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Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting cefalexin, cefadroxil and cefradine

An enzyme-linked immunosorbent reagent, cefadroxil technology, applied in the field of drug residue analysis and immunology, can solve the problems of lack of specificity of methods, differences in sensitivity of antimicrobial drugs, expensive instruments, etc., and achieve simple sample processing methods and harm to health. Small, time and cost saving effect

Active Publication Date: 2015-04-29
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among these residue analysis methods, the instrumental method is not conducive to the screening and on-site detection of large batches of samples due to its expensive equipment, complicated operation, and high detection cost.
Although the microbial method shows good performance in high-throughput screening of residues, the method lacks specificity and cannot confirm the type of residues, and the bacteria used have different sensitivities to different types of antibacterial drugs, which easily lead to false negatives and false positives The production of results

Method used

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  • Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting cefalexin, cefadroxil and cefradine
  • Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting cefalexin, cefadroxil and cefradine
  • Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting cefalexin, cefadroxil and cefradine

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] The preparation of embodiment 1 immunogen and coating former

[0031] 1.1 Preparation of immunogen

[0032] 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 ℃.

[0033] 1.2 Preparation of coating agent

[0034] Dissolve 120 mg of OVA in 8 mL of phosphat...

Embodiment 2

[0035] The preparation of embodiment 2 monoclonal antibody

[0036] Preparation of hybridoma cells: referring to "Animal Immunology" by Yang Hanchun, Balb / C mice (purchased from the Experimental Animal Center of Hubei Academy of Medical Sciences) were immunized with the immunogen cephalexin-bovine serum albumin conjugate prepared in Example 1. The immunization procedure is as follows: for basic immunization, a protein emulsion containing 100 μg of immunogen emulsified with an equal volume of Freund’s complete adjuvant is injected subcutaneously at the back of the neck of the mouse; Protein emulsion of immunogen for booster immunization. From the third immunization, the tail blood was collected on the 8th day after each immunization, the serum was separated, and the serum antibody titer was detected by indirect ELISA method. Immunization qualified mice (high titer, good sensitivity) stop immunization in preparation for fusion. At the time of fusion, take a Balb / C mouse that h...

Embodiment 3

[0041] The establishment of embodiment 3 indirect competition ELISA detection method

[0042] 3.1 Preparation of reagents (the reagents used in this example were prepared by the following methods unless otherwise specified)

[0043] Phosphate buffer: NaCl 8.0g, KH 2 PO 4 0.2g, Na 2 HPO 4 12H 2 O 2.9g, KCl 0.2g, add double distilled water to 1000mL, adjust pH to 7.4;

[0044] Coating solution: Take Na 2 CO 3 1.59g, NaHCO 3 2.93g, add triple distilled water to 1000mL, adjust the pH value to 9.6;

[0045] Washing liquid: NaCl 8.0g, KH 2 PO 4 0.2g, Na 2 HPO 4 12H 2 O 2.9g, KCl 0.2g, Tween 20 0.5mL, add double distilled water to 1000mL, adjust pH to 7.4;

[0046] Blocking solution: Ovalbumin 1g dissolved in 100mL phosphate buffer;

[0047] Substrate solution A: 3,3',5',5-tetramethylbenzidinediamine (TMB) 200mg, absolute ethanol 100mL, add double distilled water to 1000mL;

[0048] Substrate B: Na 2 HPO 4 14.6g, citric acid 9.3g, 0.75% urea hydrogen peroxide 6...

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Abstract

The invention discloses a specific monoclonal antibody capable of distinguishing cefalexin, cefadroxil and cefradine, and an enzyme-linked immunosorbent assay method and kit for detecting the cefalexin, the cefadroxil and the cefradine. According to the invention, the monoclonal antibody is secreted by a hybridoma cell strain 3A6 of which the preservation number is CCTCC No.C201340. Compared with the prior art, the monoclonal antibody, prepared by the invention, can be used for distinguishing the cefalexin, the cefadroxil and the cefradine at the same time. The enzyme-linked immunosorbent assay method and kit disclosed by the invention have the advantages of high detection efficiency, high sensitivity, high precision, high accuracy and the like.

Description

technical field [0001] The invention belongs to the technical field of drug residue analysis and immunology, and in particular relates to a monoclonal antibody against cephalexin, cefadroxil and cephradine, and the invention also relates to an enzyme-linked immunosorbent method for detecting cephalexin, cefadroxil and cephradine with kit. Background technique [0002] Cefalexin, cefadroxil and cephradine belong to the cephalosporin class of veterinary drugs, which have the basic structure of β-lactam and have a wide antibacterial spectrum. They are often used to prevent and treat bacterial infections, and have been widely used in veterinary clinics and breeding industries. People abuse drugs in pursuit of economic benefits, resulting in a large amount of drug residues in animal food. After humans eat food containing drugs, it will cause a series of damage to the body, such as allergic reactions, shock and even death in severe cases. After the drugs enter the gastrointestina...

Claims

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Application Information

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IPC IPC(8): C07K16/44C12N5/20G01N33/577C12R1/91
Inventor 袁宗辉彭大鹏朱永利戴梦红王玉莲潘源虎刘振利
Owner HUAZHONG AGRI UNIV
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