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An indirect competition ELISA kit for detecting cephalosporin antibiotics in food of animal origin and its application

A technology for the detection of animals and cephalosporins, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of lack of in-depth research on antigens and antibodies of the matrix, and the detection method is difficult to meet the detection requirements, etc. short, reliable effect

Active Publication Date: 2019-05-21
HEBEI AGRICULTURAL UNIV.
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Looking at the above research status, it can be seen that the rapid detection methods for cephalosporin antibiotics at home and abroad are only for a single drug, but there are many types of cephalosporin antibiotics, and the national standard limits are only for the limit requirements of one class of substances, which is As a result, it is difficult for single-type drug detection methods to meet the detection requirements. Therefore, the development of universal antibodies and the establishment of a high-throughput multi-residue detection system can improve the timeliness of detection
Moreover, in the practical application of these analytical methods, the existing research still lacks in-depth research on the influence of the matrix on the antigen-antibody response.

Method used

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  • An indirect competition ELISA kit for detecting cephalosporin antibiotics in food of animal origin and its application
  • An indirect competition ELISA kit for detecting cephalosporin antibiotics in food of animal origin and its application
  • An indirect competition ELISA kit for detecting cephalosporin antibiotics in food of animal origin and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0048] Synthesis and identification of embodiment 1 cephalosporin antibiotic artificial antigen

[0049] 1.1 Synthesis of Artificial Antigens of Cephalosporin Antibiotics

[0050] Select cephalexin and use the active ester method to synthesize the immunogen CEX-BSA (cephalexin-bovine serum albumin conjugate) and the original coating CEX-OVA (cephalexin-ovalbumin conjugate). The specific steps are:

[0051] (1) Accurately weigh 20.3mg of CEX (cephalexin), 17.7mg of NHS (N-hydroxysuccinimide) and 23.7mg of DCC (N,N'-dicyclohexylcarbodiimide), dissolve in 600μL of DMF (di Methylformamide), mixed well to obtain a reaction mixture, and incubated at room temperature for 3 h;

[0052] (2) Put the ice cubes or ice-water mixture prepared in advance on the horizontal vibrator, and dissolve the freshly prepared BSA solution (10mg BSA in 2mL 0.1mol / L NaHCO 3 solution) placed in ice cubes, take 360 ​​μL of the above reaction mixture, slowly add it dropwise to BSA (bovine serum albumin) s...

Embodiment 2

[0061] Preparation and identification of embodiment 2 cephalosporin antibiotic universal antibody

[0062] 1.1 Preparation of universal antibody against cephalosporin antibiotics

[0063]The conjugate CEX-BSA successfully synthesized in Example 1 was selected as the immunogen, and it was injected subcutaneously at six points on the back and two points in the thigh muscle to immunize New Zealand big ear white. For the first rabbit, dissolve CEX-BSA with sterile PBS, mix it with the same amount of FCA, and fully emulsify it; for booster immunization, dissolve CEX-BSA with sterile PBS, mix it with the same amount of FIA, and fully emulsify it, carry out 4 weeks after the first rabbit, after 6 booster immunizations, with an interval of 2 weeks between each time, 10 days after the third immunization, blood was collected through the ear vein, and after each immunization, blood was collected from the ear vein, and the antiserum obtained after immunization in different periods The po...

Embodiment 3c

[0106] Embodiment 3ci-ELISA reaction system condition optimization

[0107] 1. Test method

[0108] 1.1 Basic steps of ci-ELISA method

[0109] The buffer solution used in this example is the same as Example 2.

[0110] (i) Coating: Dilute the coating buffer (CEX-OVA, synthesized in Example 1) to 10 μg / mL in the coating buffer, and coat the 96-well ELISA plate with 100 μL per hole, 4 ℃ coated overnight;

[0111] (ii) Washing: dry the solution in the microplate, wash the plate 3 times with washing buffer, each time for 3 minutes, and pat dry for later use;

[0112] (iii) Blocking: block the microtiter plate with blocking solution, 200 μL per well, incubate in a 37°C incubator for 1 hour, and the washing steps are the same as (ii);

[0113] (iv) Adding samples: add 100 μL 1×PBS to each well in the first row as a blank control, add 50 μL 1×PBS in the second row as a negative control; dilute the CEX standard with 1×PBS to different concentrations (100 μg / mL , 1 μg / mL, 10ng / mL...

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Abstract

The invention discloses an indirect-competitive ELISA (Enzyme-linked Immunosorbent Assay) kit for detecting cephalosporin antibiotics in animal derived foods and application of the kit. The kit comprises an ELISA plate coated with a coating antigen, a cephalosporin antibiotic standard substance, a cephalosporin antibiotic general antibody, an enzyme labeled secondary antibody, a dilution buffer, a washing buffer, a substrate developing solution and a stop solution, wherein the cephalosporin antibiotic general antibody is capable of specifically identifying cefalexin, cefradine, cefadroxil, cefoperazone, cefazolin or cefotaxime and cannot identify penicillin sodium. According to the cephalosporin antibiotic general antibody prepared in the invention, the general antibody and most of cephalosporin antibiotics have high cross reaction rates, while hardly have any cross reaction for the analogue penicillin sodium containing a beta-lactam ring; and therefore, the general antibody has high specificity. The kit prepared by the general antibody has the advantages of high sensitivity, capability of detecting many types of drugs, low cost, simple operation and short detection time.

Description

technical field [0001] The invention relates to the technical field of rapid detection of food safety, in particular to an indirect competition ELISA kit for detecting cephalosporin antibiotics in animal-derived food and its application. Background technique [0002] my country is a big country in the production and use of antibiotics, and the production and use of cephalosporin antibiotics have an obvious growth trend. While bringing health to human beings, antibiotics left in food have also brought potential safety hazards. In order to reduce the degree of harm, countries have carried out research and detection methods. Since there are dozens of cephalosporin antibiotics, the detection of a single substance cannot meet the detection needs of this type of antibiotics. Therefore, the development of high-throughput and multi-residue rapid detection technologies and methods is a necessary means for food safety supervision. [0003] At present, domestic and foreign detection ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/558G01N33/535
CPCG01N33/535G01N33/558
Inventor 王向红桑亚新刘卫华张雪娇张晶
Owner HEBEI AGRICULTURAL UNIV.
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