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CN-121978329-A - Prussian blue nano test strip for detecting African horse sickness virus antibody as well as preparation method and application thereof

CN121978329ACN 121978329 ACN121978329 ACN 121978329ACN-121978329-A

Abstract

The invention belongs to the technical field of biology, and particularly relates to a Prussian blue nano test strip for detecting an African horse sickness virus antibody, and a preparation method and application thereof. The detection line and the quality control line are arranged on the chromatography detection film, the detection line is coated with staphylococcus A protein, the quality control line is coated with monoclonal antibody of the African horse sickness virus VP7 recombinant protein, and the binding pad is coated with Prussian blue nanoparticle coupled African horse sickness virus VP7 delta N 1‑129 recombinant protein fragment. The invention takes Prussian blue nanometer as a coupled tracer, and fully utilizes the specific reaction of antigen and antibody to detect the African horse sickness virus antibody. The conjugate can be combined with the corresponding antibody to clearly observe the result, has good stability, high sensitivity and strong specificity, has great clinical application function and great development potential, and is an ideal antibody test strip detection material.

Inventors

  • LONG YUNFENG
  • LIU RONGCHAO
  • ZHU HE
  • JIANG YAN
  • ZHOU BIN
  • LIU DONGFANG
  • SONG HUIXIN
  • LI KUN
  • HU PO

Assignees

  • 南京海关动植物与食品检测中心
  • 南京农业大学

Dates

Publication Date
20260505
Application Date
20260211

Claims (10)

  1. 1. A Prussian blue nanometer test strip for detecting an African horse sickness virus antibody is characterized in that a sample pad, a combination pad, a chromatographic detection membrane and a water absorption pad are sequentially fixed on a supporting bottom plate, a detection line and a quality control line are arranged on the chromatographic detection membrane, a staphylococcus A protein is coated on the detection line, a monoclonal antibody of an African horse sickness virus VP7 recombinant protein is coated on the quality control line, and a Prussian blue nanoparticle coupled African horse sickness virus VP7 delta N 1-129 recombinant protein fragment is coated on the combination pad; Wherein the amino acid sequence of the recombinant protein fragment of the V7DeltaN 1-129 of the African horse sickness virus is ：GAVEVQQSGRYYVPQGRTRGGYINSNIAEVCMDAGAAGQVNALLAPRRGDAVMIYFVWRPLRIFCDPQGASLESAPGTFVTVDGVNVAAGDVVAWNTIAPVNVGNPGARRSILQFEVLWYTSLDRSLDTVPELAPTLTRCYAYVSPTWHALRAVIFQQMNMQPINPPIFPPTERNEIVAYLLVASLADVYAALRPDFRMNGVVAPVGQINRALVL.
  2. 2. The nanometer Prussian blue test strip for detecting the African horse sickness virus antibody according to claim 1, wherein the antigen epitope of the monoclonal antibody of the recombinant protein of the African horse sickness virus VP7 is PPIFPP.
  3. 3. The nanometer Prussian blue test strip for detecting the African horse sickness virus antibody according to claim 2, wherein the monoclonal antibody of the recombinant protein of the African horse sickness virus VP7 is secreted by a hybridoma cell with the preservation number of GDMCC No: 67154.
  4. 4. The African horse sickness virus antibody detection Prussian blue nanometer test strip according to any one of claims 1-3, wherein the Prussian blue nanometer particle coupled African horse sickness virus VP7 delta N 1-129 recombinant protein fragment, wherein the amount of the African horse sickness virus VP7 delta N 1-129 recombinant protein fragment on a binding pad is 1-3 mug/cm 2 , and the amount of the Prussian blue nanometer particle on the binding pad is 3-9 mug/cm 2 .
  5. 5. The Prussian blue nano-test strip for detecting the African horse sickness virus antibody according to any one of claims 1-3, wherein the amount of the staphylococcus A protein coated on the detection line is 1.5-2.5 mug/cm.
  6. 6. The nanometer Prussian-blue test strip for detecting the African horse sickness virus antibody according to any one of claims 1 to 3, wherein the amount of the monoclonal antibody of the recombinant protein of the African horse sickness virus VP7 coated on the quality control line is 1-1.2 mug/cm.
  7. 7. The Prussian blue nano test strip for detecting the antibody of the African horse sickness virus according to any one of claims 1 to 3, wherein the chromatographic detection membrane is made of a nitrocellulose membrane, and the binding pad is made of glass cellulose.
  8. 8. The method for preparing the Prussian blue nano test strip for detecting the African horse sickness virus antibody according to claim 1, wherein the method comprises the steps of fixing a water absorption pad on one side of a chromatographic detection membrane and overlapping edges, fixing a binding pad on the other side of the chromatographic detection membrane and overlapping edges, and fixing a sample pad on one side of the binding pad far away from the water absorption pad and overlapping edges; The preparation method of the Prussian blue nanoparticle coupled African horse sickness virus VP7 delta N 1-129 recombinant protein fragment comprises the steps of adding Prussian blue nanoparticle suspension into 2- (N-morpholino) ethanesulfonic acid solution, centrifuging, adding 2- (N-morpholino) ethanesulfonic acid solution into precipitate for resuspension, uniformly dispersing, adding carbodiimide and N-hydroxysuccinimide, incubating, centrifuging, adding boric acid-borax buffer solution with pH of 8.0 for resuspension, adding the African horse sickness virus VP7 delta N 1-129 recombinant protein fragment, reacting at 4 ℃, adding BSA for sealing, centrifuging, and adding the resuspension to obtain the Prussian blue nanoparticle coupled African horse sickness virus VP7 delta N 1-129 recombinant protein fragment solution; The method comprises the steps of adding heavy suspension to resuspension to obtain Prussian blue nanoparticle coupled African horse sickness virus VP7 delta N 1-129 recombinant protein fragment solution, wherein the concentration of the African horse sickness virus VP7 delta N 1-129 recombinant protein fragment is 0.025-0.075 mug/mug, and the concentration of the Prussian blue nanoparticle is 0.08-0.26 mug/mug.
  9. 9. The preparation method of the Prussian blue nano test strip for detecting the African horse sickness virus antibody according to claim 8 is characterized in that the preparation method of the Prussian blue nano particles comprises the steps of dissolving citric acid or hyaluronic acid in water to obtain a solution A, dividing the solution A into two parts for later use, adding soluble ferrous salt into one part of the solution A to obtain a solution B, adding potassium ferricyanide into the other part of the solution A to obtain a solution C, heating the solution B, and dropwise adding the solution C under high-speed stirring to obtain Prussian blue nano particles, wherein the average particle size of the Prussian blue nano particles is 70-80 nm.
  10. 10. The use of the Prussian blue nanometer test strip for detecting the African horse sickness virus antibody according to claim 1, wherein the kit for detecting the African horse sickness virus antibody further comprises a sample diluent, wherein the sample diluent comprises, by mass percent, a solution of 20-30 mM HEPES containing 0.5-1% BSA (bovine serum albumin), 0.05-0.1% sodium ascorbate, 2-4% trehalose, 2-4% sucrose, 5-8% glycerol and 0.5-1M betaine.

Description

Prussian blue nano test strip for detecting African horse sickness virus antibody as well as preparation method and application thereof Technical Field The invention belongs to the technical field of biology, and particularly relates to a Prussian blue nano test strip for detecting an African horse sickness virus antibody, and a preparation method and application thereof. Background African horse sickness (African horse sickness, AHS) is a high-mortality infectious disease caused by African horse sickness virus (African horse sickness virus, AHSV). VP7 protein is the main structural protein of the viral core and has a plurality of key functions. VP7 protein plays an important role in virus core assembly, and can form a virus core structure on a bracket provided by the secondary core protein VP3, thereby laying a foundation for virus replication and assembly. Unlike the VP7 protein of bluetongue virus (BTV), the VP7 protein of AHSV has a unique self-assembly capability, capable of forming insoluble hexagonal crystal particles, a characteristic that may affect the assembly efficiency and replication rate of the viral core. VP7 protein is also one of the main immunogenic proteins of AHSV, contains B cell and T cell epitopes, is highly conserved among a variety Orbivirus, and is an important candidate protein for vaccine development. The crystalline nature of VP7 may affect viral replication efficiency and pathogenicity by limiting its availability in viral core assembly and is associated with cytopathological changes caused by AHSV infection. Overall, VP7 proteins play an important role in viral structure, immunogenicity, replication and pathogenicity. African horse sickness is an infectious disease with great harm to equine animals, and in the aspect of diagnosis, laboratory detection methods such as real-time fluorescence quantitative RT-PCR, ELISA (enzyme-linked immunosorbent assay) and the like are accurate, but are limited by equipment and professionals, and are difficult to be widely applied to a basic layer. Therefore, it is important to develop a rapid detection method suitable for the first line. Therefore, the invention develops the Prussian blue nanoparticle material-based antibody detection test strip, which has the advantages of simple and convenient operation, rapidness, high sensitivity and the like, and can meet the field detection requirement. Disclosure of Invention The invention provides a Prussian blue nanometer test strip for detecting an African horse sickness virus antibody, which comprises a sample pad, a combination pad, a chromatography detection membrane and a water absorption pad which are sequentially fixed on a supporting bottom plate, wherein the chromatography detection membrane is provided with a detection line and a quality control line, the detection line is coated with staphylococcus A protein, the quality control line is coated with a monoclonal antibody of an African horse sickness virus VP7 recombinant protein, and the combination pad is coated with a Prussian blue nanoparticle coupled African horse sickness virus VP7 delta N 1-129 recombinant protein fragment; Wherein the amino acid sequence of the recombinant protein fragment of the V7DeltaN 1-129 of the African horse sickness virus is ：GAVEVQQSGRYYVPQGRTRGGYINSNIAEVCMDAGAAGQVNALLAPRRGDAVMIYFVWRPLRIFCDPQGASLESAPGTFVTVDGVNVAAGDVVAWNTIAPVNVGNPGARRSILQFEVLWYTSLDRSLDTVPELAPTLTRCYAYVSPTWHALRAVIFQQMNMQPINPPIFPPTERNEIVAYLLVASLADVYAALRPDFRMNGVVAPVGQINRALVL. As a preferable scheme for detecting Prussian blue nanometer test strips by the African horse sickness virus antibody, the monoclonal antibody of the African horse sickness virus VP7 recombinant protein has an epitope of PPIFPP. As a preferable scheme for detecting Prussian blue nanometer test strips by the African horse sickness virus antibody, the monoclonal antibody of the African horse sickness virus VP7 recombinant protein is secreted by hybridoma cells with the preservation number of GDMCC No: 67154. As a preferable scheme for detecting the Prussian blue nanometer test strip by the African horse sickness virus antibody, the Prussian blue nanometer particle coupled recombinant protein fragment of the African horse sickness virus VP7 delta N 1-129, wherein the amount of the recombinant protein fragment of the African horse sickness virus VP7 delta N 1-129 on a binding pad is 1-3 mug/cm 2, and the amount of the Prussian blue nanometer particle on the binding pad is 3-9 mug/cm 2. As a preferable scheme for detecting Prussian blue nanometer test strips by the African horse sickness virus antibody, the quantity of staphylococcus A protein coated on the detection line is 1.5-2.5 mug/cm. As a preferable scheme for detecting Prussian blue nanometer test strips by using the African horse sickness virus antibody, the quantity of the monoclonal antibody of the African horse sickness virus VP7 recombinant protein coated on the quality control line is 1-1.2 mug/cm. As a preferable s