WO2021028802A1 - Test de bandelette pour la détection de la leucose bovine enzootique et procédé de détection de la leucose bovine enzootique à l'aide du test de bandelette - Google Patents

Test de bandelette pour la détection de la leucose bovine enzootique et procédé de détection de la leucose bovine enzootique à l'aide du test de bandelette Download PDF

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Publication number
WO2021028802A1
WO2021028802A1 PCT/IB2020/057467 IB2020057467W WO2021028802A1 WO 2021028802 A1 WO2021028802 A1 WO 2021028802A1 IB 2020057467 W IB2020057467 W IB 2020057467W WO 2021028802 A1 WO2021028802 A1 WO 2021028802A1
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WIPO (PCT)
Prior art keywords
test
sample
strip test
detecting
milk
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Ceased
Application number
PCT/IB2020/057467
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English (en)
Inventor
Mirosława SKUPIŃSKA
Agnieszka BELTER
Andrzej Rapak
Justyna Kutkowska
Małgorzata GRUDZIEŃ
Marcin CZERWIŃSKI
Ewa JAŚKIEWICZ
Radosław KACZMAREK
Agata ZERKA
Katarzyna SZYMCZAK-KULUS
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Bioscientia Sp Z OO
Instytut Immunologii I Terapii Doswiadczalnej Im Ludwika Hirszfelda Polskiej Akademii Nauk
Original Assignee
Bioscientia Sp Z OO
Instytut Immunologii I Terapii Doswiadczalnej Im Ludwika Hirszfelda Polskiej Akademii Nauk
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Application filed by Bioscientia Sp Z OO, Instytut Immunologii I Terapii Doswiadczalnej Im Ludwika Hirszfelda Polskiej Akademii Nauk filed Critical Bioscientia Sp Z OO
Priority to EP20775931.7A priority Critical patent/EP4014044A1/fr
Publication of WO2021028802A1 publication Critical patent/WO2021028802A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus, feline leukaemia virus, human T-cell leukaemia-lymphoma virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Definitions

  • the object of the invention is a strip test for detecting enzootic bovine leukosis by assaying anti-BLV antibodies in a cow's milk sample.
  • the invention also relates to a method for detecting enzootic bovine leukosis with the use of this strip test.
  • Strip tests have been successfully used for many years in both medical and veterinary diagnostics, as well as in agriculture, the food industry and environmental protection. They are characterized by a simple design, high sensitivity and a low price. In veterinary medical practice, tests are used to test both farm animals (cows, poultry, sheep, pigs) and pets, mainly dogs and cats. Tests are used to detect bacteria, viruses, allergic diseases, but also to check, for example, reproductive ability.
  • Enzootic bovine leukosis is an infectious disease in cattle caused by a C-type retrovirus called Bovine Leukemia Virus (BLV), and consisting in lymphatic system cell hyperplasia.
  • BLV Bovine Leukemia Virus
  • the disease is characterized by a long incubation period (up to 7 years) and in most cases (approx. 60%) is asymptomatic, but this is not related to virus latency since some cells have been shown to express the virus (Powers et al., 1992).
  • Some adult cattle (10-30%) develop lymphoceles, 1-10% develop lymphatic sarcomas on various internal organs with a marked increase in the number of B lymphocytes near the sarcoma.
  • the most common symptoms are weight loss, decreased milking, digestive disorders, enlarged lymph nodes, posterior paralysis (Polat et al., 2017).
  • Enzootic bovine leukosis is one of the causes of significant financial losses in dairy herds. Studies show a decrease in the milk yield of BLV-infected cows from 2.5 to 2.7% and a 7% decrease in the number of conceptions (Ott et al., 2003).
  • PL214884 describes an ELISA test for detecting enzootic bovine leukosis characterized in that it contains a highly purified gp51 BLV antigen obtained from a completely bovine serum-free cell culture medium.
  • PL217257 discloses a method for detecting enzootic bovine leukosis and an ELISA detection kit characterized in that the presence of antibodies against antigen BLV proteins, in particular gp51 or p24 antigens, is detected in a sample collected from a tested animal, in particular in its serum or milk.
  • Non-patent literature describes an immunochromatographic strip test using colloidal gold- labelled gp51 antigen and a monoclonal antibody immobilized on a test membrane.
  • the test is based on competitive binding of gp51 antigen to bovine serum antibodies and monoclonal antibodies on the membrane.
  • the test sensitivity is low, i.e. 1:10 as compared to the E05 reference serum (Kim et al. 2016).
  • Chinese application No. CN101303351 describes a strip test for detecting enzootic bovine leukosis in blood serum using gp51 protein and colloidal gold-based immunochromatography, the p51 antigen being expressed in a bacterial system and thus not being a glycoprotein.
  • the object of the invention was to develop a simple and rapid test that could be performed under field conditions even by unskilled individuals, e.g. stock breeders.
  • the object of the invention is a strip test for detecting enzootic bovine leukosis by assaying anti-BLV antibodies in a sample, characterized in that it contains the gp51 antigen obtained in a baculovirus system and immobilized on a nitrocellulose membrane and A, G, A/G proteins labelled with coloured nanoparticles, as well as glass fibre filter paper for filtering a sample, wherein the analysed sample is cow's milk.
  • the glass fibre filter paper is Whatman GF, MF1, AFI934 or Fusion 5 glass fibre filter paper saturated with a solution of 20 mM Tris pH 7.5 with 0.2% casein and 0.1% Tween 20 added.
  • the strip test of this invention is characterized in that it contains the gp51 antigen optionally obtained in a non-baculovirus system.
  • the strip test of this invention is characterized in that the gp51 antigen is obtained in FLK-BLV cells.
  • coloured nanoparticles are colloidal gold nanoparticles with a diameter of 20-50 nm, carbon nanoparticles with a diameter of 10-20 nm, or coloured polystyrene nanoparticles with a diameter of 50-200 nm.
  • a further object of the invention is a method for detecting enzootic bovine leukosis with the use of the strip test of this invention, comprising the following steps: a. A milk sample is diluted three times with 20 mm T ris-HCI buffer pH 7.5 containing 0.3 % casein and 0.15% Tween 20; b. The milk sample is instilled into the window on the test cassette and incubated for 10 minutes; c. The test result is read in the test window, one bar or spot indicating a negative result and two bars or spots indicating a positive result.
  • the advantages of the developed test of this invention include: a) use of a milk sample for assays (with the elimination of the stage of blood sampling and serum separation); b) prevention of the spread of infections; c) minimising of economic losses; d) monitoring of milk purity for BLV infection.
  • the test is characterized by high sensitivity and specificity and ensures good compatibility of the results with commercial ELISA tests.
  • the test gives a negative result with sera positive for other bovine viruses: BIV, BVD and BVH.
  • the object of the invention is illustrated in a figure showing a photograph of the developed strip test and its results with standard anti-BLV E05 serum diluted in normal milk.
  • Mimic Sf9 cells (Life Technologies, USA) were infected with a baculovirus with a subcloned gene encoding the gp51 protein.
  • the virus titre was determined by a serial dilution method.
  • the baculovirus construct was expressed in 1000 ml in a serum-free medium (CCM3, Becton-Dickinson or ESF921, Expression Systems). 48 hours following the infection with the baculovirus, the cells were centrifuged and the post-culture medium was dialysed for several dozen hours against phosphate buffer. The preparation was applied to an NiNTA-agarose bed and the bound protein was eluted with a step gradient of imidazole.
  • the analysis of collected fractions was performed by dot-blot methods with the 9E10 antibody.
  • the fractions with the highest gp51 concentration were combined (fractions eluted with 50, 100 and 200 mM imidazole) and dialysed against TBS buffer.
  • the resulting preparations were then concentrated using Amicon cartridges (Merck Millipore, Germany).
  • the purification degree of the preparations was tested by SDS-PAGE electrophoresis and CBB staining.
  • the preparations eluted with 100 and 200 mM imidazole were characterized by the highest purification degree.
  • the labelled A/G protein was applied to M illipore’s GFDX Glass Fiber Conjugate Pads glass fibre paper.
  • a solution of gp51 antigen (test line) and bovine immunoglobulins (control line) were applied, as a line or spot, to Millipore's HF120 or HF135 nitrocellulose membrane.
  • the paper with the coloured conjugate was glued at the start of the nitrocellulose paper, while the glass fibre paper for applying the sample was glued to the conjugate.
  • plain cellulose paper was placed.
  • the test strip was placed in a special plastic cassette.
  • a milk sample was diluted three times with 20 mM Tris-HCI buffer pH 7.5 containing 0.3 % casein and 0.15% Tween 20. 200 pi of the sample was instilled into the window on the test cassette and left for 10 minutes. The appearance of one bar or spot in the test window indicates a negative result, while the appearance of two bars or spots indicates a positive result.
  • the tests developed should become a valuable diagnostic tool for monitoring the health status of cattle directly in the field and can contribute to improving the efficiency of cow breeding economy.
  • the test may also be used to monitor the purity of milk and meat.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Urology & Nephrology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Virology (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'objet de l'invention est un test de bandelette pour la détection de la leucose bovine enzootique par dosage d'anticorps anti-BLV dans un échantillon, caractérisé en ce qu'il contient l'antigène gp51 obtenu dans un système de baculovirus et immobilisé sur une membrane de nitrocellulose et des protéines A, G, A/G marquées avec des nanoparticules colorées, ainsi que du papier filtre en fibres de verre pour filtrer un échantillon, l'échantillon analysé étant du lait de vache. Un autre objet de l'invention est un procédé de détection de la leucose bovine enzootique à l'aide du test de bandelette selon l'invention, comprenant les étapes suivantes : a. un échantillon de lait est dilué trois fois avec un tampon Tris-HCI de 20 mm à un pH de 7,5 contenant 0,3 % de caséine et 0,15 % de Tween 20 ; b. l'échantillon de lait est introduit dans la fenêtre sur la cassette de test et incubé pendant 10 minutes ; c. un résultat de test est lu dans la fenêtre de test, une barre ou un point indiquant un résultat négatif et deux barres ou points indiquant un résultat positif.
PCT/IB2020/057467 2019-08-12 2020-08-07 Test de bandelette pour la détection de la leucose bovine enzootique et procédé de détection de la leucose bovine enzootique à l'aide du test de bandelette Ceased WO2021028802A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP20775931.7A EP4014044A1 (fr) 2019-08-12 2020-08-07 Test de bandelette pour la détection de la leucose bovine enzootique et procédé de détection de la leucose bovine enzootique à l'aide du test de bandelette

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
PLP.430863 2019-08-12
PL430863A PL239791B1 (pl) 2019-08-12 2019-08-12 Test paskowy do wykrywania enzootycznej białaczki bydła i sposób wykrywania enzootycznej białaczki bydła z wykorzystaniem tego testu paskowego

Publications (1)

Publication Number Publication Date
WO2021028802A1 true WO2021028802A1 (fr) 2021-02-18

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PCT/IB2020/057467 Ceased WO2021028802A1 (fr) 2019-08-12 2020-08-07 Test de bandelette pour la détection de la leucose bovine enzootique et procédé de détection de la leucose bovine enzootique à l'aide du test de bandelette

Country Status (3)

Country Link
EP (1) EP4014044A1 (fr)
PL (1) PL239791B1 (fr)
WO (1) WO2021028802A1 (fr)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BE896752A (fr) * 1983-05-17 1983-09-16 Region Wallone Representee Pat Procede et chaine reactionnelle pour la diagnostic de la leucose bovine enzootique.
DD212389A3 (de) * 1982-05-14 1984-08-08 Univ Berlin Humboldt Verfahren der gp51-antigenherstellung zur diagnose der rinderleukose
JPH01123152A (ja) * 1987-11-07 1989-05-16 Mitsui Toatsu Chem Inc 牛白血病ウイルス由来の診断薬を用いた診断方法
EP1933144A1 (fr) * 2006-12-11 2008-06-18 The Jordanian Pharmaceutical Manufacturing Co. Détection rapide immunochromatographique par amplification du signal d'or colloïdal
EP1933146A1 (fr) * 2006-12-11 2008-06-18 The Jordanian Pharmaceutical Manufacturing Co. Détection rapide immunochromatographique par amplification du signal d'or colloïdal
PL217257B1 (pl) * 2011-04-29 2014-06-30 Inst Immunologii I Terapii Doświadczalnej Pan Sposób i zestaw do wykrywania enzootycznej białaczki bydła
CN108896765A (zh) * 2018-07-04 2018-11-27 山东农业大学 一种检测a亚群禽白血病病毒的胶体金试纸条及其制备方法

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DD212389A3 (de) * 1982-05-14 1984-08-08 Univ Berlin Humboldt Verfahren der gp51-antigenherstellung zur diagnose der rinderleukose
BE896752A (fr) * 1983-05-17 1983-09-16 Region Wallone Representee Pat Procede et chaine reactionnelle pour la diagnostic de la leucose bovine enzootique.
JPH01123152A (ja) * 1987-11-07 1989-05-16 Mitsui Toatsu Chem Inc 牛白血病ウイルス由来の診断薬を用いた診断方法
EP1933144A1 (fr) * 2006-12-11 2008-06-18 The Jordanian Pharmaceutical Manufacturing Co. Détection rapide immunochromatographique par amplification du signal d'or colloïdal
EP1933146A1 (fr) * 2006-12-11 2008-06-18 The Jordanian Pharmaceutical Manufacturing Co. Détection rapide immunochromatographique par amplification du signal d'or colloïdal
PL217257B1 (pl) * 2011-04-29 2014-06-30 Inst Immunologii I Terapii Doświadczalnej Pan Sposób i zestaw do wykrywania enzootycznej białaczki bydła
CN108896765A (zh) * 2018-07-04 2018-11-27 山东农业大学 一种检测a亚群禽白血病病毒的胶体金试纸条及其制备方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HUANG SHIWEN ET AL: "Nanotechnology in agriculture, livestock, and aquaculture in China. A review", AGRONOMY FOR SUSTAINABLE DEVELOPMENT, SPRINGER PARIS, PARIS, vol. 35, no. 2, 31 December 2014 (2014-12-31), pages 369 - 400, XP035476163, ISSN: 1774-0746, [retrieved on 20141231], DOI: 10.1007/S13593-014-0274-X *
MERZA M ET AL: "Immunoaffenity purification of two major proteins of bovine leukemia virus (gp51 and p24) and their use for discrimination between vaccinated and infected animals", JOURNAL OF VIROLOGICAL METHODS, ELSEVIER BV, NL, vol. 33, no. 3, 1 August 1991 (1991-08-01), pages 345 - 353, XP023794780, ISSN: 0166-0934, [retrieved on 19910801], DOI: 10.1016/0166-0934(91)90034-W *
MUKANTAYEV ET AL: "Immunochromatographic assay for diagnosis of bovine leukemia virus infection in cows usin g the recombinant protein gp51", VETERINARIJA IR ZOOTECHNIKA 2018 LIETUVOS VETERINARIJOS AKADEMIJA LTU, vol. 76, no. 98, 1 January 2018 (2018-01-01), pages 34 - 40, XP002800850, ISSN: 1392-2130 *

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Publication number Publication date
PL430863A1 (pl) 2021-02-22
PL239791B1 (pl) 2022-01-10
EP4014044A1 (fr) 2022-06-22

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