Enzyme-linked immuno sorbent assay (ELISA) method for detecting sulfonamides and quinolones simultaneously and kit thereof

A technology for quinolones and drugs, which is applied in the field of ELISA and kits for simultaneous detection of sulfonamides and quinolones, and can solve problems such as inability to simultaneously detect sulfonamides and quinolones residues, cumbersome process, and greater sensitivity impact

Inactive Publication Date: 2013-02-13
北京维德维康生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Existing detection methods for sulfonamides and quinolones are mostly chromatographic methods. The sensitivity of these methods is greatly affected by steps such as sample purification and concentration. Moreover, these methods require complex instruments, and the process is cumbersome, and they are not suitable for a large number of samples on site. Screening, and the existing rapid detection products have certain limitations, unable to detect residues of sulfonamides and quinolones at the same time

Method used

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  • Enzyme-linked immuno sorbent assay (ELISA) method for detecting sulfonamides and quinolones simultaneously and kit thereof
  • Enzyme-linked immuno sorbent assay (ELISA) method for detecting sulfonamides and quinolones simultaneously and kit thereof
  • Enzyme-linked immuno sorbent assay (ELISA) method for detecting sulfonamides and quinolones simultaneously and kit thereof

Examples

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Effect test

Embodiment 1

[0044] Embodiment 1, prepare the enzyme-linked immunosorbent assay kit that simultaneously detects sulfonamides and quinolone drugs

[0045] The kit is composed as follows: ELISA plate (coated with the conjugate of sulfonamide drug and carrier protein and the conjugate of quinolone drug hapten and carrier protein), antibody working solution (containing monoclonal antibody of sulfonamide drug and monoclonal antibody of quinolone drug cloned antibody), enzyme-labeled anti-antibody working solution (containing horseradish peroxidase-labeled anti-sulfa drug monoclonal antibody anti-antibody and alkaline phosphatase-labeled anti-quinolone drug monoclonal antibody anti-antibody), concentrated washing solution , sample diluent, sample extract, standard solution 1-6 containing different gradient concentrations of sulfamethazine and norfloxacin, substrate chromogenic solution A, substrate chromogenic solution B, substrate chromogenic solution C. Stop Solution A and Stop Solution B.

...

Embodiment 2

[0109] The using method of embodiment 2, embodiment 1 kit

[0110] 1. Sample pretreatment

[0111] 1. Obtaining the sample solution for meat or egg testing: Accurately weigh 1±0.01g homogenized meat or egg sample into a 50mL centrifuge tube; add 20mL sample extract; at room temperature (23-27°C), high-speed Vortex for 1min; centrifuge at 4000g or more for 10min; take 50μL supernatant for analysis.

[0112] 2. Obtaining the milk test sample solution: Take a milk sample of more than 3000g and centrifuge it for 10 minutes, lightly suck the middle layer, dilute it 5 times with the sample diluent, and take 50 μL for analysis.

[0113] Two, use embodiment 1 kit to detect

[0114] 1. Take the microtiter plate (coated with the conjugate of sulfonamide drug and carrier protein and the conjugate of quinolone drug hapten and carrier protein) and insert it into the microtiter plate rack, and record the position of each standard and sample. Make 3 parallels for each sample, seal the unu...

Embodiment 3

[0137] The specificity, sensitivity and precision detection of embodiment 3, embodiment 1 kit

[0138] 1. Specific detection (cross-reactivity rate)

[0139] 1. Add 50 μL of the structural analogue standard solution of sulfamethazine to each hole in the enzyme label plate of the kit of Example 1 (the solvent is PBS buffer solution, and the concentration of the structural analogues of sulfamethazine is 0.5 μg / L, 1.5μg / L, 4.5μg / L, 13.5μg / L, 40.5μg / L, the wells that only added PBS buffer were used as control wells) or the structural analogue standard solution of norfloxacin (solvent For PBS buffer, the concentrations of the structural analogs of norfloxacin were 0.3 μg / L, 0.9 μg / L, 2.7 μg / L, 8.1 μg / L, and 24.3 μg / L, and the wells that were only added to PBS buffer As a control well), 3 replicate wells were set for each concentration. Structural analogs of sulfamethazine and quinolones are shown in Table 1.

[0140] 2, with step 3 to step 15 in embodiment 2 step 2, wherein, rec...

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Abstract

The invention discloses an enzyme-linked immuno sorbent assay (ELISA) method for detecting sulfonamides and quinolones simultaneously and a kit thereof. The kit uses conjugate of sulfonamide hapten and carrier proteins and conjugate of carbostyril hapten and carrier proteins as the coating antigen, uses the mixture of a sulfonamide specific antibody and a carbostyril specific antibody as the detection antibody and uses antiantibody of horse radish peroxidase marked sulfonamide-resisting specific antibody and antiantibody of alkaline phosphatase marked carbostyril-resisting specific antibody as the second enzyme-labeled antibody. By means of the ELISA method, the kit is used; in developing, the substrate of alkaline phosphatase is firstly used for developing, and then the substrate of horse radish peroxidase is used for developing; and the sulfonamides and the quinolones can be detected simultaneously in one step. The ELISA method and the kit thereof are used for self quality control of food production enterprises, food security monitoring and regular screening of detection mechanisms and can be used as supplementary means for animal pharmacology and toxicology study in scientific research.

Description

technical field [0001] The invention relates to an ELISA method and a kit for simultaneously detecting sulfonamides and quinolones. Background technique [0002] Sulfonamides, the English name is Sulfonamides, are artificially synthesized sulfonamide derivatives, mainly used for the prevention and treatment of bacterial infectious diseases. Representative drugs are: sulfamethazine, sulfadiazine, sulfamethoxazole, sulfamethoxine, sulfaquinoxaline, etc. Sulfonamide drugs are generally white or light yellow crystalline powder, which is easy to deteriorate when exposed to light, and the color gradually becomes darker. Most of these drugs are not easy to dissolve in water, but are easily soluble in dilute alkali. After forming sodium salt, it is easily soluble in water, and its aqueous solution is strongly alkaline. Sulfonamide drugs can inhibit Gram-positive bacteria and some negative bacteria, and can treat a variety of bacterial infections. They are widely used in veterinary...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/535G01N21/31
Inventor 吴小平王世恩杨铮何丹婷苏丽芳李淑芳
Owner 北京维德维康生物技术有限公司
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