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Magnetic immunochromatography for quickly detecting L. monocytogenes and preparation of test strip for detection

A technology of Listeria monocytogenes and magnetic immunochromatography, which can be applied to measurement devices, analytical materials, instruments, etc., can solve the problems of inability to detect Listeria monocytogenes quickly and easily, and achieve high sensitivity and short time consumption. Effect

Inactive Publication Date: 2012-03-28
SHANGHAI OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The purpose of the present invention: to provide a magnetic immunochromatographic method for rapid detection of Listeria monocytogenes using immunomagnetic beads as a marker for the shortcomings and defects that the existing detection technology cannot detect Listeria monocytogenes quickly and easily and preparation of magnetic bead-labeled chromatography test strips

Method used

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  • Magnetic immunochromatography for quickly detecting L. monocytogenes and preparation of test strip for detection
  • Magnetic immunochromatography for quickly detecting L. monocytogenes and preparation of test strip for detection
  • Magnetic immunochromatography for quickly detecting L. monocytogenes and preparation of test strip for detection

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Experimental program
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preparation example Construction

[0041] A. Preparation and purification of antibodies:

[0042] The UV-inactivated Listeria monocytogenes was used as the antigen to immunize BALB / C mice. The monoclonal antibody cell lines were prepared and screened according to the conventional hybridoma technology and limiting dilution method, and then the specificity of the anti-Listeria monocytogenes The antibody cell line is proliferated and cultured, and injected into BALB / C mice to prepare ascites. After the prepared ascites is concentrated by 50% weight / volume ratio of ammonium sulfate, the antibody is purified using a commercial Protein-G affinity chromatography column; When specifically performing affinity chromatography purification, follow the attached product operating instructions.

[0043] The UV-inactivated Listeria monocytogenes was used as the antigen to immunize New Zealand white rabbits. The resulting antiserum was concentrated by 50% weight / volume ratio of ammonium sulfate, and then desalted using a commercial ...

Embodiment example 1

[0058] Implementation case 1 Preparation of test strips for magnetic detection of Listeria monocytogenes

[0059] (1) Preparation of specific immunomagnetic beads for Listeria monocytogenes

[0060] EDC / NHS coupling

[0061] The Listeria monocytogenes specific murine monoclonal antibody is coupled with carboxyl modified nanomagnetic beads (with a particle size of 200nm) to prepare Listeria monocytogenes specific immunomagnetic beads, that is, magnetic labeled antibodies. Take the pH5.0 MEST solution (0.05% Tween-20) as the activation buffer solution, take 2mg of carboxyl magnetic beads into a 2mL centrifuge tube, add 500μL activation buffer, mix well on the vortex shaker, and then place the centrifuge tube in On the magnetic separation rack, after the magnetic beads are completely adsorbed, extract the supernatant with a miniature desktop vacuum pump; after re-washing the magnetic beads twice with 500μL activation buffer, adjust the volume of EDC and NHS solution to 500μL with activ...

Embodiment example 2

[0065] Implementation of case 2 qualitative testing of samples

[0066] Add 100 μL of sample to the sample pad of the test strip, and after reacting at room temperature for 20 minutes, observe the test result with naked eyes. See figure 2 , The positive result shows two obvious bands or the T-line band is lighter than the C-line band, while the negative result only shows one band at the C-line, and there is no band at the T-line. If there is no band at the C line during the test, there is a problem with the test strip system, and the test result is deemed invalid.

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Abstract

The invention discloses a magnetic immunochromatography for quickly detecting L. monocytogenes. The method comprises the following steps of: screening a specific reaction rat monoclonal antibody of the obtained L. monocytogenes by using a hybridoma technique, using the specific reaction rat monoclonal antibody as a magnetic bead labeled antibody, and coupling the monoclonal antibody to the surface of a magnetic nanomaterial in a chemical bonding mode to obtain an immunomagnetic bead; constructing a magnetic immunochromatographic test strip by using a sample pad, a magnetic bead labeled conjugate pad, a chromatographic film pre-coated with a detection line T for rabbit antiserum and a control line C for goat anti-mouse (IgG) for the L. monocytogenes, and an absorbent pad; and during detection, judging macroscopic color development belts formed in the detection line T area and the control line C area according to the color of the magnetic nanomaterial on the surface of the immunomagnetic bead, or putting the used test strip on a magnetic signal detector, detecting the detection line T area and the control line C area formed after immunochromatography to obtain quantitative reaction data, so that the L. monocytogenes are quickly qualitatively or quantitatively detected.

Description

Technical field [0001] The method belongs to biological detection technology, especially a magnetic immunochromatographic method for rapid detection of Listeria monocytogenes and the preparation of detection test strips, and is suitable for the detection method of food-borne pathogenic bacteria Listeria monocytogenes. Background technique [0002] Listeria Monocytogenes (Listeria Monocytogenes, referred to as Listeria monocytogenes) is a zoonotic food-borne pathogen, referred to as Listeria monocytogenes. Listeria monocytogenes is a Gram-positive Brevibacterium that does not produce spores. The main clinical manifestations are sepsis, meningitis, etc. It is especially susceptible to pregnant women, newborns, elderly people and other immunocompromised people and immunodeficiency patients. , Pregnant women can cause bacteremia after infection, leading to premature delivery, dead babies, etc., so they are paid attention to in the field of food safety. [0003] Listeria monocytogenes ...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/569G01N33/558
Inventor 卢瑛林婷婷潘迎捷
Owner SHANGHAI OCEAN UNIV
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