Detection test paper for forest encephalitis antibody, test paper preparation method and detection kit

A forest encephalitis and antibody detection technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of time-consuming detection process, reduced amount of labeled antibody, high reagent cost, etc., to achieve accurate and reliable detection results and stable detection reagent solution , The effect of low reagent cost

Active Publication Date: 2016-05-04
CHINESE ACAD OF INSPECTION & QUARANTINE
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Problems solved by technology

[0002] At present, the detection technologies for forest encephalitis virus antibodies are mainly immunological methods such as ELISA and chemiluminescence. Among them, the ELISA detection method needs repeated cleaning and finalizing, and requires the scenery photometer to detect the absorbance; the fluorescent reagent required in the chemiluminescence method Relatively expensive, the detection requires specific fluorescence analysis instruments; these methods require expensive instruments, the operation is cumbersome, and the entire detection process takes a long time, and requires the detection personnel to have certain professional qualities
[0003] One of the most critical factors in immunological detection methods such as ELISA and chemiluminescence is the selection of antigens and antibodies. If the antigen is prepared directly from the forest encephalitis virus culture solution, there are complex antigen components, low content of effective antigen, many foreign proteins, and poor immunogenicity. And the high risk of biosafety, and when used for immunological detection, it is easy to lead to positive results
[0004] In addition, the colloidal gold detection method is suitable for on-site detection, but the detection sensitivity is limited, mainly because the colloidal gold is very unstable after the antibody to be labeled or the protein to be labeled is connected, and the antibody is easily detached due to the change of charge, causing each colloid The amount of gold-labeled antibody is greatly reduced, resulting in a decrease in sensitivity
Moreover, the preparation of the colloidal gold reagent uses chloroauric acid, and the cost of the reagent is relatively high, resulting in a relatively high overall cost of the immunochromatography test paper.

Method used

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  • Detection test paper for forest encephalitis antibody, test paper preparation method and detection kit
  • Detection test paper for forest encephalitis antibody, test paper preparation method and detection kit
  • Detection test paper for forest encephalitis antibody, test paper preparation method and detection kit

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Embodiment 1

[0049] The preparation of embodiment 1 forest encephalitis antigen

[0050] The specific steps of preparing forest encephalitis antigen are as follows:

[0051] a. Construction of recombinant plasmids

[0052] To the selection of forest encephalitis antigen, the inventor of the present invention carries out multiple tests, and finally determines to select forest encephalitis (TBE) outer membrane protein, its nucleotide sequence (GenBank sequence number: HM133639.1), according to Escherichia coli EscherichiacoliO127: The H6 preferred codon was used to modify the gene sequence, and the secondary structure of the recombinant protein was screened through bioinformatics (see the reference materials for substantive review of the screening scheme, which will not be repeated here), and after multiple experiments, the final gene was determined The sequence is shown in SEQ ID NO.1.

[0053] Entrust Shanghai Yingjun Company to synthesize according to the sequence shown in SEQIDNO.1, co...

Embodiment 2

[0058] Embodiment 2 prepares the detection test strip of forest encephalitis virus antibody

[0059] A. SPA labeling magnetic nanoparticles, and preparing magnetic nanoparticle carrier pads:

[0060] Take 100 μl ferroferric oxide magnetic nanoparticle solution (the particle size of ferric oxide tetroxide is preferably 12nm), add 700 μl water, 200 μl 25% glutaraldehyde solution, shake for 3 hours; wash 1-2 times with 1ml water, and wash 2 times in PBS , that is to carry out carboxylation treatment on ferroferric oxide magnetic nanoparticles; add 500 μl, 2 mg / ml SPA, shake for 3 hours, and the pH value is 7.6; add 500 μl, 0.5% BSA, shake for 30 minutes, and block; add 0.01% Tween -20 concentration of 0.01M PBS, wash 3-4 times; add 1mL resuspension, wherein the resuspension contains 0.1% Tris base, 1% BSA and 5% sucrose by weight, mix well, Spray on glass fiber membrane and dry at 37°C for 4 hours;

[0061] B, coated nitrocellulose membrane: the forest encephalitis virus recomb...

Embodiment 3

[0066] The detection test paper of the forest encephalitis antibody prepared by the present invention and the sensitivity detection of colloidal gold immunochromatography test paper

[0067] Wherein, the preparation method of the detection test paper of the forest encephalitis antibody prepared by the present invention sees embodiment 2;

[0068] Preparation method of colloidal gold immunochromatography test paper:

[0069] Colloidal gold immunochromatographic test paper was prepared by conventional methods, wherein SPA-labeled colloidal gold particles were used, the detection zone on the nitrocellulose membrane was coated with forest encephalitis virus recombinant antigen, and the quality control zone was coated with goat anti-rabbit IgG.

[0070]The test strips prepared in Example 2 were used to detect forest encephalitis antibody serum respectively, and diluted to 1:10, 1:100, 1:1000, 1:10000, 1:100000, 1:1000000 with fetal bovine serum, and simultaneously Take bovine seru...

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Abstract

The invention discloses a piece of detection test paper of a forest encephalitis antibody. The detection test paper consists of a sample pad, an Fe3O4 magnetic nanoparticle carrier pad, a nitrocellulose membrane and a water absorbing pad which are in lap joint and adhered to a substrate card in sequence, wherein the Fe3O4 magnetic nanoparticle carrier pad is a glass fiber membrane on which staphylococcus aureus A protein (SPA) is fixed to mark magnetic nanoparticles; the nitrocellulose membrane is provided with a detection zone and a quality control zone; the detection zone is coated by forest encephalitis antigens; and the quality control zone is coated by an antibody which can be combined with SPA. A detection kit for detecting the forest encephalitis virus antibody is prepared from the detection test paper and a peroxidase substrate developing liquid. The kit is capable of effectively detecting the forest encephalitis virus antibody, is stable and reliable in detection result, is high in specificity, is simple to operate, and is high in sensitivity, that is, the sensitivity is 10-100 times higher than that of a piece of ordinary colloidal gold detection test paper, and no detection instrument is needed but the detection result can be obtained with naked eyes.

Description

technical field [0001] The invention relates to the field of biological detection reagents, in particular to a detection test paper for forest encephalitis antibody, a preparation method of the test paper and a detection kit thereof. Background technique [0002] At present, the detection technologies for forest encephalitis virus antibodies are mainly immunological methods such as ELISA and chemiluminescence. Among them, the ELISA detection method needs repeated cleaning and finalizing, and requires the scenery photometer to detect the absorbance; the fluorescent reagent required in the chemiluminescence method It is relatively expensive, and the detection requires specific fluorescence analysis instruments; these methods require expensive instruments, cumbersome operations, and the entire detection process takes a long time, and requires the detection personnel to have certain professional qualities. [0003] One of the most critical factors in immunological detection meth...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/558
CPCG01N33/558
Inventor 杨宇韩辉曾婷婷赵婷婷王静徐宝梁
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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