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Competitive monoclonal antibody-based trichinosis antibody detection kit and preparation method thereof

A monoclonal antibody and antibody detection technology, applied in the field of fluorescence immunodetection, can solve the problems of sensitivity and accuracy limitations, and achieve the effects of easy storage, strong practicability, and wide promotion space.

Active Publication Date: 2020-08-07
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Conventional immunolabeling is to use colloidal gold to label antibodies or antigens, but due to the limitation of sensitivity and accuracy of colloidal gold detection, it can only be used in some detection items with low requirements

Method used

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  • Competitive monoclonal antibody-based trichinosis antibody detection kit and preparation method thereof
  • Competitive monoclonal antibody-based trichinosis antibody detection kit and preparation method thereof
  • Competitive monoclonal antibody-based trichinosis antibody detection kit and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1. Trichinellosis antibody detection kit based on competitive monoclonal antibody.

[0039] The trichinellosis antibody detection kit described in this embodiment contains a test strip and a competitive detection reagent; the test strip includes a sample pad, a chromatographic membrane, an absorbent pad and a bottom plate, and the chromatographic membrane is provided with a detection line And the quality control line, wherein the detection line is sprayed with rabbit anti-Ts-WN10 polyclonal antibody, and the quality control line is sprayed with rabbit anti-mouse IgG; the competitive reagent contains the monoclonal antibody secreted by the hybridoma cell line WN10-1H9 Ts-WN10-1H9 and recombinant antigen Ts-WN10; the microbial preservation number of the hybridoma cell line WN10-1H9 is: CGMCC No.18316, and the cell line was preserved in China Microorganism Culture Collection Management on August 15, 2019 The General Microbiology Center of the Committee is located a...

Embodiment 2

[0042] Example 2. The preparation method of the trichinellosis antibody detection kit based on the competitive monoclonal antibody.

[0043] 1) Preparation of Trichinella spiralis Ts-WN10 recombinant antigen: The recombinant Escherichia coli BL21(DE3)-pET28a-Ts-WN10 was induced to express and purified by one-step on-column renaturation on a Ni column to obtain the Trichinella spiralis Ts-WN10 recombinant antigen. The specific method is as follows:

[0044] ①.Primer design

[0045] According to the registered Ts-WN10 gene sequence (accession number: EU263325) in Genbank, design PCR amplification primers, the sequence is as follows:

[0046] Ts-WN10-EcoRI-atg:5'-TAACGAATTCATGCAGATACTTGGTGA-3' (as shown in SEQ ID No.1)

[0047] Ts-WN10-XhoI-tta:5'-GACGCTCGAGTTAACATTCAACA-3' (as shown in SEQ ID No.2)

[0048] The underlined part is the introduced EcoRI and XhoI restriction sites, and the expected length of the amplified product is 1187bp.

[0049] ② Extraction and reverse tran...

Embodiment 3

[0082] Example 3. Sensitivity, specificity and stability detection of the trichinellosis antibody detection fluorescent test strip prepared by the present invention.

[0083] Using the detection method described in Example 2, the sensitivity, specificity and stability detection of the kit prepared by the present invention were investigated.

[0084] 1. Sensitivity test:

[0085] Trichinella spiralis immunochromatography test strips were used to detect anti-trichinella antibodies in pig serum under different infection conditions, and the detection results were compared with commercial enzyme-linked immunosorbent antibody detection method ES ELISA (Qiagen). Make sure the test strips used in the test are from the same batch. The commercialized ES ELISA kits are the same batch of products, and the detection steps and result judgment standards are carried out according to the kit instructions.

[0086] The results showed that ES ELISA detected 30 sera were positive, and test stri...

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Abstract

The invention discloses a competitive monoclonal antibody-based trichinosis antibody detection kit and a preparation method thereof, and belongs to the technical field of fluorescence immunoassay. Theinvention discloses a method for detecting trichina infection more quickly, stably and accurately. The invention provides the competitive monoclonal antibody-based trichinosis antibody detection kit,a test strip comprises a sample pad, a chromatography membrane, a water absorption pad and a bottom plate, the chromatography membrane is provided with a detection line and a quality control line, the detection line is sprayed with a rabbit anti-Ts-WN10 polyclonal antibody, and the quality control line is sprayed with rabbit antimouse IgG; a competitive reagent contains a monoclonal antibody Ts-WN10-1H9 secreted by a hybridoma cell strain WN10-1H9 and a recombinant antigen Ts-WN10; and the microbiological preservation number of the hybridoma cell strain WN10-1H9 is CGMCC No. 18316. The kit prepared by the invention is high in specificity and sensitivity, and can be used for quickly detecting the swine trichinopathy.

Description

technical field [0001] The invention belongs to the technical field of fluorescent immunoassay, and in particular relates to a trichinellosis antibody detection kit based on a competitive monoclonal antibody and a preparation method thereof. Background technique [0002] Trichinellosis is a very serious zoonotic disease. It not only causes huge economic losses to animal husbandry production, but also poses a huge threat to human health. People or animals mainly contain trichinosis through raw or semi-raw food. Caterpillar meat (mainly pork) and disease. [0003] For the inspection of trichinellosis in slaughtered animals, the inspection methods of the regulations of the International Organization for Animal Health (OIE) are microscope inspection and sample digestion method, and these two methods are also being used in my country at present. However, the above two methods have certain disadvantages. Microscopic examination is time-consuming and laborious and has poor sensiti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/558G01N33/533
CPCG01N33/533G01N33/558G01N33/577G01N2333/4353
Inventor 刘明远刘晓雷刘琰杨勇
Owner JILIN UNIV
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