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ELISA detection method for doxycycline remnant, kit and application

A technology of doxycycline and detection method, applied in the field of immunochemical analysis, can solve the problems of high detection cost, inability to operate on site, false positives, etc.

Inactive Publication Date: 2010-02-03
HUAZHONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, microbial methods have long detection time and lack of specificity, which easily lead to false positives and false negatives.
Instrument testing mainly has defects such as expensive instrument purchase, complicated sample pretreatment, cumbersome and time-consuming procedures, high testing costs, and inability to operate on-site, so its application in production is limited
There is no report on doxycycline ELISA detection method and kit

Method used

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  • ELISA detection method for doxycycline remnant, kit and application
  • ELISA detection method for doxycycline remnant, kit and application
  • ELISA detection method for doxycycline remnant, kit and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Antigen Preparation

[0033] 1.1 Synthesis of immunogen

[0034] Immunogen (DOX-PABA-BSA) of the present invention is synthesized according to the attached figure 1 The technical synthetic route shown. The specific method is: take 27.4 mg (0.2 mmol) of p-aminobenzoic acid and dissolve it in 4 mL of 1mol / L HCl. In an ice-water bath, add the freshly prepared 1mol / L NaNO dropwise while stirring 2 0.4 mL, react at 4°C in the dark for 10 minutes, and stir while reacting; take a small amount to react with aniline, if the reaction system turns dark yellow, it indicates that the diazotization reaction is successful, and the solution A is obtained and set aside. Weigh 0.088g (0.2mmol) of doxycycline, dissolve it in 3mL triple-distilled water, add liquid A dropwise into the doxycycline solution, adjust the pH to 8-9, and react with magnetic stirring for 2h, centrifuge to take the supernatant, Add 6.8 ul of tributylamine to the reaction solution, mix and then add 3.8...

Embodiment 2

[0037] Example 2 Preparation of rabbit anti-polyclonal antibody

[0038] 2.1 Animal immunity

[0039]New Zealand white rabbits were immunized with the immunogen (DOX-PABA-BSA). During basic immunization, dissolve and dilute the immunogen with sterilized PBS, add an equal volume of Freund's complete adjuvant to fully emulsify, and inject subcutaneously at multiple points on the back of the neck. From the third booster immunization, on the 8th day after immunization, ear vein blood was collected to determine the antibody titer and specificity. After a certain titer was reached, whole blood was collected from the heart, and the antiserum was separated (first at room temperature for 1 hour, then placed in a refrigerator at 4°C for several hours, then refrigerated and centrifuged at 4000r / min for 10 minutes). The purified anti-DOX polyclonal antibody was obtained by fractional precipitation with saturated ammonium sulfate.

[0040] 2.2 Purification of DOXpAb

[0041] According ...

Embodiment 3

[0042] Embodiment 3 indirect ELISA method establishment

[0043] 3.1 Coating concentration and antibody titer test

[0044] The coating concentration and the corresponding antibody titer were preliminarily obtained by Founder titration as the working concentration for establishing the method. According to the ELISA method, the optimal coating concentration of the original coating is 0.8ug / ml, and the antibody titer is 1:32000.

[0045] 3.2 Establishment of standard curve and minimum detection line

[0046] Accurately measure an appropriate amount of DOX standard stock solution, release DOX into a series of concentrations of 0, 2.5, 5, 10, 20, 40, and 80 μg / L with PBS, repeat 5 wells for each experiment, and use indirect competition ELISA to determine the different pairs of DOX mAbs. Inhibition absorbance value of concentration DOX standard substance, repeated determination 3 times. The average absorbance value of the standard obtained is divided by B 0 Standard absorbance ...

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Abstract

The invention belongs to the technical field of immunological analysis, in particular to an ELISA method for detecting doxycycline (DOX) remnant and a kit. The method mainly comprises the preparation of immunogen, coatingen and antibody, the pretreatment of the sample and the establishment of the ELISA detection method. The kit of the invention mainly comprises anti DOX specific antibody, a DOX standard substance, and an elisa plate coated with DOX-OVA conjugate. The kit and the method of the invention are characterized by simplicity, convenience, high speed, sensitivity, accuracy and the like and are suitable for detecting DOX remnant in animal edibility tissue.

Description

technical field [0001] The invention belongs to the technical field of immunochemical analysis. It specifically relates to a doxycycline residue enzyme-linked immunosorbent immunoassay (ELISA) detection method, kit and application, and is suitable for rapid detection of doxycycline drug residues in edible animal tissues (muscle, liver, etc.). Background technique [0002] Doxycycline is a broad-spectrum antibiotic that can inhibit or kill many kinds of Gram-positive and Gram-negative bacteria, such as: Streptococcus, Bacillus anthracis, certain Staphylococcus, Bacillus tetani, Pasteurella, Salmonella, Brucella, Klebsiella and Haemophilus etc. In addition, doxycycline also has a certain inhibitory effect on some chlamydia, mycoplasma, rickettsia, spirochetes, etc. But doxycycline also has many side effects, such as: induced hypoglycemia, intracranial hypertension, photosensitivity, and liver damage. Doxycycline residues in animal tissues will threaten food safety and cause...

Claims

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Application Information

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IPC IPC(8): G01N33/53G01N33/543C07K14/765C07K1/113C07K16/44
Inventor 毕丁仁乐涛王喜亮石徳时郭延成巴巴卡肖运才沈亚安李自力
Owner HUAZHONG AGRICULTURAL UNIVERSITY