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Time-resolved fluoroimmunoassay kit for detecting olaquindox and application thereof

A time-resolved fluorescence and immunoassay technology, used in analytical materials, biological tests, immunoglobulins, etc. Simple process and high detection accuracy

Pending Publication Date: 2020-04-24
杭州佰昕科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the instrument method mainly includes spectroscopy, chromatography and liquid mass spectrometry, etc. The instrument analysis has high accuracy and precision, but the sample pretreatment process is complex and tedious, time-consuming, requires professional and technical personnel to operate, and the price of instrument reagents, etc. Expensive and cannot be greatly promoted at the grassroots level

Method used

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  • Time-resolved fluoroimmunoassay kit for detecting olaquindox and application thereof
  • Time-resolved fluoroimmunoassay kit for detecting olaquindox and application thereof
  • Time-resolved fluoroimmunoassay kit for detecting olaquindox and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Synthesis of artificial antigen of olaquindox

[0061] (1) Synthesis of olaquindox hapten

[0062] (1.1) Dissolve 1mmol olaquindox in 2-5mL THF, add 1.1-1.2mmol NaH under ice bath (0°C), stir for 1 hour, then add 1mmol ethyl 5-bromo-2,4-dienevalerate (A), reflux reaction at 65°C for 6 hours, after the reaction is complete, add 30mL H 2 O, after extraction with ethyl acetate, combined organic phase, washed with saturated brine, rotary evaporation to remove solvent, separated by column chromatography to obtain OLA-A 1 , A 1 for -CH=CH-CH=CH-COOCH 2 CH 3 ; The eluent is: petroleum ether: ethyl acetate=1:1;

[0063] (1.2) Add 1mmol OLA-A 1 Dissolve in 10mL of methanol and water mixture (the volume of methanol and water are both 5mL), add 1.5mmol of lithium hydroxide, react at room temperature for 2h; after the reaction is complete, use 1mol / L hydrochloric acid solution to adjust the pH to 5-6 , extracted twice with 50 ml of ethyl acetate, the organic phase was washe...

Embodiment 2

[0087] Determination of antiserum titer:

[0088] The artificial antigens prepared in Example 1 and Comparative Example 1 were used to immunize BALB / C mice respectively. The artificial antigens were emulsified with complete Freund's adjuvant for the initial immunization. Booster immunization every day, a total of 3 booster immunizations, emulsified with incomplete adjuvant for booster immunization, immunological dose is 150 μg / mouse, after 14 days (days) after booster immunization, blood was collected from the tail of the mouse to measure the titer of multiple antiserum. The titer of antiserum was determined by ELISA method after doubling dilution with blocking solution, the mouse serum before immunization was used as negative control, and the OD of positive serum was used as 450nm Value and Negative Serum OD 450nm The dilution where the value ratio is greater than 2.1 is the antiserum titer, and the results are shown in Table 1. Finally, final immunization was carried out, ...

Embodiment 3

[0099] The preparation of embodiment 3 olaquindox monoclonal antibody

[0100] (1) Immunization of mice:

[0101] Select BALB / C female mice aged 6-8 weeks and weighing 18-20 g. Mix and emulsify the prepared immunogen (OLA-A-BSA) with an equal volume of complete Freund's adjuvant with a syringe, then inject at multiple points in the abdomen and underarms, with a dose of 100-200 μg per mouse, and then every 21 days Booster immunization was carried out, and blood was collected after 3 times of booster immunization to determine the titer. The titer was determined by indirect ELISA method. After the serum was diluted with blocking solution, the antiserum titer was determined by ELISA method. The mouse serum before immunization was used as a negative control. , with positive serum OD 450nm The dilution where the ratio of the value to the negative serum is greater than 2.1 is the antiserum titer. When the titer was no longer significantly increased, the dose was doubled for final ...

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Abstract

The invention discloses a time-resolved fluoroimmunoassay kit for detecting olaquindox and application thereof. The time-resolved fluoroimmunoassay kit has high precision and accuracy and has high specificity to olaquindox; the kit can qualitatively and quantitatively detect olaquindox in water, feed and fish tissues; and the sample pretreatment process is simple, convenient and fast, and the detection accuracy is high.

Description

technical field [0001] The invention belongs to the technical field of time-resolved fluorescent immunoassay in biotechnology, and in particular relates to a time-resolved fluorescent immunoassay kit for detecting olaquindox and its application. Background technique [0002] Olaquindox (OLA) is an antibacterial and growth-promoting agent, which was widely used in aquaculture and was once called "aquatic lean meat extract". The toxicity and side effects of olaquindox should not be underestimated, and there are obvious genotoxicity and cumulative toxicity. Therefore, strict usage regulations and residue limit standards have been formulated successively at home and abroad. For example, the United States and the European Union prohibit the use of olaquindox, and Japan stipulates that the maximum residue limit (MRL) of olaquindox in animal tissues and viscera is 300 μg kg -1 In 2001, the Ministry of Agriculture of my country issued Announcement No. 168 "Code for the Use of Feed ...

Claims

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Application Information

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IPC IPC(8): G01N33/58G01N33/577C07K16/44G01N33/533
CPCG01N33/9446G01N33/94G01N33/582G01N33/577C07K16/44G01N33/533Y02A50/30
Inventor 王赛赛金仁耀翟璐郭建军
Owner 杭州佰昕科技有限公司
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