Fluorescent microsphere immunochromatographic test strip for detecting olaquindox residues and application thereof

A technology of immunochromatography test strips and fluorescent microspheres, which is applied in the direction of fluorescence/phosphorescence, material analysis through optical means, and measuring devices, can solve problems such as false positives, false negatives, and unsatisfactory sensitivity, and achieve high precision and Good sensitivity, dispersibility and stability, and the effect of improving analytical sensitivity

Pending Publication Date: 2020-03-27
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

Currently commonly used immunoassay methods mainly include enzyme-linked immunoassay, colloidal gold immunochromatography, fluorescent immunoassay, etc., but there are problems such as unsatisfactory sensitivity, false positives and false negatives.

Method used

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  • Fluorescent microsphere immunochromatographic test strip for detecting olaquindox residues and application thereof
  • Fluorescent microsphere immunochromatographic test strip for detecting olaquindox residues and application thereof
  • Fluorescent microsphere immunochromatographic test strip for detecting olaquindox residues and application thereof

Examples

Experimental program
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Effect test

preparation example Construction

[0055] The preparation method of the time-resolved fluorescent microsphere immunochromatographic test strip for detecting residual olaquindox mainly comprises the following steps:

[0056] 1) Preparation of the sample binding pad 1: use the time-resolved fluorescent microspheres produced by Bangs Laboratories to label the anti-oquinethanol monoclonal antibody, and dilute it with a buffer system, soak the sample binding pad 1 in the dilution buffer, and Prepared after vacuum freeze-drying;

[0057] The fluorescent microsphere is a microsphere with a diameter of 100-300nm, which is coated with a polystyrene-coated fluorescent substance, and -COOH groups are connected on its surface, and the fluorescent substance is a europium complex.

[0058] 2) Preparation of nitrocellulose membrane 2: Spray the olaquindox hapten-carrier protein conjugate on the detection area range on the nitrocellulose membrane 2 to make detection area 4; spray goat anti-mouse secondary antibody onto the nit...

Embodiment 1

[0061] Synthesis of artificial antigen of olaquindox

[0062] 1) Synthesis of olaquindox hapten

[0063] Accurately add 2.106g of olaquindox and 2.274g of oxocane-2,8-dione into a three-neck round-bottomed flask, add 85mL of pyridine, reflux at 115°C for 6h, then distill off pyridine under reduced pressure, add 60mL of ice-distilled water to the remaining mixture, 2mol L -1 Adjust the pH to 2.0-3.0 with HCl, and place it overnight at 4°C. Suction filtration under reduced pressure and washing with ice distilled water and drying, the obtained substance is the olaquindox hapten OLA-A, -A represents -CO(CH 2 ) 5 COOH; specific synthetic routes such as image 3 shown.

[0064] 2) Preparation of olaquindox-coated antigen and immunogen

[0065] Dissolve 0.04mmol OLA-A in 0.8mL N,N-dimethylformamide (DMF), add 0.04mmol N-hydroxysuccinimide (NHS) and 0.04mmol dicyclohexylcarbodiimide (DCC) , Stir at room temperature in the dark for 12 hours and then 2000r·min -1 Centrifuge for ...

Embodiment 2

[0086] Determination of antiserum titer:

[0087] 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, ...

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Abstract

The invention discloses a fluorescent microsphere immunochromatographic test strip for detecting olaquindox residue and application thereof, the test strip can qualitatively and quantitatively detectolaquindox in water and feed, the sample pretreatment process is simple, convenient and fast, the detection time is short, and the test strip has very high precision and sensitivity; in addition, theolaquindox-resistant monoclonal antibody prepared by the invention is high in specificity; an olaquindox monoclonal antibody marked by fluorescent microspheres is adopted; the luminous intensity and the detection signal of the fluorescent microsphere can be enhanced along with the enhancement of the exciting light intensity, so that the fluorescent microsphere labeled olaquindox monoclonal antibody can effectively improve the analysis sensitivity of an immunochromatographic technique, and compared with the traditional colloidal gold immunochromatographic method, the immunochromatographic method provided by the invention has higher sensitivity; meanwhile, as the fluorescent microspheres have relatively stable morphological structures, the microspheres are uniform in granularity, good in dispersity and stability, high in luminous efficiency and good in repeatability, and dye fluorescence quenching is greatly reduced.

Description

technical field [0001] The invention belongs to the technical field of time-resolved fluorescent immunoassay in biotechnology, and in particular relates to a fluorescent microsphere immunochromatographic test strip for detecting residual olaquindox and an application thereof. 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....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/558G01N33/543G01N33/533G01N21/64
CPCG01N33/577G01N33/558G01N33/54313G01N33/533G01N21/6428
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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