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Immunochromatography test paper for testing ochratoxin A based on M13 phage and preparation method of immunochromatography test paper

A technology of immunochromatography test paper and phage, which is applied in biological testing, measuring devices, analysis materials, etc.

Active Publication Date: 2014-08-06
HENAN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the immunological detection method is based on a toxic detection system based on labeled toxins or conjugated toxins as competing antigens, which has defects such as expensive toxin standard products, harmful to the health of production and operators, and poor repeatability of coupling reactions. Restricting its application and promotion

Method used

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  • Immunochromatography test paper for testing ochratoxin A based on M13 phage and preparation method of immunochromatography test paper
  • Immunochromatography test paper for testing ochratoxin A based on M13 phage and preparation method of immunochromatography test paper

Examples

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preparation example Construction

[0054] The preparation process of the test paper of the present invention includes: preparation of OTA monoclonal or polyclonal antibody, screening of M13 phage displaying OTA mimic epitope, preparation of gold-labeled antibody fiber layer, preparation of adsorption fiber layer, preparation of cellulose membrane layer And the assembly of the test paper.

[0055] 1. Preparation of OTA monoclonal antibody or polyclonal antibody

[0056] Monoclonal antibody preparation: including OTA artificial antigen preparation, immunization of animals (mouse), cell fusion, monoclonal antibody screening, monoclonal antibody preparation; polyclonal antibody preparation: including OTA artificial antigen preparation and immunization of animals (White rabbit), polyclonal antibody screening and polyclonal antibody preparation.

[0057] (1) Preparation of OTA artificial antigen

[0058] Dissolve 1mg OTA in ethanol, add 3mg DCC and 2mg NHS, stir for 12h at 4°C in the dark, centrifuge at 10000rpm for 15min, ...

Embodiment 1

[0086] Example 1: Immunochromatographic test paper for rapid detection of ochratoxin A based on M13 phage, see figure 1 , figure 2 . In the figure, 1 is the support layer, made of plastic sheet strips, 2 is the adsorption fiber layer, made of glass fiber cotton, gold-labeled antibody fiber layer 3 is adsorbed with anti-OTA monoclonal antibody gold-labeled antibody glass fiber cotton, fiber The plain membrane layer 4 is made of nitrocellulose membrane, and the absorbent material layer 5 at the handle end is made of absorbent filter paper. The absorbent fiber layer 2, the gold-labeled antibody fiber layer 3, the cellulose membrane layer 4, and the water absorbent material layer 5 are from left to The right ones are pasted and fixed on the supporting layer 1 in turn, and the fibers cross and penetrate each other at the junctions. Invisible detection blot 6 is provided on the cellulose membrane layer 4. The invisible detection blot is made of M13 phage displaying OTA mimotopes; t...

Embodiment 2

[0092] Example 2: The structure of the test paper is basically the same as that of Example 1. The difference is that the gold-labeled antibody fiber layer 3 is adsorbed with anti-OTA polyclonal antibodies, the adsorption fiber layer 2 is made of nylon membrane, and the cellulose membrane layer 4 is made of pure The cellulose membrane, the invisible detection band and the invisible control band are all "ten", and the handle end protective film 8-2 is blue.

[0093] Detection of flour: The sample preparation method is the same as in Example 1.

[0094] Result judgment: (a) If it is positive if there is a red mark with "ten" on the cellulose membrane, it means that the test result is positive, indicating that the sample to be tested contains OTA; (b) if it is negative if there is a sign on the cellulose membrane Two red bands with "ten" indicate that the test result is negative, indicating that there is no OTA in the sample to be tested; (c) Failure If there is no red band displayed o...

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Abstract

The invention discloses an immunochromatography test paper for testing ochratoxin A based on an M13 phage and a preparation method of the immunochromatography test paper. A test paper adsorbing layer comprises an adsorbing fiber layer, a gold-labeled antibody fiber layer, a cellulose membrane layer and a water absorbing material layer, wherein a testing blot used for showing OTA mimic epitope and blotted by the M13 phage and a contrasting blot using a goat anti-mice IgG antibody solution are arranged on the cellulose membrane layer; a gold-labeled antibody is a collaurum-labeled OTA monoclonal antibody or polyclonal antibody. According to the invention, the application of immunochromatography technique based on the M13 phage on quick OTA residue testing is realized, OTA conjugate blotted on the testing blot of a traditional test strip is replaced with the M13 phage shown with the OTA mimic epitope, harm of the OTA conjugate to a test paper producer, an experimenter and environment is avoided, and the test paper is strong in specificity and high in sensitivity during testing, and is simple and convenient to test, straightforward, accurate, low in cost, and wide in application range.

Description

technical field [0001] The invention relates to an immunochromatographic test paper, in particular to an immunochromatographic test paper for rapid detection of ochratoxin A by using M13 phage displaying an OTA mimetic epitope as a detection mark and a preparation method thereof. Background technique [0002] Ochratoxin is a secondary metabolite produced by certain strains of Aspergillus and Penicillium. It contains seven compounds with similar structures, among which the toxicity is the most, the most widely distributed, the highest toxin production, and the most polluting crops Ochratoxin A (OTA) is the most serious and most closely related to human health. Its target organs are mainly the kidney and liver. It is a strong kidney and liver toxin, and has strong carcinogenic and carcinogenic Teratogenic and mutagenic effects. Since OTA widely exists in agricultural products and livestock products, and can enter the human body through the food chain, causing harm to human l...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/558
CPCG01N33/558G01N33/56911G01N2333/38G01N2333/385
Inventor 胡骁飞张改平邢广旭王耀王方雨赵东邓瑞广
Owner HENAN ACAD OF AGRI SCI
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