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Chemiluminescence detection kit for furaltadone metabolite and applications of the kit

A technology of chemiluminescence detection and furatanone, applied in the field of immunological detection, can solve the problems of cumbersome process, slow detection speed, easy occurrence of false positive results, etc., and achieve the effect of high sensitivity

Inactive Publication Date: 2014-04-02
BEIJING KWINBON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Microbiological method is a classic method for the determination of antibiotic residues. It has the advantages of simple pretreatment, economy, and large batches, but its accuracy, accuracy, and specificity are not high.
Chromatography is widely used, with high accuracy and sensitivity, but it is difficult to achieve on-site detection and popularization due to reasons such as cumbersome procedures, expensive equipment, and slow detection speed.
Enzyme-linked immunoassay (ELISA) has many advantages such as simplicity, rapidity, large sample size, high sensitivity, and strong specificity. It is widely used in residue detection, but it is prone to various problems in practice, such as adding samples Whether the time is consistent, whether the washing plate is thorough, whether the sample volume is consistent, etc., resulting in the disadvantages of false positive results

Method used

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  • Chemiluminescence detection kit for furaltadone metabolite and applications of the kit
  • Chemiluminescence detection kit for furaltadone metabolite and applications of the kit
  • Chemiluminescence detection kit for furaltadone metabolite and applications of the kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1: the preparation of hapten, antigen and monoclonal antibody

[0045] (1) AMOZ hapten synthesis

[0046] Dissolve 0.5 g of AMOZ, 0.50 g of phthalic anhydride and 2 mL of pyridine in 20 mL of DMSO, and stir and react at 60°C for 4 hours. After the reaction is completed, heat to remove the solvent, and purify by column chromatography to obtain phthalic acid Mono-AMOZ ester.

[0047] (2) Synthesis of immunogen (AMOZ-BSA)

[0048] A, take 6mg hapten and dissolve it in 1mL DMF;

[0049] B. Dissolve 30mg of EDC and NHS in 0.2mL of water, add to the hapten solution, and stir at room temperature for 24 hours to obtain reaction solution A;

[0050] C. Weigh 50 mg of BSA, fully dissolve it in 3.8 mL of PBS (pH 7.2), slowly add the reaction solution A to the protein solution drop by drop, and stir at room temperature for 24 h;

[0051] D. Dialyze with 0.01mol / L PBS at 4°C for 3 days and change the dialysate 3 times a day to remove unreacted small molecules;

[005...

Embodiment 2

[0059] Example 2: Preparation of enzyme-labeled antibody

[0060] A, Weigh 2 mg HRP and dissolve in 0.5 mL double distilled water; add 0.5 mL freshly prepared 0.06 mol / L NaIO 4 solution, at 4°C in the dark for 30 min;

[0061] B, add 0.5 mL of 160 mmol / L ethylene glycol, and react at room temperature for 30 min;

[0062] C. Add 2 mg of AMOZ monoclonal antibody, mix well, put it into a treated dialysis bag, dialyze in 1000 mL of 0.05 mmol / L sodium carbonate buffer, and overnight at 4°C;

[0063] D. Aspirate the dialysate into a 10 mL centrifuge tube, add 0.25 mL of newly prepared 5g / L NaBH 4 After mixing, place at 4°C for 2 h; add an equal volume of saturated ammonium sulfate solution, act at 4°C for 30 min, centrifuge at 3000 rpm for 25 min at 4°C, and discard the supernatant;

[0064] E, Dissolve the precipitate in 1.5mL 0.02 mol / L pH 7.4 PBS, inhale into the dialysis bag, dialyze in 0.02mol / L pH 7.4 PBS, overnight at 4°C (replace PBS 3 times in the middle);

[0065] F, s...

Embodiment 3

[0066] Embodiment 3: the establishment of CLEIA detection method

[0067] (1) Optimal concentration of antibody and coated antigen (square array method)

[0068] Coat the chemiluminescence plate longitudinally with serial dilutions of each coating antigen (AMOZ-OVA) at 80.0, 40.0, 20.0, 10.0, 5.0, 2.5, 1.25, 0.625 μg / mL, 100 μL / well, and place at a constant temperature of 37 °C After 2 hours in the oven, pat dry; block with 150 μL / well blocking solution, place in a 37°C incubator for 2 hours, wash the plate once, and pat dry; add 50 μL / well of a series of diluted enzyme-labeled AMOZ monoclonal antibodies (1:1000 to 1 :512000), incubate at room temperature (20-25°C) for 15 min, wash the plate five times, and pat dry the last time; add 50 μL / well of chemiluminescence A and B solutions respectively, and measure the luminescence intensity. Specificity determination was carried out with the coating antigen concentration and antibody dilution having obvious gradient changes in the ...

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Abstract

The invention discloses a chemiluminescence detection kit (CLEIA) for a furaltadone metabolite (AMOZ). The kit comprises a kit body, a chemiluminescence plate disposed in the kit body and reagents disposed in the kit body. Apertures of the chemiluminescence plate are coated with an AMOZ coupling antigen. The reagents comprise enzyme labeled antibody concentrate, enzyme labeled antibody diluent, AMOZ series standard substance solution, chemiluminescence A liquid, chemiluminescence B liquid, concentrated washing liquid, concentrated redissolution liquid, and a derivatization agent. The detection kit has characteristics of high sensitivity, simpleness, rapidness and high accuracy. Compared with a traditional ELISA method, the operation time of the detection kit provide by the invention is largely reduced. The detection kit can be used for tissue (muscle and liver) detection of livestock and poultry, and used for AMOZ residue detection in aquatic products.

Description

technical field [0001] The invention relates to a chemiluminescent immunoassay kit for detecting furaltadone metabolites (AMOZ), which is used for detecting the AMOZ content or residue in livestock and poultry tissues (muscle, liver) and aquatic products. It belongs to the field of immunological detection. Background technique [0002] Furaltadone belongs to nitrofuran antibacterial drugs, and it is mainly used clinically for the treatment of various intestinal infectious diseases such as pullorum, chicken colibacillosis, chicken coccidiosis and as a growth-promoting agent for pigs, poultry and aquatic products. additive. However, relevant studies have proved that furaltadone and its metabolites have considerable toxicity and side effects, can induce genetic mutations and teratogenicity in organisms, and can induce cancer. [0003] The European Union promulgated Regulation 2377 / 90 / EEC as early as 1990, listing nitrofuran drugs and their metabolites as Class A banned drugs,...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N21/76
CPCG01N21/76G01N33/53G01N33/535G01N33/577
Inventor 何方洋冯才伟冯月君杨秀贤崔海峰冯静白莉李海静
Owner BEIJING KWINBON BIOTECH
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