Immunochromatographic test paper and preparation method for detecting ochratoxin a based on m13 phage

An immunochromatographic test strip and bacteriophage technology, applied in biological tests, measuring devices, analytical materials, etc., can solve the problems of poor repeatability of coupling reactions, harmful health of production and operators, restricting application and promotion, etc., and achieves strong specificity. , the effect of high sensitivity

Active Publication Date: 2016-05-25
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

Method used

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  • Immunochromatographic test paper and preparation method for detecting ochratoxin a based on m13 phage
  • Immunochromatographic test paper and preparation method for detecting ochratoxin a based on m13 phage

Examples

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preparation example Construction

[0053] 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 mimetic epitope, preparation of gold-labeled antibody fiber layer, preparation of adsorption fiber layer, preparation of cellulose film layer And the assembly of test paper and other steps.

[0054] 1. Preparation of OTA monoclonal antibody or polyclonal antibody

[0055] Preparation of monoclonal antibodies: including preparation of OTA artificial antigens, immunization of animals (mice), cell fusion, screening of monoclonal antibodies, preparation of monoclonal antibodies; preparation of polyclonal antibodies: preparation of OTA artificial antigens, immunization of animals (white rabbit), screening of polyclonal antibodies and preparation of polyclonal antibodies.

[0056] (1) Preparation of OTA artificial antigen

[0057] Dissolve 1 mg OTA in ethanol, add 3 mg DCC and 2 mg NHS, stir for 12 hours at ...

Embodiment 1

[0085] 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, which is made of plastic sheet strips, and 2 is the adsorption fiber layer, which is made of glass fiber cotton. The gold-labeled antibody fiber layer 3 is adsorbed with gold-labeled antibody glass fiber cotton of anti-OTA monoclonal antibody. The plain membrane layer 4 is made of nitrocellulose membrane, and the water-absorbing material layer 5 at the handle end is made of water-absorbing filter paper. The right side is pasted and fixed on the support layer 1 in turn, and the fibers at the junction between each other cross and penetrate each other. On the cellulose membrane layer 4, a stealth detection blot 6 is provided, and the stealth detection blot is made with M13 phage displaying OTA mimetic epitopes; the stealth control blot 7 is blotted on the cellulose membrane with a goat anti-mouse IgG antibod...

Embodiment 2

[0091] Embodiment 2: The structure of the test paper is basically the same as that of Embodiment 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 film, the invisible detection imprinted band and the invisible contrast imprinted band are all "ten", and the protective film 8-2 at the handle end is blue.

[0092] Detection of flour: the sample preparation method is the same as in Example 1.

[0093]Result judgment: (a) positive if there is a red blot "ten" on the cellulose membrane, it means that the test result is positive, indicating that OTA is contained in the sample to be tested; (b) negative if there is a red blot on the cellulose membrane Two red blots 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 n...

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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 teratogenic effects. 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 life a...

Claims

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Application Information

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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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