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Bovine echinococcosis granulosa time-resolved fluorescence immunochromatographic assay test strip and preparation method thereof

A technology for time-resolved fluorescence and echinococcosis granulosus, applied in botany equipment and methods, biochemical equipment and methods, analytical materials, etc., can solve the problems of inconspicuous antibody response, few animals, and low accuracy , to achieve good sensitivity and accuracy, rapid diagnostic method, and good specificity

Active Publication Date: 2020-08-11
SHENYANG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

With the development of molecular technology and immune technology, there are molecular diagnosis, such as LAMP, immunological diagnosis, such as ELISA kit method, colloidal gold, etc., but it is found from clinical application that the accuracy and convenience of these detection methods are not high. Strong, unable to facilitate the operation and detection of grassroots workers
[0004]Although the diagnosis of echinococcosis in humans has been extensively studied, little has been done in diagnosing animals, and when animals are naturally infected with the disease , the antibody reaction is not obvious, which can easily lead to diagnostic errors

Method used

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  • Bovine echinococcosis granulosa time-resolved fluorescence immunochromatographic assay test strip and preparation method thereof
  • Bovine echinococcosis granulosa time-resolved fluorescence immunochromatographic assay test strip and preparation method thereof
  • Bovine echinococcosis granulosa time-resolved fluorescence immunochromatographic assay test strip and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Preparation of recombinant multi-epitope antigen (named Eg-H1)

[0042] The selected peptides are as follows:

[0043] B1 (antigen B subunit 1): DDGLTSTSRSVMKMFGEVKYFFERDPLGQK;

[0044] B2 (antigenB subunit 2): KDEPKAHMGQVVKKRWGELRDFFRND;

[0045] B3 (antigenB subunit4): LGEIRDFFRSDPLGQKLVALGRRDLTAI;

[0046] B4 (EG95): KGMGVETRTTETPLRKHFNLTPVGSQGI;

[0047] B5 (antigenprotein): TGNCDQGKAQSANVTG;

[0048] B6(Ag5):ECSPHTCLEHRYRRCVD;

[0049] B7 (EPC1): GKISCAELKSALQSCSA.

[0050] In a series manner, the above-mentioned peptides were linked together using a linker GGGGSGGGGSGGGGS. As a preference, the above-mentioned peptides are linked together in sequence, and the amino acid sequence thereof is:

[0051] MDDGLTSTSRSVMKMFGEVKYFFERDPLGQKGGGGSGGGGSGGGGSKDEPKAHMGQVVKKRWGELRDFFRNDGGGGSGGGGSGGGGSLGEIRDFFRSDPLGQKLVALGRDLTAIGGGGSGGGGSGGGGSKGMGVETRTTETPLRKHFNLTPVGSQGIGGGGSGGGGSGGGGSTGNCDQGKAQSANVTGGGGGSGGGGSGGGGSECSPHTCLEHRYRRCVDGGGGSGGGGSGGGGSGKISCAELKSALQSCSA(SEQ ID NO....

Embodiment 2

[0105] Such as figure 1 As shown, the bovine echinococcosis time-resolved fluorescent immunochromatographic detection test strip of this embodiment includes a PVC base plate 1, and a sample pad 2, a binding pad 3, and a nitrocellulose base plate sequentially arranged on the PVC base plate. Membrane 4 and absorbent pad 5, binding pad 3 is coated with goat anti-bovine IgG antibody labeled with time-resolved fluorescent microspheres, nitrocellulose membrane 4 is provided with separated detection line 6 and quality control line 7, detection line 6 It is coated with recombinant multi-epitope antigen Eg-H1, and the quality control line 7 is coated with rabbit anti-goat IgG secondary antibody.

[0106] Its preparation method comprises the following steps:

[0107] Preparation of S1 binding pad: label time-resolved fluorescent microspheres with goat anti-bovine IgG antibody at a labeling amount of 50 μg / mL, and then add fluorescent working solution (0.01M phosphate buffer) at a volum...

Embodiment 3

[0114] In order to improve the accuracy of bovine echinococcosis time-resolved fluorescent immunochromatographic test strip detection, the following conditions were explored:

[0115] Preconditions for optimizing time-resolved fluorescent microsphere immunochromatographic test strips: other conditions remain unchanged, and only one of the variables is changed each time. The reaction conditions were as follows: the positive sample and the negative sample (both collected in the Zoige county slaughterhouse) were added with a sample volume of 75 μL (the volume ratio of serum to sample buffer was 1:2), and the reaction time was 15 minutes. The coating parameter is 0.75 μL / cm and the spraying parameter is used to coat the antigen or antibody on the nitrocellulose membrane to form the detection line (T) and the quality control line (C).

[0116] Determination of C-line coating amount: Spray 1mg / mL Eg-H1 on the T-line with a three-dimensional gold-spraying device, and prepare two grou...

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Abstract

The embodiment of the invention discloses a bovine echinococcosis granulosa time-resolved fluorescence immunochromatographic assay test strip and a preparation method thereof, and belongs to the technical field of biological assay. A combination pad of the test strip is coated with a goat anti-bovine IgG antibody marked by time-resolved fluorescent microspheres. The nitrocellulose membrane is provided with a detection line (6) and a quality control line (7) which are separated from each other, the detection line is coated with a recombinant multi-epitope antigen, the quality control line is coated with a rabbit anti-goat IgG secondary antibody, and the recombinant multi-epitope antigen is formed by splicing seven peptide fragments (named as Eg-H1). The test strip disclosed by the inventionhas the advantages of simplicity and convenience in operation and good sensitivity and accuracy; overcomes the defects of incapability of field operation and low accuracy of ELISA, colloidal gold andother diagnosis methods, also has good specificity, provides a rapid diagnosis method for epidemiological investigation of bovine echinococcosis granulosa and diagnosis of the disease, and has wide application prospects.

Description

technical field [0001] The embodiment of the present invention relates to the technical field of biological detection, in particular to a bovine echinococcosis time-resolved fluorescent immunochromatographic detection test strip and a preparation method thereof. Background technique [0002] Echinococcus granulosus (hydatid disease) is a serious global zoonotic disease. Humans or ruminants mainly get sick by eating the eggs excreted by Echinococcus granulosus. cancer". Up to now, my country still lists the disease as one of the animal diseases that are mainly controlled and protected. [0003] At present, there is little investment in research on diagnostic techniques for echinococcosis granulosus, and the most commonly used diagnostic method is dissection and inspection of parasites. With the development of molecular technology and immune technology, there are molecular diagnosis, such as LAMP, immunological diagnosis, such as ELISA kit method, colloidal gold, etc., but i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/58G01N33/558G01N33/52C07K14/435C12N15/70C12N15/12
CPCG01N33/6893G01N33/582G01N33/558G01N33/52C07K14/43554C12N15/70G01N2333/43543
Inventor 杨娜陈启军姜宁丁莹莹桑晓宇冯颖陈冉王新一
Owner SHENYANG AGRI UNIV
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