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WO2017109763A1 - Peptides sythétiques, méthode et trousse pour le diagnostic de la leishmaniose muqueuse humaine, et utilisation - Google Patents

Peptides sythétiques, méthode et trousse pour le diagnostic de la leishmaniose muqueuse humaine, et utilisation Download PDF

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Publication number
WO2017109763A1
WO2017109763A1 PCT/IB2016/057976 IB2016057976W WO2017109763A1 WO 2017109763 A1 WO2017109763 A1 WO 2017109763A1 IB 2016057976 W IB2016057976 W IB 2016057976W WO 2017109763 A1 WO2017109763 A1 WO 2017109763A1
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Prior art keywords
diagnosis
leishmaniasis
peptides
human
protein
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English (en)
Portuguese (pt)
Inventor
Eduardo ANTONIO FERRAZ COELHO
Carlos ALBERTO PEREIRA TAVARES
Lourena EMANUELE COSTA
Denise UTSCH GONÇALVES
Daniel Menezes SOUZA
Beatriz CRISTINA SILVEIRA SALLES
Luiz Ricardo Goulart Filho
Emilia REZENDE VAZ
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Universidade Federal de Minas Gerais
Universidade Federal de Uberlandia UFU
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Universidade Federal de Minas Gerais
Universidade Federal de Uberlandia UFU
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Priority claimed from BR102015032494-4A external-priority patent/BR102015032494B1/pt
Application filed by Universidade Federal de Minas Gerais, Universidade Federal de Uberlandia UFU filed Critical Universidade Federal de Minas Gerais
Publication of WO2017109763A1 publication Critical patent/WO2017109763A1/fr
Anticipated expiration legal-status Critical
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    • 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
    • A61K39/002—Protozoa antigens
    • A61K39/008—Leishmania antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • 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

  • the present technology addresses 6 (six) highly specific and reactive synthetic peptides with samples from patients with human mucosal leishmaniasis, as well as a method and kit for the diagnosis of symptomatic and asymptomatic mucosal leishmaniasis, which use the 6 synthetic peptides, isolated or associated; or bacteriophage clones containing such peptides, isolated or associated.
  • Leishmaniasis are neglected diseases caused by protozoan parasites of the genus Leishmania, belonging to the Trypanosomatidae family and order Kinetoplastida. The disease is found in 98 countries worldwide and an estimated 12 million people are clinically affected, with an annual incidence of 700,000 to 1,200,000 cases of cutaneous leishmaniasis (LT) (DESJEUX, P. worldwide Trans R Soe Trop Med Hyg, v. 95, no. 3, pp. 239-43, May-Jun 2001 A. ISSN 0035-9203 Leishmaniasis: current situation and new perspectives Comp Immunol Microbiol Infect Dis, v. 27, No. 5, pp. 305-18, 2004. ALVAR, J.
  • L (V) braziliensis and L. (L.) amazonensis are the most pathogenic to man (SILVEIRA, FT; LAINSON, R .; CORBETT, CE. Clinical and immunopathological spectrum of American cutaneous leishmaniasis with special reference to the disease in Amazonian. Brazil: the review Mem Inst Oswaldo Cruz, v. 99, no. 3, pp. 239-51, 2004).
  • LC cutaneous leishmaniasis
  • ML mucocutaneous or mucosal leishmaniasis
  • LCD diffuse cutaneous leishmaniasis
  • L, brazi ⁇ iensis is the main etiologic agent of LC in South America. It is characterized by chronicity and latency aspects, in addition to developing mucosal metastases that lead to disfiguring clinical pictures of LM (MARSDEN, PD Mucosal). leishmaniasis ("espund ⁇ a” EscomeL 191.) Trans R Sound Trop Med Hyg, v. 80, no. 6, pp. 859-76, 1986. ISSN 0035-9203 (Print), Mucosal leishmaniasis due to Leishmania (Viann ⁇ a) brazi ⁇ ensi L (V) b in Tres Bradictions, Bahia-Brazil Rev. Soe Bras Med Trop, v.
  • Lesions may be infiltrative, atrophic-crusted, ulcer-vegetating, ulcer-destructive, mutilating, polyposing and may cause partial or total destruction of the naso-pharyngeal region of patients (Desjeux P 2001. The increase in risk factors for leishmaniasis wor! dwide. Transactions of the Royai Society (Tropical Medicine and Hygiene, 95, 239-243),
  • the definitive diagnosis of the disease is based on clinical manifestations associated with detection of parasites by examination of smears stained with material collected from lesion biopsies, culture in a sterile environment or inoculation in laboratory animals.
  • the epidemiological relationship between individuals in an endemic area is evident and is important information for the diagnosis of travelers living in non-endemic areas who eventually came into contact with endemic areas of the disease.
  • early diagnosis is critical to prevent permanent damage and severe functional sequelae to patients. Symptoms of SCI may be similar to those of other infectious diseases such as tuberculosis, paracoccidioidomycosis and leprosy.
  • Non-infectious diseases such as granuloma and neoplasms
  • SC Commonly-infectious diseases
  • Non-infectious diseases such as granuloma and neoplasms
  • Antibody subclass profile against Leishmania braziliensis and Leishmania amazonensis in the diagnosis and follow-up of mucosal leishmaniasis Diagnostic microbiology and infectious disease, 47, 477-485).
  • Detection of the causative agent of all infectious disease is a determining step for clinical management and / or disease monitoring.
  • Leishmaniasis is a challenge for clinical and laboratory diagnosis and a cause of constant concern for public health worldwide, as the number of cases of therapeutic failure with conventional treatment has increased (Alvar J, Croft S, Olliaro P 2006. Chemotherapy in the treatment and contrai of leishmaniasis (Advances in parasitology, 61, 223-274).
  • Direct diagnostic methods which demonstrate the presence of the parasite in the sick individual, are considered ideal for the diagnosis of the disease.
  • the practice has limitations that can be compensated by indirect methods based on immune response of infected hosts (Kar K 1995. Serodiagnosis of leishmaniasis. Critical reviews in microbiology, 21, 123-152).
  • the sensitivity in smear observation depends on the patient's time of disease, age and gender; the time of existence of the lesion; the parasite species; besides its distribution in the lesion, which may not be homogeneous (Boggild AK, Ramos AP, Espinosa D, Valencia BM, Veland N, Miranda-Verastegui C, Arevalo J, Low DE, Llanos-Cuentas A 2010. Clinical and demographic stratification of test performance: a pooled analysis of five laboratory diagnostic methods for American cutaneous leishmaniasis (The American Journal of Tropical Medicine and Hygiene, 83, 345-350).
  • Serological diagnosis is based on the detection of parasite-specific antibodies and / or antigens by the infected host, and is an alternative for the diagnosis of SCI.
  • One of the disadvantages of these tests has been their low sensitivity, observed in patients with low parasite-specific antibody titers, or their specificity, due to the antibody cross-reactivity of patients with other leishmaniasis-related diseases. (Guimar ⁇ es MC, Celeste BJ, Franco EL 1986. Evaluation of dot enzyme-linked immunosorbent assay for mucocutaneous leishmaniasis and comparison with microplate enzyme immunoassay.
  • the humoral response in SCI is of moderate intensity compared to the immune response observed in patients with VC.
  • the humoral response in SCI is generally associated with elevated circulating antibody levels; however, it is associated with an ineffective cellular response to eliminate the infection.
  • the ELISA technique is considered an important tool for the diagnosis of SCI, although this test uses antigens obtained from crude fractions and / or semi-purified parasite molecules. The technique allows for improved sensitivity in order to differentiate SCI patients from those with diseases that can cause cross-reactivity (Junqueira-Pedras et al., 2003). Other reasons why ELISA has enormous potential as a diagnostic method is the possibility of technological innovations that improve its effectiveness.
  • Phage display technology has been used in several studies for the research of new antigens, so that in the form of bacteriophage clones expressing peptides of interest, or in the form of synthetically produced individual peptides; can be tested for laboratory diagnosis of various diseases, such as viral infections, cancer and autoimmune diseases; as well as in the production of recombinant vaccines (Manoutcharian K, Terrazas LI, Gevorkian G, Acero G, Petrossian P, Rodrigues M, et al.
  • Phage-displayed T-cell epitope grafted into immunoglobulin heavy-chain complementarity-determining regions an effective vaccine design tested in murine cysticercosis Infect Immun 1999; 67: 4764-4770; Noya, O; Patarroyo, ME; Guzman, F.; Alarcon De Noya, B.; Immunodiagnosis of parasitic diseases with synthetic peptides. Sci., V. 4, pp. 299-308, 2003; Manoutcharian K, Diaz-Orea A, Gevorkian G, Fragoso G, Acero G, Gonzalez E, et al., Recombinant bacteriophage-based multiepitope vaccine against Taenia solium pig cysticercosis.
  • Phage display technology is based on the expression of peptides, proteins or antibody fragments associated with proteins present on the outer surface of recombinant bacteriophages.
  • the nucleotide sequence encoding the inserted peptides is genetically fused to a sequence encoding some bacteriophage proteins, resulting in a hybrid product, which is then exposed on the outer surface of viral particles (Smith GP Filamentous fusion phage: novel expression vectors que display cloned antigens on the virion surface (Science, v. 228, pp. 1315-1317, 1985).
  • Bacteriophage libraries expressing exogenous peptides have been used to identify various cell receptors, facilitating the identification of small molecules that bind with high affinity and mimic the interaction with their natural ligands, as well as the identification of antibody-interacting peptides without need for prior knowledge of the antigenic region recognized by the antibody.
  • the selection of high affinity molecules with respect to a given target receptor, adhered to a solid surface is performed by consecutive selection steps called "bio-panning cycles". The number of biohazard cycles performed on the phage display determines the degree of enrichment of the phages that bind to a target receptor.
  • a population of high affinity phage clones with respect to a given receptor can be obtained by performing 3 to 5 cycles of bio-panning (Crameri R, Suter M. Display of biologically active proteins on the surface of filamentous phages: a cDNA cloning system for selection of functional gene products linked to the genetic information responsible for their production (Gene 1993; 137: 69-75, 1993).
  • bio-panning cycles can be tailored according to the research group that mastered such technology. For example, in the case of the present invention, IgG class antibodies from patient sera to be investigated were fused to the surface of microspheres (called "beads") and such a hybrid was subjected to negative or positive uptake (selection) processes. to phage clones present in the recombinant library used.
  • Patent document BR102013027542 entitled “Canine Visceral Leishmaniasis Vaccine Composition, Synthetic Peptides and Use" describes a vaccine composition for the prevention and or treatment of canine visceral leishmaniasis, using as peptides expressed on the external surface of bacteriophages. non-harmful to the mammalian host, or synthesized, either alone or in combination.
  • the technology also describes the use of these peptides in the preparation of vaccine compositions against visceral leishmaniasis. These peptides were selected by the phage display technique and induce Th1-specific immune response, highlighted by the higher production of IFN-gamma.
  • This technology differs from the present invention in that it comprises peptides other than the six peptides described herein. Furthermore, in the present invention, the 6 antigens were identified by antibodies in the sera of human patients. with LM, which widens the range of possibilities for using them both in humans and in other mammals.
  • Patent Document BR102013033627 entitled “Synthetic Peptides, Method and Kit for Immunodiagnosis of Canine Visceral Leishmaniasis and Human Tegumentary and Visceral Leishmaniasis” describes peptides, a method and kit for immunodiagnosis of leishmaniasis in humans and / or dogs, and use of peptides derived from B cell epitopes to identify individuals and animals infected with visceral and cutaneous leishmaniasis in diagnostic method and diagnostic kit.
  • This technology differs from the present invention in that it comprises peptides other than the six peptides described herein.
  • the 6 antigens have been identified using sera from human SCI patients, which broadens the range of possibilities for use in both humans and other mammals.
  • the experimental strategy was performed using dog sera.
  • Patent document PI0804859-2 entitled “Synthetic peptides for obtaining protein polymer for immunization against leishmaniasis, products and their uses” refers to two synthetic antigenic peptides (epitopes / mimotopes) selected and synthesized by the techniques. of phage display and spot synthesis, and the process of obtaining a protein polymer obtained by conjugating these peptides, the use of this protein polymer, the vaccine composition produced with it, the vaccination method, the disease diagnosis method and the diagnostic kit.
  • This technology differs from the present invention in that it comprises peptides other than the six peptides described herein. Also, as described in the present invention, the 6 antigens were identified using human patient sera.
  • the present technology deals with 6 (six) highly specific and reactive synthetic peptides with human mucosal leishmaniasis patient samples, as well as a method and kit for the symptomatic and asymptomatic mucosal leishmaniasis, which use the 6 peptides as antigens. synthetic, isolated or associated, or bacteriophage clones containing such peptides, isolated or associated.
  • the present technology deals with 6 (six) highly specific and reactive synthetic peptides with samples from patients with human mucosal leishmaniasis, as well as a method and kit for the symptomatic and asymptomatic mucosal leishmaniasis, which use the 6 peptides as antigens.
  • the present invention addresses the peptides identified by the following amino acid sequences: ASFLKNR (Seq ID No. 1), SSPFLFS (Seq ID No. 2), RSMEIDR (Seq ID No. 3), LEKVFSP (Seq ID No. 4), KFTLKAR (Seq ID No. 5), MKFTLNA (Seq ID No. 6), of bacteriophages capable of expressing such peptides; and the use of such peptides and / or bacteriophages in the diagnosis of human mucosal leishmaniasis.
  • ASFLKNR Seq ID No. 1
  • SSPFLFS Seq ID No. 2
  • RSMEIDR Seq ID No. 3
  • LEKVFSP Seq ID No. 4
  • KFTLKAR Seq ID No. 5
  • MKFTLNA MKFTLNA
  • step (b) exposing a sample to at least one of the peptides defined by the amino acid sequences SEQ ID NOS. 1 to 6, or to at least one of the bacteriophage clones expressing each of the peptides defined by the amino acid sequences SEQ ID NOS. 1 to 6, such peptides or bacteriophages being attached to a solid support or a carrier;
  • B addition of a secondary antibody or protein which is conjugated to an enzyme or label and which binds to the antibodies of the sample from step (a);
  • step "a" the samples used are selected from the group consisting of blood, serum, plasma and / or other body fluid.
  • the secondary antibody may be IgG, IgM, IgA, IgE or their subclasses;
  • the protein may be Protein A and / or Protein G;
  • the enzyme which is conjugated to the secondary antibody or protein is selected from the group comprising peroxidase, alkaline phosphatase, beta-galactosidase, urease, xanthine oxidase, glucose oxidase and penicillinase.
  • the marker is selected from the group comprising enzymes, radioisotopes, biotin, chromophores, fluorophores and chemiluminescent.
  • the enzyme or label detecting reagent is selected from the group comprising any chromogen substrate that is recognized by any of the above enzymes, or any of the above markers.
  • the kit for the diagnosis of human mucosal leishmaniasis is characterized by: a. solid or carrier support containing at least one of the synthetic peptides defined by amino acid sequences SEQ ID NO: 1 to 6 or at least one of the bacteriophage clones expressing at least one of the peptides defined by amino acid sequences SEQ ID NO: 1 to 6; B. secondary antibody or protein, conjugated to an enzyme or a marker; ç. reagent to detect the enzyme or label.
  • the solid support of item "a” may be selected from the group of materials comprising nitrocellulose, nylon, latex, polypropylene and / or polystyrene. Preferably, it should consist of 96-well microtiter plates, tubes, beads or nitrocellulose and / or nylon papers.
  • the secondary antibody may be IgG, IgM, IgA, IgE and / or their respective subclasses and the protein may be protein A and / or protein G.
  • the enzyme that is conjugated to the secondary antibody or protein may be selected from the group comprising peroxidase, alkaline phosphatase, beta-galactosidase, urease, xanthine oxidase, glucose oxidase and penicillinase.
  • the marker may be selected from the group comprising enzymes, radioisotopes, biotin, chromophores, fluorophores and / or chemiluminescent.
  • the reagent for detecting the enzyme or marker of item "c” may be selected from the group comprising chromogen substrates that are recognized by any of the above enzymes, or any of the above-mentioned markers.
  • the microspheres adsorbed with the antibodies were washed 3 times in 0.1 M MES pH 5.0 buffer to remove the non-adherent IgG antibodies.
  • the ea / s-antibody system was washed 2 times with 1 ml of 0.2 M triethanolamine buffer pH 8.2 and resuspended in 1 ml of covalent binding buffer (20 mM dimethylpimelinidate / HCl in triethanolamine buffer) for 30 min under constant stirring at room temperature.
  • the reaction was neutralized by incubating the system with 1 ml of 50 mM Tris buffer pH 7.5 for 15 min at room temperature.
  • the incorporated microspheres were washed 3 times in TBS-T 0.1% Tween 20 buffer and blocked with blocking solution (5% BSA in TBS-T 0.05% Tween 20) for 1 h at 37 °. C, then resuspended in 200 ⁇ T_ TBS buffer.
  • 5 ⁇ _ of the IgG-coated beads were incubated for 1 h at 37 ° C with the anti-human IgG antibody (1: 5,000 dilution). After incubation, the beads were washed 3 times with 0.1% TBS-Tween and the reaction was revealed by the addition of the tetramethylbenzidine substrate. The reaction was then stopped by the addition of 2 N sulfuric acid and the absorbance reading was taken at 450 nm in a microplate reader (Titertek Multiskan Plus, Flow Laboratories, USA).
  • Bio-Panning Cycles For performing the bio-panning cycles, 1 x 10 12 viral particles from a recombinant library containing filamentous phage fused peptides (Ph.D.-C7C commercial library obtained from New England, BioLabs ® , USA) were used. They were diluted in 190 ⁇ _ 0.1% TBS-Tween for selection of ligands in the IgG antibody-incorporated microspheres.
  • the recombinant bacteriophage library was incubated for 30 minutes at room temperature with 100 ⁇ _ of the IgG-coupled microspheres from healthy individuals. Then the microspheres were precipitated by magnetic attraction to the Dynal Biotech support (no. 12020), being the The supernatant containing the non-IgG adherent clones was transferred to a new tube containing 100 ⁇ _ of the IgG-coupled microspheres of the CD patient group. The microspheres were then precipitated by magnetic attraction to the Dynal Biotech support (No. 12020), and the supernatant containing only phage clones that did not adhere to such antibodies was recovered.
  • the supernatant collected and recovered during negative selection was transferred to a new tube containing the IgG-coupled microspheres of SCI patients, and incubated for 30 minutes at room temperature.
  • Microspheres containing the ea / s-antibody system precipitated from the antibody phage bio-panning tubes of the LM patients containing the phages of interest were washed 10 times with 0.1% TBS-Tween 20 and eluted at 500 ⁇ . of 0.2 M glycine buffer pH 2.0, such solution being neutralized by the addition of 75 ⁇ . of Tris-base pH 9.0. Phage clones were recovered to perform the second step of the positive selection process.
  • microspheres containing the ea / s-antibody system precipitated in the antibody phage bio-panning tubes of the LC patients containing the phages of interest were washed 10 times with 0.1% TBS-Tween 20 and eluted at 500 ⁇ . of 0.2 M glycine buffer pH 2.0, such solution being neutralized by the addition of 75 ⁇ . of Tris-base pH 9.0. The process was repeated two more times, totaling 3 bioselection cycles. After such procedure, the clones were individually titrated.
  • the culture was transferred to new tubes containing 3 ml Top agar (10 g Bacto-Tryptone, 5 g yeast extract, 5 g NaCl, 7 g agarose, 1 g MgCl 2 6H20 per 1 L of deionized water) and spread in a Petri dish containing solid LB medium plus IPTG (0.5 mM) / Xgal (40 pg / mL) and ampicillin (100 mg / mL). For each titration, a culture plate was made. The plates were incubated at 37 O C for 16 h. Afterwards, the blue and individualized colonies were manually quantified. To estimate the titre values, the total number of counted colonies was multiplied by the dilution factor of each plate.
  • E. coli ER2738 colonies containing the selected bacteriophages were collected for DNA extraction.
  • 1.2 ml of early-log phase E. coli ER2738 cell culture (OD6oonm ⁇ 0.3) was distributed into each well of a deepwell plate. With sterile toothpicks, 96 blue colonies were removed from the Petri dish and transferred to the culture dish. To each well, only one phage colony was added, totaling 96 colonies on the plate. It was sealed and incubated for 5 h under constant agitation at 37 ° C. After incubation, the plate was centrifuged for 20 min at 2,250 xg and the supernatant transferred to another plate.
  • the 35-cycle reaction occurred in a plate thermocycler under the following conditions: denaturation at 95 S C for 20 sec, annealing at 58 S C for 15 sec and extension at 60 S C for 60 sec.
  • the generated amplicons from the sequencing reaction were precipitated with 1 L of ammonium acetate and 27.5 L of absolute ethanol.
  • the plate was centrifuged for 45 min at 2,432 xg and the supernatant was discarded.
  • 150 l of 70% ethanol was added to the pellet and the material was centrifuged again for 10 min at 2,432 xg and the supernatant discarded.
  • the plate was inverted on a paper towel and in this position was centrifuged at 486 xg for 1 min.
  • EXAMPLE 3 - ELISA Assays for Selection of Specific Bacteriophages for Diagnosis of Human Mucosal Leishmaniasis
  • 96-well ELISA plates (Jet Biofil) were sensitized for 16 h at 37 S C with 100 ⁇ ⁇ - of a solution containing 1 x 10 9 phages from each of the 48 phage clones and diluted in carbonate buffer. bicarbonate pH 9.6.
  • Table 1 Results of the absorbances found for each phage clone tested in reaction with a sera pool consisting of samples from patients with (positive) LM. The result obtained using a wild phage clone as a control was used. Such a clone does not express exogenous peptides of interest, and serves as phage control. The value considered "real" was obtained by the difference found between the absorbance value found for each clone minus the value obtained by the reaction of wild phage with the pool of positive sera. As ELISA tests were performed on the same day and at the same time, the value found for the wild clone was unique.
  • Table 2 Absorbance results found for each phage clone tested in reaction with a sera pool consisting of samples from healthy (negative) individuals. The result obtained using a wild phage clone as a control was used. Such a clone does not express exogenous peptides of interest, and serves as phage control. The value considered "real" was obtained by the difference found between the absorbance value found for each clone minus the value obtained by the reaction of wild phage with the pool of negative sera. As ELISA tests were performed on the same day and at the same time, the value found for the wild clone was unique.
  • Table 3 Ratio of actual absorbance values for each phage clone evaluated. The ratio was determined by dividing the reaction values with positive and negative serum pools for each clone evaluated. SLA L. braziliensis was used as a control. Clones with a ratio greater than or equal to that found for the SLA (1, 7) were selected ( * ).
  • Table 4 Sequence of the 6 peptides specific for human ML serodiagnostics.
  • EXAMPLE 4 Evaluation of the efficiency and specificity of the diagnostic test for human mucosal leishmaniasis using bacteriophages expressing each of the six selected peptides
  • Table 5 Diagnostic performance of phage clones selected for mucosal leishmaniasis serodiagnosis. Abbreviations: If: Sensitivity. Sp: specificity. 95% CI: confidence interval. AUC: area over the curve. SLA: soluble L. braziliensis antigen. FS: wild phage.

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Abstract

La présente technologie concerne 6 (six) peptides synthétiques hautement spécifiques et réactifs avec des échantillons de patients atteints de leishmaniose muqueuse humaine, outre une méthode et une trousse pour le diagnostic de la leishmaniose muqueuse symptomatique et asymptomatique, utilisant comme antigènes les 6 peptides synthétiques, isolés ou associés, ou des clones de bactériophages exprimant ces peptides, isolés ou associés.
PCT/IB2016/057976 2015-12-23 2016-12-23 Peptides sythétiques, méthode et trousse pour le diagnostic de la leishmaniose muqueuse humaine, et utilisation Ceased WO2017109763A1 (fr)

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Citations (5)

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Publication number Priority date Publication date Assignee Title
BR102013027542A2 (pt) * 2013-10-25 2015-09-08 Univ Minas Gerais composição vacinal contra leishmaniose visceral canina, peptídeos sintéticos e uso
BR102013033627A2 (pt) * 2013-12-27 2015-09-22 Univ Minas Gerais peptídeos sintéticos, método e kit para imunodiagnóstico da leishmaniose visceral canina e das leishmanioses tegumentar e visceral humana
BR102013013069A2 (pt) * 2013-05-27 2015-11-10 Univ Minas Gerais método, kit para teste imunodiagnóstico de leishmaniose visceral canina e vacina
BR102014004107A2 (pt) * 2014-02-21 2015-11-17 Univ Minas Gerais método e kit para diagnóstico das leishmanioses utilizando peptídeos sintéticos
BR102014013193A2 (pt) * 2014-05-30 2019-02-26 Univ Minas Gerais método e kit para diagnóstico das leishmanioses utilizando peptídeos sintéticos derivados do gene codificador da proteína quinase ativada por mitógeno 3 (putativa)

Patent Citations (5)

* Cited by examiner, † Cited by third party
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