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Oxoquinoxaline-2-carboxylic acid remained enzyme linked immunity detecting method and reagent kits

A quinoxaline and kit technology, applied in the field of enzyme-linked immunosorbent methods and kits, to achieve high sensitivity, high specificity, and convenient use

Inactive Publication Date: 2007-05-30
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no reports on the ELISA detection method and kit of carbadox residue marker quinoxaline-2-carboxylic acid (QCA) at home and abroad

Method used

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  • Oxoquinoxaline-2-carboxylic acid remained enzyme linked immunity detecting method and reagent kits

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Effect test

Embodiment 1

[0034] Embodiment 1, antigen preparation

[0035] 1.1 Synthesis of immunogen

[0036] The pharmaceutical transformation and immunogen preparation of the present invention follow the technical route shown in Figure 1. The specific steps are: add 0.0348g of acid chloride QCA to 1.0mL N,N-dimethylformamide (DMF), add γ-aminobutyric acid solution dropwise under stirring, and stir for 3h. Centrifuge to remove the precipitate, this is liquid A. Dissolve 340mg of bovine serum albumin (BSA) in 5mL of physiological saline, which is liquid B. Add liquid A dropwise to liquid B, then add 0.023g of N-hydroxysuccinimide (NHS), 0.0434g of N,N-dicyclohexylcarbodiimide (DCC), react overnight at 4°C, centrifuge to remove the precipitate, and take The supernatant was then dialyzed with normal saline for 6 days, and the dialysate was changed every 12 hours. The resulting product was freeze-dried and stored in a -20°C refrigerator for immunization. Its UV spectrum is shown in Figure 2.

[003...

Embodiment 2

[0039] Embodiment 2, the preparation of antibody

[0040] 2.1 Immunization of animals

[0041] New Zealand white rabbits were used as immunized animals, and QCA was reacted with γ-aminobutyric acid, and the reaction product was coupled with ovalbumin to form an immunogen. Freund's complete adjuvant was mixed to make an emulsifier, and it was injected subcutaneously at multiple points on the back of the neck. The same dose of immunogen plus an equal amount of Freund's incomplete adjuvant was mixed and emulsified at an interval of 2 to 4 weeks, and the booster immunization was performed once, and a total of 6 immunizations were performed. To monitor serum antibody titer and specificity, no adjuvant was added for the last time. Blood was collected 7-10 days after the last immunization, blood was exsanguinated from the carotid artery, and the purified QCA polyclonal antibody was obtained by fractional precipitation with ammonium sulfate.

[0042] 2.2 Antibody purification

[00...

Embodiment 3

[0052] Embodiment 3, establishment of sample pretreatment method

[0053] (1) Take 2 g of porcine muscle or liver tissue homogenate, add 5 mL of 5% metaphosphoric acid and 10% methanol solution, shake for 2 min, centrifuge at 4800 r / min for 10 min, and take the supernatant; repeat the extraction once again according to the above steps, and combine two extracts;

[0054] (2) Add 6 mL of ethyl acetate to the extract of step (1), shake for 2 minutes, centrifuge at 4800 r / min for 10 minutes, take the upper layer of ethyl acetate layer; repeat the extraction once for the lower layer of liquid according to the above steps, and combine the ethyl acetate layer ;

[0055] (3) Add 5mL of 0.5mol / L phosphate buffer (pH7.0) to the ethyl acetate layer of step (2) for back extraction, shake for 1min, let stand for 10min, take the lower aqueous phase; The above steps were repeated for extraction once, and the aqueous phase was combined;

[0056] (4) Activate the MAX column with 3mL of meth...

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Abstract

The invention belongs to fields of immunochemistry analysis. It discloses an enzyme-uniting immune method and reagent box used in detection of quinoxaline-2-carboxyl acid residues, the method mainly contain medicine reconstruction, the preparation of immunogen, peridium antigen and antibody and pretreatment of sample and building of ELISA detecting method. The reagent box comprises quinoxaline-2-carboxyl acid (QCA) specificity antibody, QCA standard substance, enzyme target-object peridiumed with coupling substance of egg albumin and reaction production of QCA and gamma-aminobutyric acid. The sample is hydrolysed by metaphosphoric acid to discharge QCA, and cleaned by MAX column, derivatized by butyl amine, indirect competition ELISA is adopted in detection. The method and reagent box in the invention has advantages of simpleness, speediness, sensitivity and accuracy, large-lot samples can be detected fast and simultaneously, lowest detecting limit is 0.6 mug / kg.

Description

technical field [0001] The invention belongs to the technical field of food immunochemical analysis. It specifically relates to an enzyme-linked immunosorbent method and kit for the detection of quinoxaline-2-carboxylic acid (QCA) residues, which is suitable for the determination of quinoxaline, a marker for carbadoxy residues, in edible tissues of food animals such as muscle and liver - Residual amount of 2-carboxylic acid (QCA). Background technique [0002] Carbadox is a quinoxaline drug, which is a synthetic broad-spectrum antibacterial drug, and has been widely used as a growth-promoting agent in livestock and poultry breeding. Toxicology studies have found that carbadox is carcinogenic and mutagenic. In order to protect the health of consumers and expand the trade of animal foods, it is imperative to strengthen the detection of carbadox residues in animal foods. [0003] Carbadox is metabolized into quinoxaline-2-carboxylic acid (QCA) in animals. QCA combined with t...

Claims

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Application Information

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IPC IPC(8): G01N33/544G01N21/78
Inventor 袁宗辉张泽英彭大鹏王玉莲谢长清陶燕飞陈冬梅黄玲利戴梦红刘振利
Owner HUAZHONG AGRI UNIV
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