EP0954614A1 - Methode et composition pour diagnostiquer l'anemie infectieuse equine virale au moyen de la proteine recombinante (p26) de la capside virale - Google Patents

Methode et composition pour diagnostiquer l'anemie infectieuse equine virale au moyen de la proteine recombinante (p26) de la capside virale

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Publication number
EP0954614A1
EP0954614A1 EP97953596A EP97953596A EP0954614A1 EP 0954614 A1 EP0954614 A1 EP 0954614A1 EP 97953596 A EP97953596 A EP 97953596A EP 97953596 A EP97953596 A EP 97953596A EP 0954614 A1 EP0954614 A1 EP 0954614A1
Authority
EP
European Patent Office
Prior art keywords
antigen
equine
virus
enzyme
recombinant
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP97953596A
Other languages
German (de)
English (en)
Inventor
P. César Apart.201 PEREGRINO FERREIRA
Erna Geessien Kroon
Jenner Karlisson Pimenta Dos Reis
Isabella Bias Fortes Ferraz
Rômulo CERQUEIRA LEITE
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universidade Federal de Minas Gerais
Original Assignee
Universidade Federal de Minas Gerais
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universidade Federal de Minas Gerais filed Critical Universidade Federal de Minas Gerais
Publication of EP0954614A1 publication Critical patent/EP0954614A1/fr
Withdrawn legal-status Critical Current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
    • G01N33/56983—Viruses

Definitions

  • the present invention relates to a method of detecting antibodies against core antigen of equine infectious anemia virus (EIAV), using as antigen the non glycosilated recombinant protein (rgp26) in immunoenzymatic assays. More particularly, it relates to the use of recombinant protein p26 in kits for diagnosis of equine infectious anemia (EIA).
  • EIAV equine infectious anemia virus
  • EIA equine infectious anemia
  • LIGNEE Rec. Med. Vet, 20:30, 1843
  • VALLEE and CARRE Acad. Sci. ,139:331-333,1904
  • the disease affects exclusively the members of the family Equidae presenting a worldwide distribution and of great economical importance consequently .
  • the EIA virus (EIAV) is classified as a lentivirus of the Retroviridae family (CHARMAN et al. J. Virol. 19(2): 1073 -1076,1976), it is genetic and antigenically related to the other lentiviruses which are characterized by developing persistent infection in host.
  • the EIA has played a specially important role in comparative virology and in the studies of the acquired immunodeficiency syndrome (AIDS). Besides their morphological identity, both viruses are similar in terms of nucleotide sequences that code for structural surface proteins. These group of virus present genetic and antigenic variants during persistent infections, which is associated to immune response scape (MONTAGNIER et al. Ann.
  • EIAV The transmission of EIAV occurs mainly through bite of arthropods vectors (tabanideos) which inoculate the virus into the animal's blood stream (mechanical transmission) when feeding themselves.
  • the way of transmition is responsible for the high prevalence of EIA in areas favorable to the life cycle of vectors (ISSEL et al. Vet.. 17:251-286, 1988).
  • the EIAV can also be transmitted by the placenta and colostra of mares with high virus levels, and by needles and surgical instruments contaminated with blood (COGGINS Comparative diagnosis of viral diseases.NY, 4:646-658, 1981 ).
  • the laboratory diagnosis plays a decisive role in the control and prevention of EIA if considering the high prevalence of assymptomatic carriers, non conclusive and possibility to confuse clinical diagnosis with other diseases as the trypanosomiasis, pyroplasmosis, leptospirosis, hepatitis and by parasites .
  • the diagnose of EIAV has been done through the detection of specific antibodies against surface antigen of virus present in the serum of affected animals by using the Coggins or agar gel diffusion test (U.S.Pat, nro.3,929,982 and U.S.Pat. No. 3,932,601 ).
  • the Coggins test the antigen and sample serum is placed side by side in an agarose gel plate. If EIA antibodies are present in the test serum, they will form a precipitin line when diffusing toward the agarose gel .
  • Porter discloses a method for detecting the EIA virus using a competitite enzyme-linked immunoabsorbent assay incorporating a purified viral antigen and a monoclonal antibody.
  • the EIAV must first be cultured.
  • the antigen used was p26 capsid protein of the EIAV and is obtained through (purification of the cultured virus by a variety of means) well known in the art.
  • the technique of virus tissue cultures increases the possibility of assay yield false positive results since the virus may be contaminated with other forms of protein or even another virus. Additionally, the EIAV is hard to culture, making Porter's approach very difficult for large scale production.
  • Figure 1 shows schematically the method of diagnosis
  • Figure 2 shows the titration of positive and negative sera in Elisa with the recombinant protein rgp26 as antigen.
  • Figure 3 demonstrates the distribution of the optical density (OD) in
  • an object of the present invention to provide a method of immunodiagnosis for EIA disease that uses the recombinant protein p26 corresponding derived from viral envelope of EIAV.
  • the method consists of binding the recombinant antigen to solid supports (microtiter plates, tubes, beads or nitrocellularlose or nylon papers or any kind that allow protein binding) and to proceed the analysis of the sera (presence of antibodies) from animals suspected of infection with the EIAV.
  • the recombinant protein p26 is added to a solid phase support and incubated for sufficient time to ensure that protein was bound to the support.
  • the equine test sample is added to the support and incubated for a period of time sufficient to permit that any EIA-antibodies are removed from sample.
  • Labeled conjugate is added which binds to the protein-antibody complex. Following enough time to allow such binding, any unbound labeled conjugate is removed by washing. Labeled conjugate is added wich binds to the protein-antibody complex. Following enough time to allow such binding, any unbound labeled conjugate is removed by washing. High level of bound conjugate indicates a positive result, which mean presence of EIA viral antibodies. A low level of bound conjugate indicates a negative result which mean ausence or undetectable level of EIA viral antibodies..
  • a variety of commercially available solid phase supports may be used for protein binding.
  • the direct binding of equine antibodies present in the test serum to the solid phase support is likely to result in a false positive reading.
  • the blocking solution is used to fill any empty binding sites on the support which did not bind antibodie-protein. Any substance which will not react with EIA viral antibodies and antigen will function as a blocker.
  • a conjugate is something which will recognize and bind with the test serum EIA viral antibody.
  • the conjugate may be labeled using a variety of labeling means, including but not limited to: enzyme labeling, fluorescent labeling, and magnetic labeling. If enzymatic labeling is the labeling means chosen, the conjugate is labeled with an enzyme preferably select from the group consisting of horseradish peroxidase and alkaline phosphatase. Other enzymes may be used.
  • an enzyme label is used, the labeled conjugate is detected by adding an amount of a substrate which will recognize and react with the enzyme label to form a product that will produce a color change visible to the naked eye. The presence of color indicates a sufficient level of test serum antibodies to indicate infection. An absence of color is an indicator of a lack of infection, as the animal did not produce a significant number of antibodies to the virus.
  • the labeled conjugate had few antibodies, if any, to bind with and was subsequently removed from the support.
  • peroxidase and phosphatase substrates which will react with horseradish peroxidase and alkaline phosphatase enzymes, respectively to form a colored product.
  • a preferred peroxidase substrate is an ortho- phenylenediamine/hydrogen peroxide solution.
  • the intensity of the color of the product may be quantified using a spectrophotometer to read absorvance. Howewer, measuring the absorvance is not necessary to obtain an accurate reading of the results of the assay.
  • Figure 2 shows the detection of antibodies anti-p26 in the ELISA test using dilutions of the serum from 1 :4 to 1 :256 and obtaining from 0.800 to 0.400 OD.
  • the negative controls demonstrate that there is a non specific reaction.
  • the support was washed for 3 to 6 times with buffer solution (0.01-0.02 M NaH 2 P04 , 0.01-0.02 MNa 2 HPO4 , 0.02-0.04M KCI, 0.85-0.9% NaCI pH 7.0-7.5),and then with 0.05-0.1% of tween-20 (Buffer- Tween).
  • buffer solution 0.01-0.02 M NaH 2 P04 , 0.01-0.02 MNa 2 HPO4 , 0.02-0.04M KCI, 0.85-0.9% NaCI pH 7.0-7.5
  • Buffer- Tween Buffer- Tween
  • the positive and negative control and the serum samples were diluted in Tween buffer, to bound to the antigen linked to the solid support (3), and incubated at 23°C-37°C.
  • the conjugate was added, where the anti- equine immunoglobuline binds to the antibodies that are tied up to the antigens (4).
  • Conjugate can be an equine anti- immunoglobuline conjugated to the enzyme peroxidase or any other enzyme as acetylcolinesterase, lactate desidrogenase, galactosidase, glicose oxidase, alkaline fosfatase, or another.
  • This conjugate was diluted in Tween buffer in agreement with its title and added to the support and then incubated at 23°C- 37°C for 30-60 min. A new wash of the support with Tween buffer and the development of the reaction was proceeded (5) with the enzyme of the conjugate, transforms the substrate of colorless to a red-faced product.
  • the developing solution is composed of the substrate of the enzyme used in the conjugate that for the peroxidase for example is the ortofenilenodiamino diluted in phosphate or citrate buffer 0.1-0.2 M, pH 5.0-8.0.
  • solution of acid was used (sulfuric acid) for stop-reaction (6), where the acid interrupts the previous reaction.
  • the measurement(7) of the color intensity formed in each reaction (sample) was made. This reading was made visually or in espectrophotometer, in absorbance, with a specific filter for the color formed by the developing solution.

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

Abstract

L'invention concerne une méthode et une trousse qui permettent de détecter des anticorps dans des échantillons cliniques d'animaux infectés par le virus de l'anémie infectieuse équine au moyen d'un immunodiagnostic avec l'antigène viral recombinant p26. L'antigène a été fixé sur des supports solides (plaques de microtitrage, tubes, billes, feuilles de nitrocellulose ou de nylon) et mis à réagir avec le sérum à tester. Après incubation avec un conjugué immunoglobuline anti-équidé/enzyme, la réaction a été révélée avec une solution composée du substrat de l'enzyme utilisée dans le conjugué (chromogène). Après développement de la réaction (apparition de couleur), celle-ci a été arrêtée avec une solution acide, puis mesurée. Le dosage immunologique peut être un dosage immunologique direct utilisant un second anticorps, ou un dosage immunologique en sandwich à deux étapes.
EP97953596A 1996-12-18 1997-12-19 Methode et composition pour diagnostiquer l'anemie infectieuse equine virale au moyen de la proteine recombinante (p26) de la capside virale Withdrawn EP0954614A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
BR9606273A BR9606273A (pt) 1996-12-18 1996-12-18 Processo para o teste imunoenzimático com proteína P26 recombinante do capsídio viral no diagnóstico da anemia infecciosa equina
BR9606273 1996-12-18
PCT/BR1997/000081 WO1998027231A1 (fr) 1996-12-18 1997-12-19 Methode et composition pour diagnostiquer l'anemie infectieuse equine virale au moyen de la proteine recombinante (p26) de la capside virale

Publications (1)

Publication Number Publication Date
EP0954614A1 true EP0954614A1 (fr) 1999-11-10

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EP97953596A Withdrawn EP0954614A1 (fr) 1996-12-18 1997-12-19 Methode et composition pour diagnostiquer l'anemie infectieuse equine virale au moyen de la proteine recombinante (p26) de la capside virale
EP97953598A Expired - Lifetime EP0951646B1 (fr) 1996-12-18 1997-12-19 DOSAGE IMMUNO-ENZYMATIQUE POUR DIAGNOSTIQUER L'ANEMIE INFECTIEUSE EQUINE AU MOYEN DE LA PROTEINE RECOMBINANTE (gp90) DERIVEE DU VIRUS DE L'ANEMIE INFECTIEUSE EQUINE

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EP97953598A Expired - Lifetime EP0951646B1 (fr) 1996-12-18 1997-12-19 DOSAGE IMMUNO-ENZYMATIQUE POUR DIAGNOSTIQUER L'ANEMIE INFECTIEUSE EQUINE AU MOYEN DE LA PROTEINE RECOMBINANTE (gp90) DERIVEE DU VIRUS DE L'ANEMIE INFECTIEUSE EQUINE

Country Status (4)

Country Link
EP (2) EP0954614A1 (fr)
AU (1) AU736224B2 (fr)
BR (1) BR9606273A (fr)
WO (1) WO1998027231A1 (fr)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6670142B2 (en) 2001-10-26 2003-12-30 The Regents Of The University Of California Method for screening combinatorial bead library, capturing cells from body fluids, and ligands for cancer cells
US7262269B2 (en) 2001-10-26 2007-08-28 The Regents Of University Of California Method for screening combinational bead library; ligands for cancer cells
CN104020294B (zh) * 2014-05-30 2016-03-30 中国农业科学院哈尔滨兽医研究所 用于检测马传染性贫血病毒p26蛋白的试剂盒及其用途

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3932601A (en) * 1971-04-07 1976-01-13 Cornell Research Foundation, Inc. Method and composition for the diagnosis of equine infectious anemia virus infection by using agar-gel-immunodiffusion reaction
US4806467A (en) * 1985-10-21 1989-02-21 Fermenta Animal Health Company Method for the detection of equine infectious anemia and other retrovirus infections using a competitive enzyme-linked immunoabsorbent assay and reagents useful in the same
US5427907A (en) * 1990-02-23 1995-06-27 Virginia Commonwealth University Assay for equine infectious anemia virus

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9827231A1 *

Also Published As

Publication number Publication date
AU5743198A (en) 1998-07-15
BR9606273A (pt) 1998-12-15
AU736224B2 (en) 2001-07-26
WO1998027231A1 (fr) 1998-06-25
EP0951646B1 (fr) 2004-06-23
EP0951646A1 (fr) 1999-10-27

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