody and ELISA method and kit for detecting neomycin, amikacin and paromomycin; CN 102608319A discloses a method and kit for detecting gentamicin and sisal The monoclonal antibody of neomycin and ELISA method and test kit, above two inventions can not detect above five kinds of aminoglycoside antibiotics simultaneously, and detection sensitivity is lower, and the minimum detection limit of the former to neomycin in pig muscle ( LOD) was 15.90 μg / L, and the latter had a minimum detection limit (LOD) of gentamicin in chicken muscle of 34.09 μg / L

Method used

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  • Antibody chip kit and method for detecting aminoglycoside antibiotic residues in food
  • Antibody chip kit and method for detecting aminoglycoside antibiotic residues in food
  • Antibody chip kit and method for detecting aminoglycoside antibiotic residues in food

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Embodiment 1

[0039] The synthesis of embodiment 1 aminoglycoside coating former

[0040] 1) Synthesis of Amikacin Coating Progen

[0041] Weigh 148.0 mg of amikacin and 40.0 mg of OVA and dissolve in 12 mL of pure water (pH7.4), stir evenly, then add dropwise EDC430.00 mg dissolved in 4 mL of pure water, and stir for 8 hours at room temperature. Finally, the reaction solution was transferred into a dialysis bag, and dialyzed in PBS solution (pH7.4) at 4° C. for 4 days. Freeze-dry after centrifugation and store at -20°C for later use.

[0042] 2) Synthesis of sisomycin coating agent

[0043] Dissolve sisomycin 20.0mg and OVA 200.0mg in 20mL PBS solution (pH7.4), stir well, then slowly add 80.00mgEDC dissolved in 1mL pure water. The reaction was stirred at room temperature for 2 hours. Finally, the reaction solution was transferred into a dialysis bag, and dialyzed in PBS (pH7.4) solution at 4° C. for 4 days. Freeze-dry after centrifugation and store at -20°C for later use.

Embodiment 2

[0044] The selection of embodiment 2 chip parameters

[0045] 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 clearly seen that the relative fluorescence signal intensity of the polymer substrate G is the highest, indicating that the polymer substrate G has the best adsorption to our sample; from Figure 2-2 It can be seen that the background value is lower than that of the aldehyde group. Considering the two factors of adsorption and background value, the commercial crystal core polymer substrate G was finally selected as the substrate.

[0046] 2) Selection of different spotting order: according to the following four ways respectively: B is from right to left, from bottom to top; C is from le...

Embodiment 3

[0054] Embodiment 3 Preparation of antibody chip

[0055] 1) Two kinds of aminoglycoside coating agents and monoclonal antibodies used in the assessment

[0056] Determine the titer of the antibody according to the ELS1A operating procedure, dilute the original coating solution into a series of working concentrations with carbonate buffer, coat overnight at 4°C, shake off the coating solution, add 250 μL of washing solution to each well, and let it stand for 30 seconds , shake out the washing solution, pat dry, repeat washing 3 times, pat dry, add 250 μL of blocking solution to each well, place in a 37°C wet box for sealing for 1 hour, shake off the blocking solution, wash 3 times, and pat dry. Dilute the antibody to 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, add 50 μL PBS to each well, and 50 μL to dilute a series of antibody concentrations, 37 Incubate in a wet box at ℃ for 30 min, shake off the liquid in the well, wash 3 times, pat dry, dilute the HRP-lab...

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Abstract

The invention discloses an antibody microarray kit for detecting the residue of aminoglycoside antibiotics in food. The antibody microarray kit comprises a microarray, an antibody, a secondary antibody marked by a Cy3, and an extraction reagent, wherein the antibody consists of a neomycin monoclonal antibody and a gentamycin monoclonal antibody, wherein the neomycin monoclonal antibody is secreted by a hybridoma cell EDC / 5G04 of which the preservation number is CCTCC NO:C201144; the gentamycin monoclonal antibody is secreted by a hybridoma cell EDC / 2D5 of which the preservation number is CCTCC NO:C201145, and the microarray fixes an amikacin coating antigen and a sisomicin coating antigen. The invention further discloses an antibody microarray method for detecting the residue of the aminoglycoside antibiotics in the food. The method can be used for detecting 5 aminoglycoside antibiotics at the same time, so that the method which is quick, is sensitive and is high in flux is provided for detecting the residue of the aminoglycoside antibiotics in the food, and the method has the characteristics of high accuracy, high precision, high efficiency and the like.

Description

technical field [0001] The invention belongs to the technical field of biochips, and in particular relates to an antibody chip kit and method for detecting residues of aminoglycoside antibiotics in food. Background technique [0002] Aminoglycoside antibiotics have a broad antibacterial spectrum, especially have a strong killing effect on Gram-negative bacteria, and are commonly used in the treatment of bacterial infections, bacterial enteritis and mastitis in veterinary clinics. In livestock breeding, it is also added to feed for prevention and growth promotion. Irrational drug use leads to the accumulation of drugs in edible tissues of animals. After human consumption, vestibular dysfunction, cochlear and auditory nerve damage, decreased renal function, and even respiratory arrest occur, directly endangering human health. Therefore, my country has stipulated the maximum residue limit (MRL) for aminoglycoside antibiotics. It is stipulated that the MRL of neomycin in milk a...

Claims

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Application Information

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Patent Type & Authority Patents(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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