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Test bar and method for quantitatively detecting micromolecular compound in sample

A technology for small molecule compounds and test strips, applied in the field of detection, can solve the problems affecting the accuracy of test results and the high background of detection

Inactive Publication Date: 2015-03-18
SHANGHAI VENTURE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main reason is that the nitrocellulose membranes in the immunochromatographic detection materials will emit different degrees of fluorescence under the action of excitation light, and at the same time, the detection samples such as blood, urine samples and saliva all contain proteins, nucleic acids and sugars, all of which have fluorescence effects. Therefore, the background of the detection is high, which affects the accuracy of the detection results.

Method used

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  • Test bar and method for quantitatively detecting micromolecular compound in sample
  • Test bar and method for quantitatively detecting micromolecular compound in sample
  • Test bar and method for quantitatively detecting micromolecular compound in sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] Preparation of fluorescent latex-labeled ketamine antibody (antibody complex)

[0091] Take 100 μl of 10% europium latex microspheres with activated carboxyl groups on the surface (200 nm, purchased from Thermo Fisher, USA), add 900 μl of MES (0.05M, pH6.1) to wash, centrifuge at 13000 rpm for 15 minutes, and discard the supernatant. Add 1ml marker solution (0.05M MES pH6.1, 2.0mg / ml N-hydroxysuccinimide NHS, and 0.5mg / ml EDC.HCl (1-ethyl-3-(3-dimethylaminopropyl )-carbodiimide EDC)) resuspended, sonicated, and reacted at room temperature for 1 hour. The activated latex was centrifuged at 13,000 rpm for 15 minutes, and the supernatant was discarded. Resuspend with 1ml 0.05M MES buffer pH6.1. After sonication, 80 μg / ml of ketamine antibody was added, mixed evenly, and reacted at room temperature for 2 hours. The labeled latex was centrifuged at 13,000 rpm for 15 minutes, and the supernatant was discarded. Add 1ml of 0.05M MES buffer pH6.14%BSA to block, sonicate, and...

Embodiment 2

[0093] Preparation of immunochromatographic detection reagent plate

[0094] (1) Preparation of binding pad (marker pad)

[0095] The antibody complex and mouse IgG prepared in Example 1 were mixed with 10 mM phosphate buffer solution at pH 7.4 containing 0.5% surfactant Tetronic-1037 (purchased from BASF) to prepare a solution with a concentration of 0.5 mg / ml. Coat evenly on glass cellulose paper or polyester film, the coating amount is 50μl / cm 2 , vacuum dried. A conjugated pad is prepared.

[0096] (2) Detection area and quality control area

[0097] Detection area (also known as detection line): Dissolve ketamine bovine serum albumin (BSA) conjugate in 0.01M pH7.3 phosphate buffer containing 10% sucrose, spray on the nitrocellulose reaction membrane, Coating amount is 10μl / cm 2 ; Then dry at 15-35°C for 20 hours to make a detection area;

[0098] Quality control area (also called control line or control area): Dissolve goat anti-mouse IgG in 0.01M pH7.3 phosphate bu...

Embodiment 3

[0111] detection

[0112] (1) Draw a standard curve

[0113] Prepare 1.0ml of ketamine solution with a concentration of 1000ng / ml in PBS, take 500μl and dilute with PBS to obtain different concentrations of ketamine solutions, as follows: 10000ng / ml, 5000ng / ml, 2500ng / ml, 1250ng / ml, 625ng / ml , 312.5ng / ml, 156ng / ml, 78ng / ml, 39ng / ml, 0ng / ml. PBS was used as blank control. Take 200 μl of sample and add it directly to the sample pad, react at room temperature for 5 minutes, then insert the test strip into the detection device for detection, obtain the fluorescence values ​​of the T line in the detection area and the C line in the quality control area, and calculate the T / C ratio. The results are as follows Table 1 shows.

[0114] Table 1 Fluorescence detection data

[0115] Ketamine concentration (ng / ml)

T-line fluorescence value

C-line fluorescence value

T / C ratio

10000

5017

17724

0.283063

5000

5914

17730

0.333559

...

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Abstract

The invention provides a test bar and a method for quantitatively detecting a micromolecular compound in a sample. The detection is performed by adopting the test bar containing fluorescence emulsion particles and a micromolecular compound antibody on a binding pad and using a time resolution fluorescence immunochromatographic assay, and a specific value is compared with a standard curve, so that whether the micromolecular compound exists or not is determined or the content of the micromolecular compound is detected. The test bar can be used for quantitatively detecting the micromolecular compound in the sample, can be used for greatly eliminating the non-specific combination so that the signal to noise ratio of a detection signal is remarkably improved, and test bar has the advantages of rapidness, high sensitivity, high specificity and the like.

Description

technical field [0001] The present invention relates to the field of detection. Specifically, the present invention relates to a test strip and method for detecting small molecule compounds in a sample. Background technique [0002] Immunochromatographic test (immunochromatographic test), also known as lateral flow immunoassay (LFIA), is a rapid detection technology that emerged in the 1980s. After years of development, this technology has been widely used in clinical diagnosis, drug detection and food safety and other fields. [0003] Most small molecule compounds are detected, usually using competitive inhibition assays. In the immunochromatography method, the complete antigen of the small molecular substance is immobilized on the detection area (solid-phase antigen) on the nitrocellulose membrane, and the small molecular substance (free antigen) in the sample solution to be tested competes with the solid-phase antigen to bind to the colloidal gold. Or colored latex mic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/566G01N33/577
CPCG01N33/558G01N33/577
Inventor 胡小龙丁国荣
Owner SHANGHAI VENTURE BIOTECH CO LTD
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