WO1999041610A1 - Diagnostic serologique de la maladie de chagas - Google Patents

Diagnostic serologique de la maladie de chagas Download PDF

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Publication number
WO1999041610A1
WO1999041610A1 PCT/BR1998/000006 BR9800006W WO9941610A1 WO 1999041610 A1 WO1999041610 A1 WO 1999041610A1 BR 9800006 W BR9800006 W BR 9800006W WO 9941610 A1 WO9941610 A1 WO 9941610A1
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WO
WIPO (PCT)
Prior art keywords
buffer
plates
pbs
test
epex
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Application number
PCT/BR1998/000006
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English (en)
Inventor
Luiz Rodolpho Raja Gabaglia Travassos
Igor Correia Almeida
Dimas Tadeu Covas
Original Assignee
Fundação Hemocentro de Ribeirão Preto
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.)
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Application filed by Fundação Hemocentro de Ribeirão Preto filed Critical Fundação Hemocentro de Ribeirão Preto
Priority to PCT/BR1998/000006 priority Critical patent/WO1999041610A1/fr
Priority to CA002309705A priority patent/CA2309705A1/fr
Priority to US09/554,312 priority patent/US6682900B1/en
Publication of WO1999041610A1 publication Critical patent/WO1999041610A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • 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
    • G01N33/56905—Protozoa
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Chagas 'disease is characterized by a short-term acute phase, with very few clinical symptoms, and a long-term chronic phase, usually accompanied by severe gastrointestinal and/or cardie complications which result in permanent physical disability or death.
  • Chagas 'disease is an endemic disease caused by the flagellate Trypanosoma cruzi. In Latin America, approximately 16 to 18 miHion individuals are already infected and as many as 90 million individuals are at risk of infection (W.H.O., 1991). The disease is transmitted in Nature by Triatominae vectors. As a result of effective public health measures for the control of the vector in most countries, blood transfusion is quantitatively the most important form of transmission of the disease today. In Latin America, blood samples with antibodies associated with Chagas 'disease represent 1-4% of the total blood samples in major Hemocenters. More recently, Chagas 'disease has also become a major public health concern in North America, owing to the increasing number of immigrants from Latin American countries, in the last decade. Recent studies estimate that there may be in the United States approximately 100,000 Trypanosoma crttzz-infected individuals with potential risk of transmitting Chagas 'disease by blood transfusion (Hagar and Rahimtoola, 1991).
  • PCR hemmagglutination
  • IIF indirect immunofluorescence
  • ELISA immunosorbent assay
  • WHO World Health Organization
  • at least two positive tests of the three cited above are necessary for the diagnosis of the disease. Blood samples that are positive to only one of the three tests are classified as "indeterminate or inconclusive" and, in consequence, discarded.
  • the indeterminate diagnosis is associated with 20 to 90% of all blood samples that gave one or more positive tests for Chagas 'disease, depending on the methods employed and how they are applied. This high percentage of indeterminate results represents a serious problem in blood banks, both in terms of volume of discarded blood and doubtful diagnosis of Chagas 'disease.
  • a blood sample with a false positive test is no longer used for transfusion or isolation of cells and other blood components.
  • Such loss of donated blood also affects the production of blood derivatives such as albumin, immunoglobulins and clotting factors which are of commercial value.
  • a blood sample with a false negative test is a dangerous source of contamination by the parasite.
  • the invention describes the purification of the A&T and EpEx antigens, and their use in a chemiluminescent enzyme-linked immunosorbent assay (CL-
  • A&T antigen is purified from trypomastigote forms of Trypanosoma cruzi according to Almeida et al, 1993 and Almeida et al., 19 4a .
  • Trypomastigote forms are obtained from infected green monkey kidney fibroblasts (LLC-MK 2 cells) cultured in Dulbecco's modified Eagle medium (D-MEM) containing 10% fetal bovine serum.
  • D-MEM Dulbecco's modified Eagle medium
  • the cell-derived trypomastigotes are collected 6-7 days later, following their release from infected cells, from the top fluid after sedimentation of the cell debris and incubation for 1.5 h at 37°C.
  • Parasites are washed 3 times in 0.15 M phosphate-buffered saline (PBS), pH 7.4, centrifuged at 12,000g, and kept at -70°C until lyophilization. Lyophilized trypomastigotes are sequentially extracted 5 times with 10 volumes of chloroform/methanol (2:1), chloroform/methanol (1:2), chloroform/methanol/water (10:20:8), for 30 min each time, at room temperature. After centrifugation at 12,000g, the organic extracts are discarded and the final delipidated pellet is dried under a stream of nitrogen. The dry pellet is then extracted 5 times with 10 volumes of 9% 1-butanol for 2 h each time, at room temperature.
  • PBS phosphate-buffered saline
  • the soluble extract corresponds to the fraction containing at A&T antigen together with some hydrophilic and hydrophobic contaminants.
  • the A&T-containing fraction is then lyophilized for 24 h and chromatographed on a column of octyl-Sepharose (Pharmacia-LKB, Upsala, Sweden), pre-equilibrated with 5% 1-propanol in 0J M ammonium acetate buffer, pH 7.2.
  • the A&T-containing fraction dissolved in 5% 1-propanol in 0J M ammonium acetate buffer, pH 7.2 is applied to the column at a low flow rate.
  • the colum is washed with 5% 1-propanol in 0J M ammoniun acetate buffer, pH 7.2 and eluted with a 1-propanol gradient (5-60%).
  • the A&T antigen are tested for immunoreactivity with a specific polyclonal antibody generated against the A&T antigen (anti-A&T antibody).
  • the A&T- positive fractions from the octyl-Sepharose column are pooled, dried and partitioned between water and 1-butanol.
  • the aqueous phase is lyophilized for 24 h, resuspended in 5% 1-propanol in ammonium acetate OJ M, pH 7.2 and applied to the phenyl-Superose column (Pharmacia-LKB, Sweden) (pre-equilibrated with 5% 1-propanol in ammonium acetate OJ M, pH 7.2).
  • the column is eluted with a 1-propanol gradient (5-60%). Material eluting in earlier fractions (column void) and containing the A&T antigen is pooled and lyophilized for 24 h.
  • the material included in the column is basically constituted of hydrophobic contaminants, mainly phospholipids.
  • A&T antigenic preparation is re-applied to a column of octyl- Sepharose (Pharmacia-LKB, Sweden), pre-equilibrated with 5% 1-propanol in OJM ammonium acetate buffer, pH 7.2.
  • the A&T-containing fraction dissolved in 5% 1-propanol in OJ M ammonium acetate buffer, pH 7.2 is applied to the column at a low flow rate.
  • the column is washed with 5% 1-propanol in OJ M ammonium acetate buffer, pH 7.2 and eluted with a 1-propanol gradient (5-70%) and eluted with a shallow 1-propanol gradient (20-40%).
  • the fractions are assayed for immunoreactivity with the anti-A&T antibody by dot-blotting and Western-blotting.
  • Antibody binding fractions are pooled, exhaustively dialyzed against deionized water, lyophilized for 48 h, redissolved in deionized water and stored at -70°C. Purification of the EpEx antigen
  • EpEx antigen is prepared from epimastigote forms of Trypanosoma cruzi, Tulahuen strain. Parasites are cultured at 28°C, in Schneider's insect medium containing 20% fetal calf serum. After 7-10 days, the parasites are collected from the culture supernatant, washed three times with 100 mM phosphate-buffered saline, pH 7.4 and centrifuged at 12,000g for 30 min, at 4°C. Pelleted parasites are immediately resuspended in 10 mM Tris-HCI buffer, pH 7.5, 0.2 mM
  • Chemiluminescent ELISA is carried out according to protocols previously described (Almeida et al, 1993, 1994b).
  • A&T at 0J5 ⁇ g dry weight/ ⁇ l deionized water
  • EpEx at 0J5 ⁇ g protein/ ⁇ l of lysis buffer
  • antigens are diluted in 50 mM sodium carbonate buffer, pH 9.6, for a final concentration of 0.2 ng/ ⁇ l and 0.8 ng/ ⁇ l, respectively.
  • Fifty microliters of each antigen are separately added to each well of milky- white 96- well Maxisorp FluoroNunc plates (Nunc, Denmark).
  • Plates are washed 5 times with PBS-T, the excess liquid removed by inversion or filter paper, and then incubated with biotinylated goat anti-human IgG (Amersham, UK), diluted 1:2,000 with PBS-TB, for 30 min at 37°C. After washing 5 times with PBS-T, a streptavidin-horseradish peroxidase conjugate (Amersham, UK) diluted 1: 1,000 with PBS-TB is added, following incubation for 30 min at 37°C.
  • the luminometer reading of a serum sample is divided by the predeterminated cutoff value.
  • a positive result is defined when the relative serum reading (RLU) is greater than 1, which represents the cutoff value.
  • RLU relative serum reading
  • a negative sample has an RLU equal or lower than 1.
  • A&T-CL-ELISA 2 1 2,000 74 0 26 0 0
  • the A&T antigen is a purified preparation of closely related molecules thar are specific of the trypomastigote stage obtained in tissue culture of mammalian
  • the A&T antigen is easily obtainable in amounts sufficient for a great number of tests in appropriate ELISA plates for chemiluminescent reading. Moreover, the purified A&T antigen is highly stable when fixed on plates for prolonged periods. 5. Since A&T antigen reacts with lytic (protective) antibodies, characteristic of active infection and present in high titers in chronic patient sera, it can be used to monitor the response of patients to chemotherapy (Fig. 2).
  • EpEx complex antigen is prepared from the epimastigote form and contains many components that are also expressed in the infective stage. It reacts with antibodies that are recognized by conventional serology for Chagas 'disease, but not with those antibodies whose reactions are due to artifacts such as blocking reagents, culture medium supplements, etc.
  • EpEx antigen is readily prepared from fast growing epimastigote culture, and although it is not as specific as A&T purified antigen, it is highly sensitive and provide complementary and confirmatory data for the positive reactions obtained with A&T antigen (Fig. 1).
  • Chemiluminescent immunoassays discrimination between the reactivities of natural and human patient antibodies with antigens from eukaryotic pathogens, Trypanosoma cruzi and Paracocidioides brasiliensis. J. Clin. Lab. Anal. 8: 424-431.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
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  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
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  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Urology & Nephrology (AREA)
  • Food Science & Technology (AREA)
  • Toxicology (AREA)
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  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Virology (AREA)
  • General Physics & Mathematics (AREA)
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  • Microbiology (AREA)
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  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
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Abstract

L'invention concerne un procédé par chimiluminescence (CL)-ELISA avec des antigènes purifiés et complexes de Trypanosoma cruzi permettant de diagnostiquer de manière spécifique et sensible la maladie de Chagas chez des patients et dans des échantillons sanguins. Les préparations utilisées sont un antigène spécifique trypomastigote (A & T) et un extrait d'épimastigote (EpEx) permettant de réguler la sensibilité. La forte sensibilité du procédé CL-ELISA permet une utilisation de quantité extrêmement petite d'antigène et une dilution de sérum dans des tests de routine de 1:2000, réduisant au minimum ainsi les réactions non spécifiques ou faux-positifs. L'utilisation de l'antigène purifié A & T élimine les réactivités croisées avec d'autres agents d'infection, détecte l'infection active et permet de surveiller la chimiothérapie chez les patients chroniques. L'utilisation de la préparation antigène EpEx ne confirme pas seulement les résultats positifs avec A & T mais suggère, lors de divergences, d'autres infections telles que la leishmaniose. Comparé à d'autres tests courants utilisés dans les banques de sang principales, le procédé CL-ELISA avec les antigènes A & T et EpEx, testé en parallèle, s'est avéré nettement plus efficace en éliminant des résultats indéterminés ou en augmentant les statistiques d'échantillons positifs diagnostiqués.
PCT/BR1998/000006 1996-08-02 1998-02-16 Diagnostic serologique de la maladie de chagas WO1999041610A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
PCT/BR1998/000006 WO1999041610A1 (fr) 1998-02-16 1998-02-16 Diagnostic serologique de la maladie de chagas
CA002309705A CA2309705A1 (fr) 1998-02-16 1998-02-16 Diagnostic serologique de la maladie de chagas
US09/554,312 US6682900B1 (en) 1996-08-02 1998-02-16 Serological diagnosis of Chagas' disease

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/BR1998/000006 WO1999041610A1 (fr) 1998-02-16 1998-02-16 Diagnostic serologique de la maladie de chagas

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005055812A3 (fr) * 2003-12-05 2005-09-15 Ciphergen Biosystems Inc Biomarqueurs seriques de la maladie de chagas
CN105548565A (zh) * 2015-12-30 2016-05-04 深圳市新产业生物医学工程股份有限公司 一种用于检测克氏锥虫抗体的试剂盒及其制备和应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BIOLOGICAL ABSTRACTS, vol. 97, Philadelphia, PA, US; abstract no. 434414, XP002083817 *
I.C. ALMEIDA ET AL.: "A highly sensitive and specific chemiluminescent enzyme-linked immunosorbent assay for the diagnosis of active Trypanosoma cruzi infection", TRANSFUSION, vol. 37, no. 8, 1 August 1997 (1997-08-01), Bethesda MD USA, pages 850 - 857 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005055812A3 (fr) * 2003-12-05 2005-09-15 Ciphergen Biosystems Inc Biomarqueurs seriques de la maladie de chagas
US8043825B2 (en) 2003-12-05 2011-10-25 Mcgill University Serum biomarkers for Chagas disease
CN105548565A (zh) * 2015-12-30 2016-05-04 深圳市新产业生物医学工程股份有限公司 一种用于检测克氏锥虫抗体的试剂盒及其制备和应用

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